Pl: MCCLUSKEY, LYNNETTE Marie 

Received: 08/05/2021 

Competition ID: FORMS-F 

1R21 DC019832-01 A1 

IPF: 676605 

Former Number: 1R21DC019832-01 

IRG/SRG: NIC 

Subtotal Direct Costs 

Year 1: 

Year 2: 

Senior/Key Personnel: 

LYNNETTE MCCLUSKEY 

Lin Gan 

Daniel Linder 

Camille King 

Michael King 

Xin-Yun Lu 

Title: Ace2 in the healthy and inflamed taste system 

Opportunity: PA-20-195 Council: 01/2022 

FOA Title: NIH Exploratory/Developmental Research Grant Program (Parent R21 

Clinical Trial Not Allowed) 

Dual: Accession Number: 4608611 

Organization: 

Department: Neuroscience and Regen. Med. 

AIDS: N 

Animals: Y 

Humans: N 

Clinical Trial: N 

Current HS Code: 10 

HESC: N 

HFT: N 

Organization: 

Expedited: N 

New Investigator: 

Early Stage Investigator: 

Role Category: 

PD/Pl 

MPI 

Co-Investigator 

Consultant 

Consultant 

Consultant

1
APPLICATION FOR FEDERAL ASSISTANCE 

SF 424 (R&R) 

1. TYPE OF SUBMISSION* 

0MB Number 4040.0010 

Expiration Date 12/31/2022 

3. DATE RECEIVED BY STATE State Application Identifier 

4.a. Federal Identifier 

DC019832 

0 Pre-application • Application 0 Changed/Corrected 

Application 

b. Agency Routing Number 

2. DATE SUBMITTED Application Identifier 

37562 

5. APPLICANT INFORMATION 

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on matters involving this application 

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Organizational DUNS*: 9666686910000 

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Small Business Organization Type 0 Women Owned O Socially and Economically Disadvantaged 

8. TYPE OF APPLICATION* 

ONew 

0 Renewal 

• Resubmission 

O Continuation O Revision 

Is this application being submitted to other agencies?* 

9. NAME OF FEDERAL AGENCY* 

National Institutes of Health 

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT* 

Ace2 in the healthy and inflamed taste system 

12. PROPOSED PROJECT 

Start Date· 

04/01/2022 

Tracking Number: GRANT13439458 

Ending Date· 

03/31/2024 

If Revision, mark appropriate box(es). 

O A. Increase Award O B. Decrease Award O C. Increase Duration 

O D. Decrease Duration O E. Other {specify) : 

OYes e No What other Agencies? 

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER 

TITLE: 

13. CONGRESSIONAL DISTRICTS OF APPLICANT 

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Page 1 

Funding Opportunity Number: PA-20-195 . Received Date: 

2021-08-05T182152.000-04:00
SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2 

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION 

Prefix: First Name*: LYNNETTE Middle Name: Marie Last Name*: MCCLUSKEY Suffix: 

Position/Title: Professor

Organization Name*: 

Department: 

Division: 

Street1*: 

Street2: 

City*: 

County: 

State*: 

Province: 

Country*: USA: UNITED STATES

ZIP / Postal Code*: 

Phone Number*: Fax Number: Email* 

15. ESTIMATED PROJECT FUNDING 

a. Total Federal Funds Requested* 

b. Total Non-Federal Funds* 

c. Total Federal & Non-Federal Funds* 

d. Estimated Program Income* 

16. IS APPLICATION SUBJECT TO REVIEW BY STATE 

EXECUTIVE ORDER 12372 PROCESS?* 

a. YES ❍ THIS PREAPPLICATION/APPLICATION WAS MADE 

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372 

PROCESS FOR REVIEW ON: 

DATE: 

b. NO ● PROGRAM IS NOT COVERED BY E.O. 12372; OR 

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR 

REVIEW 

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein 

are true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with 

any resulting terms if I accept an award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to 

criminal, civil, or administrative penalties. (U.S. Code, Title 18, Section 1001) 

● I agree* 

* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions. 

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name: 

19. AUTHORIZED REPRESENTATIVE 

Prefix: Ms. First Name*: Sheree Middle Name: Last Name*: Wright Suffix: 

Position/Title*: Interim Executive Director

Organization Name*: .

Department: 

Division: 

Street1*: 

Street2: 

City*: 

County: 

State*: 

Province: 

Country*: USA: UNITED STATES

ZIP / Postal Code*: 

Phone Number*: Fax Number Email*: 

Signature of Authorized Representative* 

Sheree Wright

Date Signed* 

08/05/2021

20. PRE-APPLICATION File Name: 

21. COVER LETTER ATTACHMENT File Name:2021.8_GanCoverLetter_ACE2.pdf 

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195 . Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 2 
424 R&R and PHS-398 Specific 

Table Of Contents 

SF 424 R&R Cover Page 1 

Table of Contents 3 

Performance Sites 4 

Research & Related Other Project Information 5 

Project Summary/Abstract(Description) 6 

Project Narrative 7 

Facilities & Other Resources 8 

Equipment 13 

Research & Related Senior/Key Person 15 

PHS398 Cover Page Supplement 42 

PHS 398 Modular Budget 44 

Personnel Justification 47 

PHS 398 Research Plan 48 

Introduction to Application 49 

Specific Aims 50 

Research Strategy 51 

PHS Human Subjects and Clinical Trials Information 57 

Vertebrate Animals 58 

Multiple PD/PI Leadership Plan 59 

Bibliography & References Cited 60 

Letters of Support 66 

Resource Sharing Plan(s) 69 

Authentication of Key Biological and/or Chemical Resources 70 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Table of Contents Page 3 
Contact PD/Pl: MCCLUSKEY, LYNNETTE Marie 0MB Number: 4040-0010 

Expiration Date: 12/31/2022 

Project/Performance Site Location(s) 

Project/Performance Site Primary Location 

Organization Name: 

Duns Number: 

Street1•: 

Street2: 

City•: 

County: 

State•: 

P

Co

rov

un

in

try•: 

ce: • 

USA: UNITED STATES 

Zip / Postal Code•: 

Project/Performance Site Co

-

ngressional District-: 

Additional Location(s) File Name: 

Tracking Number: GRANT1 3439458 

O I am submitting an application as an individual, and not on behalf of 

a company, state, local or tribal government, academia, or other type of 

organization. 

-

Page 4 

Funding Opportunity Number: PA-20-195. Received Date: 

2021-08-05T18:21 :52.000-04:00 
OMB Number: 4040-0010

Expiration Date: 12/31/2022

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No 

1.a. If YES to Human Subjects 

Is the Project Exempt from Federal regulations? ❍ Yes ❍ No 

If YES, check appropriate exemption number: 1 2 3 4 5 6 7 8 

If NO, is the IRB review Pending? ❍ Yes ❍ No 

IRB Approval Date: 

Human Subject Assurance Number 

2. Are Vertebrate Animals Used?* ● Yes ❍ No 

2.a. If YES to Vertebrate Animals 

Is the IACUC review Pending? ● Yes ❍ No 

IACUC Approval Date: 

Animal Welfare Assurance Number D16-00197

3. Is proprietary/privileged information included in the application?* ❍ Yes ● No 

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No 

4.b. If yes, please explain: 

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an 

environmental assessment (EA) or environmental impact statement (EIS) been performed? 

❍ Yes ❍ No 

4.d. If yes, please explain: 

5. Is the research performance site designated, or eligible to be designated, as a historic place?* ❍ Yes ● No 

5.a. If yes, please explain: 

6. Does this project involve activities outside the United States or partnership with international 

collaborators?* 

❍ Yes ● No 

6.a. If yes, identify countries: 

6.b. Optional Explanation: 

Filename 

7. Project Summary/Abstract* McCluskey_Abstract_2.pdf

8. Project Narrative* McCluskey_Narrative_2.pdf

9. Bibliography & References Cited McCluskey_Bibliography_FINAL.pdf

10.Facilities & Other Resources McCluskey_Facilities_2.pdf

11.Equipment McCluskey_Equipment_2.pdf

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195. Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 5 
PROJECT SUMMARY / ABSTRACT 

Taste deficits are prevalent in people infected with SARS-CoV-2, the virus responsible for the 

global COVID-19 pandemic. The loss of taste sensation negatively affects nutrition and quality 

of life and in some patients this deficit is long-lasting. The biological basis for taste loss due to 

SARS-CoV-2 is largely unknown. Our preliminary results demonstrate that the ACE2 receptor 

and TMPRSS2 which together mediate SARS-CoV-2 host cell entry are expressed in taste buds 

indicating their potential for viral infection. The function of taste cell ACE2, also a member of 

the renin-angiotensin system that regulates fluid balance, is unknown. We have developed 

three novel genetic mouse strains to overcome the limitations of currently available mouse 

models. In aim 1 we map ACE2 reporter expression to determine which taste receptor cell 

populations and pathways are potential targets of SARS-CoV-2. In aim 2 we test how lingual 

epithelium-specific ACE2 contributes to taste receptor cell dynamics and neurophysiological 

taste responses under baseline and inflammatory conditions. We will also test how taste 

function is affected by human SARS-CoV-2 spike protein in a humanized ACE2 knock-in 

mouse. Our hypothesis predicts that taste buds are SARS-CoV-2 targets, that taste ACE2 

contributes to taste function and is protective during inflammation, and that SARS-CoV-2 spike 

protein will exacerbate damage in taste buds and depress neural taste responses under 

inflammatory conditions. This R21 Exploratory / Developmental grant application addresses the 

urgent need for fundamental insights to mechanisms underlying taste dysregulation in people 

with COVID-19. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Project Summary/Abstract Page 6
NARRATIVE 

Taste loss is a common symptom of infection by SARS-CoV-2, the virus which causes COVID19 by using the angiotensin converting enzyme (ACE)2 receptor to enter host cells. We will use 

novel mouse models to map ACE2-expressing cells in the taste system and determine the 

receptor’s role in taste function under normal and inflammatory conditions. These studies will 

provide much-needed insight to mechanisms responsible for taste dysfunction due to SARSCoV-2. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Project Narrative Page 7
FACILITIES AND OTHER RESOURCES 

Intellectual Environment. There are currently 24 primary faculty in the 

. Faculty recruitment is a priority with 7 faculty hired since 2019 and searches ongoing. 

Neurologic disease is a major area of research emphasis at MCG, including over 60 neuroscience faculty. 

Neuroscientists are located in multiple clinical and basic science departments, research units, and institutes at 

the medical campus. The PIs’ home department is largely focused on neuroscience research and targeted 

recruiting has strengthened the focus on neural degeneration and regeneration. Thus, there are excellent 

opportunities for interaction with other neuroscientists including groups studying sensory systems, spinal cord 

injury, and neuroimmunology. New core facilities, including Dr. Gan’s Transgenic and Gene Editing Core 

Laboratory, used to generate novel mouse models for this application, are another benefit in the growing 

department. We also enjoy frequent interaction with external experts as part of the weekly 

seminar series, an annual Neuroscience Retreat, and numerous other 

weekly seminars and grand rounds, now held in virtual formats. Research in immunology is based in the 

Cancer, Immunology, Inflammation & Tolerance (CIT) group within the Cancer Center, and Dr. McCluskey has 

established relationships with this group as the former director of the Graduate Program in Molecular Medicine 

which focuses on immunology. Dr. McCluskey now directs the Graduate Program in Neuroscience which 

provides frequent interaction with student and faculty neuroscientists. In sum, the PIs’ research programs 

have excellent collaborative and intellectual support for both the neural and immune components of the 

proposed project. 

McCluskey Laboratory. Dr. McCluskey’s laboratory occupies ~1100 square feet in CA-3059. The laboratory 

is equipped with two neurophysiology and surgical stations, and image analysis system for fixed tissue and cell 

quantification, an image analysis system for measuring fluorescence in live cells and tissues, Bio-Tek plate 

reader, Leica cryostat, fume hood, -20oC and -80oC freezers, a water purification system, and 

molecular/biochemistry equipment. Benchtop and desk space is available for up to 10 personnel. 

Gan Laboratory. Dr. Gan’s main laboratory occupies approximate 2,000 sq. ft. in the adjacent 

within the The laboratory 

is fully equipped for tissue culture (Thermo Scientific biosafety cabinet/CO2 incubator), molecular biology and 

biochemistry (Thermo Scientific -80oC freezer, Milli-Q IQ700 water purification system, NanoDrop OneC for DNA 

and RNA quantification, Eppendorf 5804 benchtop centrifuges, Eppendorf 5424 and 5424R microcentrifuges 

Bio-Rad QX200TM Droplet Digital PCR, Bio-Rad CFX qPCR system, Bio-Rad C1000 thermal cyclers, Diagenode 

Bioruptor sonicator, Bio-Rad Gel Doc XR+, Bio-Rad semi-dry blotter, Eppendorf Vacufuge Plus, bacterial 

incubators and shakers), immunohistochemistry and in situ hybridization (Cryostar NX50 cryostat, Hybaid 

rotating/shaking ovens), embryo/tissue dissection stations (Nikon SMZ1500 and SMZ-U stereoscopes), bright 

light/DIC/pifluorescent imaging stations (Nikon TE2000U inverted microscope with DS-Ri2 camera and Zeiss 

Lumar V12 stereoscope with AxioCam camera), NEPAGENE NEPA21 in vitro & in vivo electroporator for 

cultured cells or explants. 

Animals. The maintains a centralized animal care program with 

administrative responsibility assigned to the Office of Laboratory Animal Services. Animal facilities for this project 

are located in the building adjacent to the PI's lab, accessible by a short hallway and service elevator. The facility 

and program have been accredited by the Association for Assessment and Accreditation of Laboratory Animal 

Care International (AAALAC) since May 16th , 1978. is a Registered Research Facility with 

the United States Department of Agriculture (USDA No. 57-R-0002), and has an Office of Laboratory Animal 

Welfare OLAW Assurance Statement on file (D16-00197-01; valid until August 31, 2023). Importantly for the 

proposed studies on immune-gustatory interactions, the animal facilities are specific-pathogen-free (SPF). 

The routine animal husbandry and health monitoring services are provided by experienced vivarium veterinary 

staff consisting of several laboratory technicians and full-time veterinarians. 

Offices: The PIs’ adjacent offices occupy approximately 140 square feet each in and , home 

to the The PIs’ offices are near their laboratories in a 

wing with five other neuroscientist faculty members. Additional Institute faculty offices and laboratories are 

located on the floors immediately above and below. Dr. McCluskey’s office is equipped with a new Dell Optiplex 

and a Color LaserJet Pro printer. Dr. Gan’s office is equipped with one MacBook Pro laptop computer and one

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Facilities & Other Resources Page 8 
HP Laserjet 3525DN printer. The provides staff 

support, copier, FAX, and phone services. Co-Investigators Dr. Bloomquist and Dr. Linder have offices are 

located nearby in the Dental College of Georgia and the Department of Biostatistics, respectively. 

Computers: 

The McCluskey Lab is equipped with 5 Dell Optiplexes, 3 Dell Latitude laptops, HP color printers and Dell 

laptops for each neurophysiology rig. Each computer has Photoshop, GraphPad Prism, Origin, EndNote and 

other software. One desktop computer is equipped to run Nikon’s NIS Elements software for confocal image 

analysis. The majority of computers in the department were refreshed in 2020. 

The Gan Lab is equipped with multiple common Apple and PC computers for all students and postdocs. The 

computers have software for word processing, spreadsheets, statistical analysis, graphics, and DNA analysis. 

The computers are connected to the internet and HP network laserjet M533DN printer. Each computer is 

installed with word processing, Photoshop, and other data analysis software. Two imaging microscopes are 

each equipped a dedicated PC computer, digital camera and softwares. The IT department at 

provides technical assistance for electronic data management and hardware maintenance. 

Other: The PI’s home Department provides many high end, shared equipment that are relevant and available 

for this application, including Zeiss LSM700 confocal microscope system with software, Miltenyi Biotec 

UltraMicroscope II light sheet microscope for fast imaging of large 3D biological samples, two Nikon A1R-MP 

multiphoton microscopes with Mai Tai oscillators/multimaniuplator systems/patch clamp amplifiers, Keyence BZX810 fully-motorized all-in-one fluorescence microscope, and LI-COR Odyssey® CLx Imaging System for 

western blots. In addition, the Department provides free shared common equipment such as autoclave, 

glassware washing facility, high speed and ultra centrifuges (Beckmann and Sorvall). 

Departmental Facilities: 

Transgenic and Gene Editing Core Laboratory (Dr. Lin Gan, Director) is a full-service facility using state-ofthe-art facilities and equipment for the creation of novel transgenic, knockout, knock-in, and conditional knockout 

mouse strains. The Core’s facilities and equipment are located in CA3056, next to the PI’s laboratory, and include

two Nikon TE2000-S inverted microinjection microscope setups equipped with Eppendorf FemtoJet 4i electronic 

microinjectors/Nomarski Optics/Narishige micromanipulators for microinjections, Nikon surgical microscopes for 

egg and embryo isolation and transfer, Sutter P1000 micropipette puller for microinjection pipette preparation, 

biosafety cabinet/CO2 incubator/cell counter/electroporation system for ES cell culture and gene targeting, and 

C1000 PCR for mouse genotyping analysis. The Core’s surgical and mouse housing facilities are located in Suite

CA1117 on the 1st floor of CA building. The Core maintains all mice necessary for egg donors, egg recipients, 

and vasectomized males. 

Biostatistics and Bioinformatics Core (Director, Yang Shi, PhD). The Biostatistics and Bioinformatics Core 

is a shared departmental resource for biostatistics and bioinformatics consulting and support ranging from 

project design to data analysis. It is a critical component of the research infrastructure in the Department of 

Neuroscience & Regenerative Medicine. Statistical analyses provided include statistical design of biomedical, 

translational and clinical studies, including sample size and power analysis; testing of assumptions of statistical 

tests; applying regression-based models to identify factors associated with disease outcomes; development of 

novel statistical methods and models; and bioinformatics analysis for Bioinformatics Analysis RNA-seq, 

microarray, ChIP-seq, DNA methylation, and whole genome sequencing. 

Viral Vector Core (Director, Quansheng Du, PhD) The mission of the Viral Vector Core is to provide 

technological resources to faculty members 

interested in the use of viral based vectors for basic research and preclinical applications. The core is 

supported by the DNRM Research Enrichment Funds. The vector core focuses on producing lentiviral, 

gammaretroviral and recombinant adeno-associated viral (rAAV) particles for transgene expression, gene 

silencing and gene editing in either dividing or non-dividing cells. The following viral vectors are available 

from the core: lentiviral vectors, adeno-associated viral vectors and gammaretroviral vectors. 

Microscopy Facility (Director, Sergei Kirov, PhD). The imaging core is equipped with a Nikon A1R MP+ 

Multiphoton/Confocal Microscope with four high-sensitivity detectors for multiphoton imaging, seven visible 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Facilities & Other Resources Page 9 
laser lines for confocal imaging, a MaiTai eHP DS ultrafast laser, full electrophysiology recording capabilities, 

fast imaging: 420 frames/sec maximum speed, high-speed piezo-drive focus motor, simultaneous 

photostimulation and imaging, and a best-in-class two-photon objective. Another Nikon A1R MP+ 

Multiphoton/Confocal Microscrope has four visible laser lines for confocal imaging and the same capabilities as 

A1R system above except for electrophysiology. The facility also includes a Nikon C1 Confocal with three 

visible laser lines and an imaging workstation with deconvolution software (NIS Elements) 

Light-Sheet Microscopy Core. (Director: Alexis Stranahan, PhD) The 

operates an Imaging Core Facility with a newly installed Lavision Ultramicroscope II 

light-sheet microscope (Miltenyi Biotec). The Ultramicroscope II has an infrared laser and a second tunable 

laser for acquisition of up to three excitation channels (488/560/633). This system is capable of imaging 

samples up to 8x8cm in aqueous or organic media in Zoom Body or Super Plan configuration. In the Super 

Plan configuration with the 12x objective, this system can acquire images with a maximal XY resolution of 

0.217 microns/pixel. Resolution in the z-axis is determined by sample size, clearing efficacy, and laser 

configuration, but optical sections can be acquired at user-specified intervals for subsequent registration, 

segmentation, and high-throughput morphometric analysis. File size for tiled image acquisitions can reach 

500GB per sample, and Augusta University provides unlimited cloud storage on an internal secure ftp site for 

data transfer and backup purposes. The departmental imaging facility also has two analysis workstations; one 

with Neurolucida 360 Studio for automated neuron tracing, dendritic spine and synapse detection, and a 

second with NeuroInfo software for registration onto the Allen Brain Atlas, automated segmentation, and cell 

detection (both from Microbrightfield). 

Augusta University Medical College of Georgia Shared Core Facilities: All facilities are within the Medical 

Center complex. 

Electron Microscopy and Histology Core Laboratory: The mission of the Electron Microscopy and Histology 

Core is to provide high quality services at an affordable cost to research investigators at the Augusta University 

and external research facilities. The core occupies ~3,000 sq. ft. and is located on the first floor of the Carl 

Sanders Research and Education Building (CB1113). The Core is well equipped and offers a variety of specimen 

preparation services for transmission and scanning electron microscopy, vibratome, paraffin, cryostat and JB-4 

sectioning. The Core, which operates on a fee-for-service basis, offers routine H&E staining and various 

histological special stains, PASH, Masson Trichrome, Van Gieson and many others. The Core offers 

investigators a full line of immunohistochemical services including immuno-EM studies. It is a full-service facility, 

and augments research by providing expensive, specialized equipment and experienced technical staff to 

perform ultrastructural analyses. The Core staff performs all aspects of the ultrastructural procedures from 

specimen processing to imaging (for Electron Microscopy, TEM and SEM) and processing, sectioning and 

staining for light microscopy (paraffin, cryostat, and JB-4). The core is managed by Ms. Penny Roon (HT, 

ASCP). Additional staff members include Dr. Brendan Marshall, an expert in Electron Microscopy and 

Immunohistochemistry; Ms. Elizabeth Perry (MSA Certified), an expert in Electron Microscopy (TEM, SEM and 

IEM) and Immunohistochemistry; and Ms. Donna Kumiski, an ASCP Cerified Histotechnologist. Newly added 

services include sectioning of calcified bone specimens and EM-cryosectioning for EM-cryoimmunodetection. 

The Genomics Core Laboratory is located on the campus in the Interdisciplinary building 

(CA1041). It is a multifunctional molecular biology resource facility directed through the Center of Biotechnology 

and Genomic Medicine. Services are available to all campus investigators, students and incubator tenants, as 

well as off-campus users. Instruments available to users include: Life Technologies 3730XL genetic analysis 

instrument, Illumina Bead Station micro array instrument, Nano-Drop micro spectrophotometer, Fluidigm high 

throughput microfluidic-based genetic analysis instrument, Agilent Tapestation 2200 bioanalizer, Ion Torrent 

PGM next gen sequencer, Life Technologies 7900HT high throughput real time PCR instrument, RNA-seq, ChIPseq, targeted re-sequencing, and de novo assembly, on the Illumina HiSeq and Roche 454 GS-FLX platforms. 

For automated fluorescence DNA sequencing the state-of-the-art ABI 3730 XL 96-capillary sequencer is 

available for ultra-high throughput projects and the ABI Big Dye Terminator 3.1 is the default instrument also 

available to users. The staff is qualified in assisting investigators in analysis and interpretation of their results. 

Flow Cytometry Core: The AU Campus Flow Cytometry Core Facility (CFCCF located in CA2056) is equipped 

with flow cytometer equipment of 2 types 1) Cell Sorter - operated as a service to Investigators and 2) Cell 

Analyzer(s) - generally Investigator operated but can also be provided as a service as well. The Cell Sorter is 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Facilities & Other Resources Page 10 
the Beckman Coulter MoFlo and allows for 7-color fluorescence, 4-way or multiwell plate cell sorting and has 

recently been upgraded to allow for sorting of well characterized BSL2 materials (ATCC, NIH, etc.) approved by 

the Biological Safety Office in addition to the BSL1 materials it has historically sorted. The Cell Analyzers, two 

bench-top BD (BD Biosciences) FACSCalibur instruments, are capable of 4-color fluorescent analysis. 

Computers workstations attached to the analyzers are available for data analysis, presentation and publication 

preparation when not in use for acquisition purposes; additionally, there are 2 standalone MAC’s and one PC

available in a separate analysis room as well. There are several softwares available for analysis purposes; BD 

CellQuest Pro, Treestar Flowjo, Verity Software House (VSH) ModFit in addition to MS Office to aid in 

presentation preparation. Core personnel are available to aid in experiment design and implementation as well 

as data preparation. 

Cell Imaging Core: The mission of the cell imaging core is to offer investigators the use of state-of-the-art 

imaging equipment at a reasonable cost with expert training assistance if needed. The core, which is located in 

CB2201, offers confocal microscopy, multiphoton microscopy, digital light microscopy, computation, ratiometric 

analysis, image enhancement, 3D reconstruction, deconvolution and microinjection photo-uncaging and super 

resolution microscopy. The state-of-the art Carl Zeiss confocal microscopes offer imaging of live cells, thick 

sections and whole mouse studies. The Nikon Eclipse Ti super resolution scope equipped with STORM 

technology was recently installed. The specific instruments and their capabilities include: (1) Zeiss LSM510 

META Axiovert 200M confocal microscope, LSM software; (2) Zeiss 780 Upright Confocal Microscope with 

GaAsp detectors, modules include - 3D, Deconvolution, HDR, Tiling, atmosphere (CO2/O2) and temperature 

control (for live cells); (3) Zeiss 780 Upright Confocal Microscope with the Definite Focus feature, modules 

include Physiology, FRET, FRAP, 3D, Tiling; (4) Zeiss 780 Upright Multiphoton Microscope, a 2-photon confocal 

instrument for visualizing thick samples. It features a fixed stage suitable for whole mouse studies. All but one 

lens are ‘dipping lenses’, (image while immersed in saline-bathing live samples). This system offers temperature 

control in 35-60 mm dishes. Its modules include: Physiology, FRAP, RICS. This application will primarily use 

the Zeiss 780 upright microscope with lasers: 405 nm, 458, 488, 514 nm, 543 nm, 633 nm. It is equipped with 

five lenses: 5x EC Plan-Neofluar(dry) - 0.16 NA, 10x EC Plan-Neufluar(dry) - 0.30 NA, 20x PlanApochromat(dry) - 0.8 NA, 40x Plan-Apo(oil) - 1.4 NA, 63x Plan-Apochromat(oil) - 1.4 NA. Owing to its two 

GaAsp detectors this instrument offers excellent resolution and outstanding sensitivity. Image analysis utilizes 

Zen 2012 imaging software. (5) Nikon Eclipse Ti research inverted microscope equipped for super resolution 

microscopy using STochastic Optical Reconstruction Microscopy (STORM) technology. The system is under 

the control of the universal NIS-Elements platform and features the SR Apochromat TIRF 100X 1.49 N.A. 

objective lens, which provides the highest quality point spread function for N-STORM super-resolution imaging. 

Expert microscopists assist investigators in the use of this equipment. They train investigators (or members of 

their lab) in the use of the instrumentation, or work directly with investigators to obtain images on a fee for service 

basis. 

Integrated Genomics Shared Resource: This resource offers both Next Generation 

Sequencing (NGS) and Microarray technologies and data analysis. For NGS analysis, the facility equipped with 

Illuminas High-Seq 2500, MiSeq , Next-Seq and a Life Technologies Ion Torrent. The HiSeq 2500 permits highthroughput sequencing output generating up to 200Gb data per run or 2 billion paired end reads per run. The 

cBot cluster generation station is also available for automated generation of clusters. For high through-put 

computational analysis the Resource is equipped an IlluminaCompute analysis and storage system with 2 Rack 

and 8 Blade servers with 36 AMD 16-core CPUs and 64GB RAM from Dell Corporate running Linux, dedicated 

of computational analysis. This cluster includes a 2-way SMP system acting as front-end cluster nodes and a 

file server, a large memory 2-way SMP system acting as a web front-end and a database server. Storage with 

approximately 60TB of raw storage capacity is shared out to the computational nodes. All file systems are built 

on top of a parity RAID scheme using RedHat XFS. The core personnel will prepare samples and libraries or 

these can be user generated. The core personnel are well-versed at running the following applications: Targeted 

Sequencing analysis, Gene Regulation Analysis, Sequencing-Based Transcriptome Analysis, SNP Discovery 

and Structural Variation Analysis, Cytogenetic Analysis, DNA-Protein Interaction Analysis (ChIP-Seq), 

Sequencing-Based Methylation Analysis, Small RNA Discovery and Analysis. The Facility also has access to a 

variety of software including the TopHat suite of analyses algorithms, ANNOVAR, BWA, GSNAP, GATK 2, Partek 

Genomics Suite and CLC Bio Genomics Workbench which can be accessed by individual users. In addition, the 

Bioinformatics core has 2 individuals assigned specifically to perform novel and downstream analysis of NextGen sequencing data. The Facility computers also contain Illumina software, Partek Genomics Suite, Ingenuity 

Pathways Analysis and a wide range of freeware analysis programs which can be accessed by individual users 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Facilities & Other Resources Page 11 
including GATK 2.0 and the Tuxedo suite of programs for RNA-Seq data. In addition, the Bioinformatics core 

has 2 individuals assigned specifically to perform novel and downstream analysis of Next-Gen sequencing data. 

The GCC Integrated Genomics Facility also provides Affymetrix microarray technologies to faculty. The 

equipment and personnel are located within the GCC Cancer Center. The Resource aims to provide researchers 

with access to microarray technology and bioinformatics at an affordable cost, and to provide training to the 

research community through educational seminars for analysis of microarray data and programs designed to 

analyze large data sets. Equipment in this facility includes an Affymetrix GeneChip Scanner 3000 7G Plus, an 

Affymetrix Hybridization Oven 640, two Affymetrix Fluidics Station 450’s, an Agilent Microarray Scanner, an

Agilent Hybridization Oven, an Agilent 2100 Bioanalyzer, an Applied Biosystems GeneAmp 9700 Thermocycler, 

and a NanoDrop 1000 spectrophotometer. DNA sequencing and synthesis is also available. The available 

softwares for data analysis include; Affymetrix Genotyping Console, Affymetrix Expression Console, Affymetrix 

GCOS, GTYPE and CNAT, Clustall, CNAG, GO, Ingenuity Pathway Analysis, OGT CytoSure Visualization 

Software, Partek Genomics Suite, TreeView and Exon Easy. The facility personnel are well versed in the use of 

these various softwares for data analysis. The Pyrosequencer, and QPCT and associated software to be used 

in the epigenomics research projects is also located in this facility. 

offers 

collaborative research and services in the areas of bioinformatics. The staff has experience and expertise in 

bioinformatics software/database development, customized data analysis, and providing bioinformatics 

infrastructure and training. Services include, but are not limited to: development of bioinformatics software and 

databases, integrative analysis of multi-dimensional high-throughput data (e.g. gene expression, array 

CHIP-chip, DNA methylation tiling arrays, SNPs, DNA sequencing data, metabolomics, proteomics etc.), 

identification of transcriptional regulatory elements, mapping of pathways and gene ontology, gene annotation, 

identification of alternative splicing forms, etc. The Resource has a seamless partnership with the Department 

or Biostatistics at MCG coordinating the effort of providing bioinformatics service/support to the local biomedical 

research community. The Resource staff are responsible for management of the HPC 

Server composed 544 total compute cores and an aggregated memory of 2.9TB. The system is composed of 

(15) PowerEdge R430 1U systems (128 GB RAM each), (1) PowerEdge R830 (1024 GB RAM) and a high-speed 

10GbE interconnect for intra-node communication. The HPCC also houses 832 TB RAW storage capacity. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Facilities & Other Resources Page 12 
EQUIPMENT 

McCluskey Lab. The McCluskey lab occupies approximately 1500 sq. ft. in CA-3059. The lab is equipped 

with two complete neurophysiology rigs including: Zeiss OPMI surgical microscope, Powerlab hardware and 

software, Grass amplifier, P511 power source, Hum Bug 60 Hz noise reducer, integrator, oscilloscope, TMC air 

table with cage, and Dell Latitude laptops with each rig. We also have a live fluorescence imaging system with 

Olympus BX51WI microscope, Dell precision T3500 computer with wide monitor, MetaFluor software 

(Universal Imaging), Warner stimulus delivery system and multiple Warner RC-50 modified epithelial imaging 

chambers with custom 1 mm insert. Another image analysis system is equipped with Olympus BX50 

microscope, digital cameras for fluorescent and brightfield microscopy (Roper Scientific), Dell Optiplex 

computer and MetaMorph software. We also have a Leica cryostat, BioTek Plate reader, gel electrophoresis 

equipment and thermal cyclers, standard laboratory benchtop and surgical equipment, a refrigerated centrifuge 

(Fisher 1R), -80o and -20oC freezers and Dell desktop computers for personnel use in addition to those used 

with equipment. We have 24-hr access to Kodak gel documentation system, cell culture facilities (laminar flow 

hoods, incubators, inverted microscope) adjacent to PI’s laboratory, and a QuantStudio 3 real-time PCR 

system (ThermoFisher). 

Gan Laboratory. Dr. Gan’s main laboratory occupies approximately 2,000 sq. ft. in the adjacent rooms CA3055 

and CA3054A on the 3rd floor within the The laboratory 

is fully equipped for tissue culture (Thermo Scientific biosafety cabinet/CO2 incubator), molecular biology and 

biochemistry (Thermo Scientific -80oC freezer, Milli-Q IQ700 water purification system, NanoDrop OneC for DNA 

and RNA quantification, Eppendorf 5804 benchtop centrifuges, Eppendorf 5424 and 5424R microcentrifuges 

Bio-Rad QX200TM Droplet Digital PCR, Bio-Rad CFX qPCR system, Bio-Rad C1000 thermal cyclers, Diagenode 

Bioruptor sonicator, Bio-Rad Gel Doc XR+, Bio-Rad semi-dry blotter, Eppendorf Vacufuge Plus, bacterial 

incubators and shakers), immunohistochemistry and in situ hybridization (Cryostar NX50 cryostat, Hybaid 

rotating/shaking ovens), embryo/tissue dissection stations (Nikon SMZ1500 and SMZ-U stereoscopes), bright 

light/DIC/pifluorescent imaging stations (Nikon TE2000U inverted microscope with DS-Ri2 camera and Zeiss 

Lumar V12 stereoscope with AxioCam camera), NEPAGENE NEPA21 in vitro & in vivo electroporator for 

cultured cells or explants. 

Department of Neuroscience & Regenerative Medicine (NDRM) Facilities 

Transgenic and Genome Editing Core Facility 

The Genome Editing Core is a full-service facility supported by , 

and user fees. The Core’s mission is to provide timely and cost-effective services in all phases of 

transgenic mouse production by pronuclear microinjection into zygotes, gene targeting in embryonic stem 

cells (ESCs), and gene editing using CRISPR/Cas9 approach to the research community at Augusta 

University as well as other academic institutions and for-profit companies. The Core also provides services 

on preserving your valuable lines by cryopreservation of sperms and embryos, and resurrecting mouse lines 

using in vitro fertilization (IVF) or embryo transfer. Additional services include rederivation to create 

pathogen-free mouse lines, developing molecular reagents for genetic engineering, full colony management 

services, and expert advice on mouse genetics and breeding schemes. 

Microscopy The imaging core is equipped with a Nikon A1R MP+ 

Multiphoton/Confocal Microscope with four high-sensitivity detectors for multiphoton imaging, seven visible 

laser lines for confocal imaging, a MaiTai eHP DS ultrafast laser, full electrophysiology recording capabilities, 

fast imaging: 420 frames/sec maximum speed, high-speed piezo-drive focus motor, simultaneous 

photostimulation and imaging, and a best-in-class two-photon objective. Another Nikon A1R MP+ 

Multiphoton/Confocal Microscrope has four visible laser lines for confocal imaging and the same capabilities as 

A1R system above except for electrophysiology. The facility also includes a Nikon C1 Confocal with three 

visible laser lines and an imaging workstation with deconvolution software (NIS Elements). 

Light-Sheet Microscopy Core. Departmental facility recently became 

operational and personnel available for training in August, 2021 

The operates an Imaging Core Facility with a newly 

installed Lavision Ultramicroscope II light-sheet microscope (Miltenyi Biotec). The Ultramicroscope II has an 

infrared laser and a second tunable laser for acquisition of up to three excitation channels (488/560/633). This 

system is capable of imaging samples up to 8x8cm in aqueous or organic media in Zoom Body or Super Plan 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Equipment Page 13 
configuration. In the Super Plan configuration with the 12x objective, this system can acquire images with a 

maximal XY resolution of 0.217 microns/pixel. Resolution in the z-axis is determined by sample size, clearing 

efficacy, and laser configuration, but optical sections can be acquired at user-specified intervals for subsequent 

registration, segmentation, and high-throughput morphometric analysis. File size for tiled image acquisitions 

can reach 500GB per sample, and Augusta University provides unlimited cloud storage on an internal secure 

ftp site for data transfer and backup purposes. The departmental imaging facility also has two analysis 

workstations; one with Neurolucida 360 Studio for automated neuron tracing, dendritic spine and synapse 

detection, and a second with NeuroInfo software for registration onto the Allen Brain Atlas, automated 

segmentation, and cell detection (both from Microbrightfield). 

Biostatistics and Bioinformatics Core The Biostatistics and Bioinformatics Core 

is a shared departmental resource for biostatistics and bioinformatics consulting and support ranging from 

project design to data analysis. It is a critical component of the research infrastructure in the 

Statistical analyses provided include statistical design of biomedical, 

translational and clinical studies, including sample size and power analysis; testing of assumptions of statistical 

tests; applying regression-based models to identify factors associated with disease outcomes; development of 

novel statistical methods and models; and bioinformatics analysis for Bioinformatics Analysis RNA-seq, 

microarray, ChIP-seq, DNA methylation, and whole genome sequencing. 

Viral Vector Core The mission of the Viral Vector Core is to provide 

technological resources to faculty members 

interested in the use of viral based vectors for basic research and preclinical applications. The core is 

supported by the DNRM Research Enrichment Funds. The vector core focuses on producing lentiviral, 

gammaretroviral and recombinant adeno-associated viral (rAAV) particles for transgene expression, gene 

silencing and gene editing in either dividing or non-dividing cells. The following viral vectors are available 

from the core: lentiviral vectors, adeno-associated viral vectors and gammaretroviral vectors. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Equipment Page 14 
OMB Number: 4040-0010 

Expiration Date: 12/31/2022 

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix:  First Name*: LYNNETTE Middle Name Marie Last Name*: MCCLUSKEY Suffix: 

Position/Title*: Professor

Organization Name*:

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*:

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  PHD Degree Year:  1996

Attach Biographical Sketch*:             File Name: McCluskey_Biosketch_R21_Aug2021.pdf

Attach Current & Pending Support:   File Name:

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195 . Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 15 
PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Lin Middle Name  Last Name*: Gan Suffix: 

Position/Title*: Professor

Organization Name*:

Department: d

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*:

Phone Number*:  Fax Number: 

E-Mail*: ligan@augusta.edu

Credential, e.g., agency login

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  PhD Degree Year:  1992, 1985

Attach Biographical Sketch*:             File Name: 2021.8_Gan_NIH-Bio_ACE2_FINAL.pdf

Attach Current & Pending Support:   File Name:

PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Daniel Middle Name F. Last Name*: Linder Suffix: 

Position/Title*: Assistant Professor

Organization Name*:

Department:

Division: gia

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*:

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Co-Investigator Other Project Role Category: 

Degree Type:  PHD,MS,BS Degree Year:  2013,2008,2006

Attach Biographical Sketch*:             File Name: Linder_biosketch.pdf

Attach Current & Pending Support:   File Name:

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195 . Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 16 
PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Camille Middle Name Tessitore Last Name*: King Suffix: 

Position/Title*:

Organization Name*:

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 1

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login

Project Role*:  Consultant Other Project Role Category: 

Degree Type:  PHD Degree Year:  1990

Attach Biographical Sketch*:             File Name: biosketch-C_King2.pdf

Attach Current & Pending Support:   File Name:

PROFILE - Senior/Key Person

Prefix:  First Name*: Michael Middle Name Stephen Last Name*: King Suffix: 

Position/Title*:

Organization Name*:

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*:

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Consultant Other Project Role Category: 

Degree Type:  PHD,AB Degree Year:  1992,1986

Attach Biographical Sketch*:             File Name: biosketch-M_King2.pdf

Attach Current & Pending Support:   File Name:

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195 . Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 17 
PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Xin-Yun Middle Name  Last Name*: Lu Suffix: 

Position/Title*:

Organization Name*:

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*:

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Consultant Other Project Role Category: 

Degree Type:  MD,PHD Degree Year:  1987,1998

Attach Biographical Sketch*:             File Name: LU_Biosketch_July_2021.pdf

Attach Current & Pending Support:   File Name:

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195 . Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 18 
OMB No. 0925-0001 and 0925-0002 (Rev. 09/17 Approved Through 03/31/2020) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Lynnette Phillips McCluskey 

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Professor 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) 

INSTITUTION AND LOCATION 

DEGREE 

(if 

applicable) 

Completion 

Date 

MM/YYYY 

FIELD OF STUDY 

Florida State University, Tallahassee, FL B.A. 12/1990 Psychology / Biology 

University of Virginia, Charlottesville, VA M.S. 06/1995 Psychobiology 

University of Virginia, Charlottesville, VA Ph.D. 04/1996 Psychobiology 

Brigham & Women’s Hospital/Harvard Medical

 School, Boston, MA Postdoc 09/2000 Neuroimmunology 

A. Personal Statement 

My long-standing interest is in taste-immune interactions. The main focus of my research group is on the 

impact of cytokines and leukocytes on regenerating taste buds. The taste system is inherently plastic, but 

mechanisms responsible for taste bud and nerve regeneration and the recovery of taste function are poorly 

understood. Insights to immune regulation of regeneration are needed to develop therapies to recover taste 

following inflammation and neural injury. My laboratory has recently found that IL-1R signaling in infiltrating 

leukocytes and taste buds is needed to restore normal neurophysiological taste responses and fully regenerate 

taste buds. As taste deficits emerged as a major symptom of COVID-19 I began studies on the SARS-CoV-2 

receptor, ACE2, in mice and collaborative work on COVID-19 in patients based on my expertise in the tasteimmune axis. My role is to advise a team of neurologists on taste and smell testing to determine whether longterm chemosensory deficits emerge in COVID-19 positive patients, half of whom are African-American and at 

greater risk of COVID-19. In a supplement to Dr. David Hess’ R01 we test the hypothesis that there will be 

clinical, racial and genetic predictors of the occurrence and severity of neurological sequelae. In the current 

R21 application my team proposes to study inflammation and SARS-CoV-2 spike protein challenge in novel 

mouse models developed by Dr. Gan to shed light on the molecular, cellular, and neurophysiological role of 

ACE2 in the taste system. Our group is well-positioned to contribute fundamental insights to taste loss due to 

SARS-CoV-2. 

Complete list of published works in My Bibliography (Note: the PI has published under the names Lynnette 

McCluskey and Lynnette Phillips) 

https://www.ncbi.nlm.nih.gov/myncbi/lynnette.mccluskey.1/bibliography/public/ 

B. Positions and Honors 

Professional Positions 

2001-2005 Assistant Professor, Dept. of Physiology, Medical College of Georgia, Augusta, Georgia 

2005-2006 Assistant Professor, Dept. of Neuroscience & Regenerative Medicine, Medical College of 

Georgia, Augusta, Georgia 

2005-2020 Associate Professor (tenured), Dept. of Neuroscience & Regenerative Medicine, Medical 

College of Georgia at Augusta University, Augusta, Georgia 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 19 
2020-present Professor, Dept. of Neuroscience & Regenerative Medicine, Medical College of Georgia at 

Augusta University, Augusta, Georgia 

2009-2020 Director, Graduate Program in Molecular Medicine 

2020-present Director, Graduate Program in Neuroscience 

Honors 

1993, 1995 Travel Award, Association for Chemoreception Sciences (AChemS) 

1996-1998 NRSA Fellowship 

1996-1999 National Multiple Sclerosis Society Fellowship 

1998 Dale McFarlin Travel Award, National Multiple Sclerosis Society (one of two awarded yearly) 

2006 Recipient, Ajinomoto Young Investigator in Gustation Award, AChemS 

2014 Distinguished Service Award, The Graduate School, MCG 

2015 Distinguished Teacher Award, The Graduate School, MCG 

Other Professional Activities 

2001-present AChemS (Student member from 1992-1996) 

2001-present Reviewer for journals including: J Neurophysiol, Exp Neurol, Neurosci, Am J Physiol, Chem 

Senses, Neurosci Lett, Dev Dynamics, NeuroImmunoModulation, J Lipid Res, Dev Biol, PLOS 

One, J Neurosci, Sci Rep, J Gene Dev, J Pharmacol Exp Therap, Genesis, Nutrients, ACS 

Pharm Translational Sci, Int J Mol Sci, Nutrients, ACS Pharm Transl Sci, Cell Prolif 

2008-2010 Councilor (Executive Committee), AChemS 

2009 Reviewer for NIAID Special emphasis panel “Immune Defense Mechanisms at the 

Mucosa”

2011 Reviewer for Thomas F. and Kate Miller Jeffress Memorial Trust to support research in 

Virginia 

2014 Ad hoc reviewer, Neural Systems Cluster, Integrative Organismal Systems Division, National 

Science Foundation, April 2014 

2013-2019 Member, MCG Institutional Animal Care & Use Committee (IACUC); Vice-Chair 2016-2019 

2017-2020 Program Committee member, AChemS 

2017 Symposium Co-Chair, “Emerging mechanisms for sensory-immune interactions”, AChemS,

Bonita Springs, FL 2017 

2018-2019 NIH Ad hoc Reviewer, Special Emphasis Panel/Scientific Review Group 2019/01 ZDC1 SRB-R, 

10/12/2018, 10/24/2018, 2/21/2019, 6/25/2019, 7/1/2019 

2019 NIH Ad hoc Reviewer, Scientific Review Group Clinical and Integrative Diabetes and Obesity 

(CIDO) 10/2019 

2020 NIH Ad hoc Reviewer, Scientific Review Group Chemosensory Systems (CSS), 10/15/2020 

2020 NIH Ad hoc Reviewer, Special Emphasis panel, COVID-19 U01 applications (NIDCD) ZRG1-

IFCN-B-51, 10/16/20 

2020 Kansas IDeA Network of Biomedical Research Excellence (K-INBRE) reviewer, Bridging Grant 

Award 

2021 NIH Ad hoc Reviewer, Special Emphasis panel, ZRG1 IFCN-J 02 M and ZRG1 IFCN-U (07) 

07/14/2021 

C. Contributions to Science 

1. My undergraduate training at Florida State University included research in taste and feeding behavior with 

Dr. James Smith, Robert O. Lawton Distinguished Professor (now Emeritus). This experience combined with 

strong didactic and laboratory training in sensory systems fostered my career-long interest in chemosensation. 

I then trained in taste neurophysiology with Dr. David Hill at the University of Virginia for graduate studies. As 

an extension of Dave’s formative studies demonstrating developmental taste plasticity, I became interested in 

adult plasticity triggered by taste bud and neural injury. We found that altering the dietary environment by 

depleting sodium induced neurophysiological changes in regenerated taste buds as during development. 

Surprisingly, the function of intact taste buds on the opposite side of the tongue was also affected by 

neighboring nerve injury. I hypothesized that diet-induced immunosuppression was responsible for 

contralateral functional deficits. Indeed, by stimulating immune function with lipopolysaccharide (LPS) normal 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 20 
neural responses were restored. This sparked my >20 year interest in taste-immune interactions which is the 

focus of the current application. Once I established my own laboratory at the Medical College of Georgia, the 

Hill lab and my team collaborated to show that exogenous aldosterone, like ACE2 a member of the reninangiotensin system (RAS) 2, impacts both taste function and macrophage responses to nerve injury. In this 

R21 proposal Dr. Gan and I test whether deletion of Ace2 in the lingual epithelium will also alter taste 

responses and local inflammatory responses. 

a. Hill, DL and LM Phillips (1994) Functional plasticity of regenerated and intact taste receptors in adult rats 

unmasked by dietary sodium restriction. J Neurosci, 14: 2904-2910. 

b. Phillips, LM and DL Hill (1996) Novel regulation of peripheral gustatory function by the immune system. Am 

J Physiol, 271: R857-R862. 

c. Guagliardo N, West K, McCluskey L, and DL Hill (2009) Attenuation of peripheral salt taste responses and 

local immune function contralateral to gustatory nerve injury: effects of aldosterone. Am J Physiol. 297: 

R1103-R1110. PMID: 19675282 

2. My postdoctoral training with Dr. Lois Lampson at Brigham & Women’s Hospital / Harvard Medical School 

provided intensive studies in neuroimmunology. Dr. Lampson’s laboratory was part of the Center for

Neurologic Disease which focuses on the immune mechanisms mediating damage in multiple sclerosis, 

Alzheimer’s disease and brain tumor. My training there, supported by NIH and the National Multiple Sclerosis 

Society, focused on the effect of local brain microenvironments on T cell and microglial responses to cytokines. 

During this period I added immunological and CNS neuroanatomical techniques to my background in sensory 

neurophysiology. 

a. Phillips, LM and LA Lampson (1999) Site-specific control of T cell traffic in the brain: T cell entry to 

brainstem vs. hippocampus after local injection of IFN-γ. J Neuroimmunol, 96: 218-227. 

b. Phillips, LM, PJ Simon, and LA Lampson (1999) Site-specific immune regulation in the brain: differential 

modulation of major histocompatibility complex (MHC) proteins in brainstem vs. hippocampus. J Comp 

Neurol, 405: 322-333. 

c. McCluskey, LP and LA Lampson (2000) Local neurochemicals and site-specific immune regulation in the 

CNS. J Neuropathol Exp Neurol, 59: 177-187. 

d. McCluskey, LP and LA Lampson (2001) Local immune regulation in the CNS by substance P and 

glutamate. J Neuroimmunol, 116: 136-146. 

3. My research program was an early contributor to the subfield of taste-immune interactions. My group has 

shown the functional significance of neutrophil, macrophage, cytokine and chemokine responses in the normal 

and injured taste system. For example, neutrophils and macrophages have opposing, dynamic effects on taste 

function. Neutrophils responding to nerve injury or inflammation are detrimental to sodium taste responses, 

while macrophages are beneficial for the return of normal function. We have also studied the effects of 

cytokines released by leukocytes on taste function and determined that IL-1 and TNF have opposite effects on 

sodium flux in polarized taste buds, with a net increase in neural sodium sensitivity. These studies are 

important for our understanding of taste changes that impact nutrition during infection, injury, and local and 

systemic inflammation. Our work has stimulated interest into the normal and pathological roles of inflammation 

in the taste system. Recently we have determined that interleukin (IL)-receptor signaling is necessary for 

regenerating taste buds and recovering normal neurophysiological taste function after chorda tympani nerve 

sectioning in mice. We are currently testing genetic models of interleukin (IL)-1 receptor deletion in leukocytes 

or taste buds to determine when and where IL-1 signaling is needed to restore taste function after nerve mice. 

a. McCluskey, LP (2004) Upregulation of activated macrophages in response to degeneration in the taste 

system: effects of dietary sodium restriction. J Comp Neurol, 479: 436-55. PMID: 15389612 

b. Shi L, He L, Sarvepalli P, and McCluskey LP (2011) A functional role for IL-1 in the injured peripheral taste 

system. J Neurosci Res 90: 816-830. PMID: 22213141. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 21 
c. He L, Yadgarov A, Sharif S, and McCluskey LP (2012) Aging profoundly delays functional recovery from 

gustatory nerve injury. Neuroscience 3:208-18. PMID: 22387273 

d. Kumarhia D and McCluskey LP (2016) Inflammatory stimuli acutely modulate peripheral taste function. J 

Neurophysiol 115: 2965-75. PMID: 27009163 

4. Animal and human studies link gut inflammation and infection with taste deficits through unknown 

mechanisms. In collaborative studies with Dr. David Pittman we used a mouse model to test the hypothesis 

that gut inflammation alters taste function and behavior. Oral gavage with lipopolysaccharide (LPS), a 

proinflammatory stimulus derived from gram-negative bacteria, acutely reduces neural and behavioral 

responses to sweet stimuli through toll-like receptor (Tlr)-4 signaling. Chronic LPS gavage prolongs these 

deficits in sweet responses and also decreases salt taste responses. Taste changes relevant to health and 

nutrition are thus triggered by localized gut inflammation. In a collaboration with MCG colleague, Dr. Ruth 

Harris, we observed similar changes in neural taste responses to sweet and salt in rats chronically ingesting 

liquid sucrose which parallels human overconsumption of sweet stimuli. Current work is aimed at identifying 

changes in taste function in mouse models of inflammatory bowel disease. 

a. Pittman DW, Dong G, Brantly AM, He L, Nelson TS, Kogan S, Powell J and McCluskey LP (2020) 

Behavioral and neurophysiological taste responses to sweet and salt are diminished in a model of subclinical 

intestinal inflammation. Sci Rep 10(1): 17611. PMID: 33077838. 

b. McCluskey LP, He L, Dong G, Harris R. (2020) Chronic exposure to liquid sucrose and dry sucrose diet 

have differential effects on peripheral taste responses in female rats. Appetite. 2020 Feb 1;145:104499. PMID: 

31669578 

c. Zhu X, He L, and McCluskey LP (2014) Ingestion of lipopolysaccharide inhibits peripheral taste responses to 

sucrose in mice. Neuroscience 258: 47-61. PMID: 2421598. 

5. Taste and smell deficits are prominent symptoms of COVID-19 viral infection caused by SARS-CoV-2. 

Though most patients recover taste and smell sensation a subset of COVID-19 patients experience longerterm sensory deficits detrimental to health and nutrition. The number of people with chronic chemosensory 

deficits is likely significant given the massive scale of the current pandemic. My interest in taste-immune 

interactions and ~25 years in chemosensory research complements the team of Medical College of Georgia 

neurologists testing neurologic symptoms of COVID-19. 

Hess DC, Rutkowski E, Morgan J and L McCluskey (2020) COVID-19 and neurological symptoms: Is the 

COVID-CoV-2 virus neurotropic? Cond Med 3(5): 241-245. PMID: 34136764 

D. Additional Information: Research Support 

Current Research Support 

R01 DC016668 National Institutes of Health (NIDCD) 5/1/18-4/30/23 

McCluskey (PI) 

Cytokines in the regenerating taste system 

The goal of this project is to determine how IL-1 signals through its receptor to rebuild taste buds and restore 

taste function after injury. 

Completed Research Support (last 3 years) 

R01 3R01NS112511-01A1S1 National Institutes of Health (NINDS) 08/01/2020 – 01/31/2021 

Hess (PI) McCluskey (Co-I) 

COVID-19 neurological Georgia cohort study 

This supplement supports studies to identify factors associated with neurological symptoms including 

chemosensory deficits in COVID-19 patients. 

PSRP 000102 Pilot Study Research Program Grant, MCG. 11/1/17-10/31/18 

McCluskey (PI) 

Taste changes in a mouse model of inflammatory bowel disease 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 22 
This project supported preliminary studies for the submission of an R01 investigating taste changes in mice 

with chronic DSS-induced colitis. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 23 
OMB No. 0925-0001 and 0925-0002 (Rev. 12/2020 Approved Through 02/28/2023) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Gan, Lin 

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Professor 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) 

INSTITUTION AND LOCATION 

DEGREE 

(if 

applicable) 

Completion 

Date 

MM/YYYY 

FIELD OF STUDY 

Zhongshan University, Guangzhou, China B.S. 07/1985 Biochemistry 

University of Texas Graduate School of Biomedical 

Sciences, Houston, TX Ph.D. 12/1992 Biochemistry and Molecular Biology 

U.T. M.D. Anderson Cancer Center, Houston, TX Postdoc. 12/1995 Developmental Biology 

Fundamental Issues in Vision Research-NEI 

sponsored summer training program at MBL - 08/1994 Vision Research 

A. Personal Statement 

The long-term objective of my research is to understand the molecular mechanisms underlying neuronal 

differentiation and disease, especially the function of transcription factors (TFs) in sensory organs such as the 

mammalian inner ear and retina. As a graduate student, I first became interested in the roles of transcription 

factors in embryonic development. My work on the transcription factors has contributed significantly to our 

understanding of the molecular mechanisms governing neural development in the retina and inner ear, such as 

the Atoh7Sox4/Sox11Isl1/Pou4f2 regulatory pathway in the development of retinal ganglion cells, and the 

roles of Pou4f3, Gata3, and Lmo4 in inner ear development. In recent years, my work on subtype specification 

of retinal neurons has revealed the roles of several transcription factors and the underlying molecular mechanism 

specifying neuronal subtypes during retinogenesis. Additionally, my research has made available to the research 

community over 100 genetically modified mouse strains and other reagents, particularly tissue-specific Cre 

deleter mouse strains such as Itga8-CreERT2, Bhlhb5-Cre, Atoh7-Cre, Gfi1-Cre, Gfi1-2A-CreERT2, Sox2-2ACreERT2, and Atoh1-Cre. 

In addition, I am an expert in mouse genetics and related transgenic/knockout technologies with over 28 years 

of experience in transgenic and knockout mouse generation and analysis. I have founded and directed 

transgenic/knockout facilities at The University of Texas M.D. Anderson Cancer Center (1996-1999), University 

of Rochester (2006-2019), and Augusta University Medical College of Georgia (2019-present). Throughout my 

research career, I have successfully developed, improved, and incorporated new transgenic, gene targeting in 

ES cells, and CRISPR/Cas9 techniques to address diverse scientific challenges. Over 1,000 knockout/knockin/conditional knockout mouse models have been created by me or under my supervision, including about 500 

by CRISPR/Cas9 method in the past 6 years. 

Specific for this application, I will assist with the generation of three novel Ace2 strains and will make my 

expertise available to project design, Ace2-expressing cell lineage tracing, Ace2 cKO phenotypic analysis, 

breeding schemes, and genotyping strategies. 

Ongoing and recently completed projects that I would like to highlight include: 

Ongoing Research Support 

1R01EY026614-01 02/18/2016-02/28/2022 (NCE) 

NIH/NEI 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 24 
Title of Project: “The Roles of LIM-Homeodomain Transcription Factors in Retinal Development”

The major goal of this proposal is to identify the role of LHX9’s role in the specification of retinal amacrine 

neuronal subtype. 

Role: PI 

B. Positions, Scientific Appointments, and Honors 

Positions and Scientific Appointments 

10/2019- present Professor, Department of Neuroscience and Regenerative Medicine, Augusta University 

Medical College of Georgia 

10/2019-present Director of Genome Editing Core, Augusta University Medical College of Georgia 

10/2019- present GRA Eminent Scholars, Georgia Research Alliance 

7/2009-9/2019 Professor of Ophthalmology, Neurobiology and Anatomy, Center for Neural Development and 

Diseases, and Center for Visual Sciences, University of Rochester 

7/2009-9/2019 Dean’s Professor (endowment/chair), University of Rochester Medical Center 

7/2009-9/2012 Director of Research, Flaum Eye Institute, University of Rochester Medical Center 

9/2006-9/2019 Director, Mouse Genome Editing Resource (formerly The Transgenic and Gene Targeting 

Core), University of Rochester Medical Center 

6/2005-6/2009 Associate Professor, Dept. of Ophthalmology, Department of Neurobiology and Anatomy, 

Center for Visual Sciences, University of Rochester Medical Center 

9/1999-5/2005 Assistant Professor, Dept. of Neurobiology and Anatomy, Center for Aging and 

Developmental Biology, University of Rochester Medical Center 

9/1996-8/1999 Assistant Professor, Dept. of Biochem. & Mol. Biol., The University of Texas M.D. Anderson 

Cancer Center. 

9/1996-8/1999 Founding Director of Gene Targeting (Knockout) Core, The University of Texas M.D. 

Anderson Cancer Center. 

1/1996-8/1996 Instructor, Dept. of Biochem. & Mol. Biol., U.T. M.D. Anderson Cancer Center. 

Selected Memberships, Editorial Boards and Advisory Committees 

2021.2-2021.6 Standing Member of the Biology and Development of the Eye (BDE) Study Section, NIH 

2017.7-2020.11 Standing Member of the Biology of the Visual System (BVS) Study Section, NIH 

2010-2015 Editorial Board: Journal of Biological Chemistry 

2007-present Member, Association for Research in Otolaryngology (ARO) 

2006-present: Ad hoc Member of the following NIH Study Sections: NDPR (Neural Differentiation, Plasticity, 

and Regeneration) Study Section (2006.2), NIH BDPE (Biology and Diseases of the Posterior 

Eye) Study Section (2008.6, 2009.2, 2009.6), NIH ZRG1 CB-G 02 S (Retinopathy Models and 

Therapy) Study Section (2009.6), NIH NCF (Neurogenesis and Cell Fate) Study Section 

(2009.10), NIH ZRG1 CB-G 90 (Retinopathy Models and Therapy) Study Section (2010.2), 

ZRG1-IFCN-B-02M (Auditory Neuroscience) Study Section (2010.10), ZRG1 BDPE-N (09) 

(Biology and Diseases of the Posterior Eye) Study Section (2011.9), Neurodifferentiation, 

Plasticity, Regeneration and Rhythmicity (NDPR) study section (2012.2), Special Emphasis 

Panel/Scientific Review Group 2013/01 ZDC1 SRB-R (38) (2012.10), Special Emphasis 

Panel/Pain and Hearing ZRG1-IFCN-B-02M (2013.6), Neurodifferentiation, Plasticity, 

Regeneration and Rhythmicity (NDPR) study section (2014.2). 

2005-present Member, Society for Neuroscience (SfN) 

2003-present Life Member, Society of Chinese Bioscientists in America (SCBA) 

2001-present Life Member, The Chinese Biological Investigators Society (formerly Ray Wu Society) Life 

Member 

1999-present Member, The American Association for the Advancement of Science (AAAS) 

1998-present Member, Association for Research in Vision and Ophthalmology (ARVO) 

Honors 

2010 Research to Prevent Blindness Senior Scientific Investigator Award 

1999-2003 Rosanne H. Silbermann Fellow 

1985 CUSBEA (China-United States Biochemistry and Molecular Biology Examination and 

Administration, aka Ray Wu Program) Scholar, Ministry of Education, Beijing, China 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 25 
C. Contributions to Science 

1. Development of retinal ganglion cells 

My early publications focused on the roles of transcription factors in retinal development, particularly the 

development of retinal ganglion cells (RGCs). I have shown that ATOH7 determines the RGC-competence of 

retinal progenitors and that POU4F2 and ISL1 are the key RGC differentiation factors acting synergistically to 

regulating the differentiation and survival of RGCs. Furthermore, we have demonstrated that SOX4 and SOX11 

act upstream of POU4F2/ISL1 and downstream of ATOH7 in this process. Thus, these TFs constitute the 

ATOH7SOX4/SOX11ISL1/POU4F2 regulatory pathway. Taken together, my work has contributed 

significantly to our currently understanding of the molecular mechanisms governing retinal ganglion cell 

development. 

a. Gan, L., Xiang, M., Zhou, L., Wagner, D.S., Klein, W.H., and Nathans, J. (1996). POU domain factor Brn3b is required for the development of a large set of retinal ganglion cells. Proc. Natl. Acad. Sci. 93, 

3920-3925. 

b. Wang, S.W., Kim, B.S., Ding, K., Wang, H., Sun, D., Johnson, R.L, Klein, W.H. and Gan, L. (2001). 

Requirement of math5 in the development of retinal ganglion cells. Genes & Dev. 15, 24-29. 

c. Pan, L., Deng, D., Xie, X., and Gan, L. (2008). ISL1 and BRN3B co-regulate the differentiation of murine 

retinal ganglion cells. Development 135, 1981-1990. PMCID: PMC2758274. (cover photograph) 

d. Jiang, Y., Ding, Q., Xie, X., Libby, R., Lefebvre, V., and Gan, L. (2013). Transcription factors SOX4 and 

SOX11 function redundantly to regulate the development of retinal ganglion cells. J. Biol. Chem. 288, 

18429-18438. PMCID: PMC3689985. 

2. Specification of neuronal subtypes in the retina 

Besides RGC development, my work on subtype specification of retinal neurons, along with the studies by 

other investigators, has begun to reveal the underlying molecular mechanism specifying neuronal subtypes in 

the retina, which unique combinations of transcription factors determine specific retinal subtypes. 

a. Feng, L., Xie, X., Joshi, P., Yang, Z., Shibasaki, K., Chow, R., and Gan, L. (2006). Requirement for 

Bhlhb5 in the specification of amacrine and cone bipolar subtypes in mouse retina. Development 133, 

4815-4825. PMID: 17092954. (cover photograph) 

b. Elshatory, Y., Deng, M., Xie, X., Barlow, R.B., and Gan, L. (2007). Islet-1 controls the differentiation of 

retinal bipolar and cholinergic amacrine cells. J. Neurosci. 27:12707-12720. PMID: 18003851. 

c. Ding, Q., Chen, H., Xie, X., Libby, R., Tian, N., and Gan, L. (2009). BARHL2 differentially regulates the 

development of retinal amacrince and ganglion neurons. J. Neurosci. 29, 3992– 4003. PMCID: 

PMC2756297. (cover photograph) 

d. Dong, X., Yang, H., Zhou, X., Xie, X., Yu, D., Guo, L., Xu, M., Zhang, W., Liang, G., and Gan, L. (2020). 

LIM-Homeodomain Transcription Factor LHX4 Is Required for the Differentiation of Retinal Rod Bipolar 

Cells and OFF-Cone Bipolar Subtypes. Cell Rep. 2020 Sep 15;32(11):108144. doi: 

10.1016/j.celrep.2020.108144. PubMed PMID: 32937137. 

3. Roles of transcription factors in the inner ear 

I was first attracted to the development of inner ear during my postdoctoral training and demonstrated 

POU4F3 (Brn3c) as an essential TF in the development and survival of inner ear hair cells (Proc. Natl. Acad. 

Sci., 94:9445-9450). In the recent years, I have focused on a group of functionally associated TFs of GATA, 

LMO, and ISL family proteins, which are uniquely expressed in the cochlear epithelium prior to and during the 

formation of prosensory domain. I have begun to show that these TFs positively or negatively regulate the 

formation of cochlear sensory domain. This body of work reveals a novel network of TFs that function upstream 

of SOX2, a master regulator of prosensory specification, to specify the prosensory domain as well as nonsensory regions in the cochlea. 

a. Deng, M., Pan, L., Xie, X., and Gan, L. (2010). Requirement for Lmo4 in the vestibular morphogenesis 

of mouse inner ear. Dev. Biol. 338, 38–49. PMCID: PMC2812651. (cover photograph) 

b. Luo, X., Deng, M., Xie, X., Huang, L., Wang, H., Jiang, L., Liang, G., Hu, F., Tieu, R., Chen, R., and Gan, 

L. (2013). GATA3 controls the specification of prosensory domain and neuronal survival in the mouse 

cochlea. Hum. Mol. Genet. 22, 3609-3623. PMCID: PMC3749857. (cover photograph) 

c. Deng, M., Luo, X., Pan, L., Yang, H., Xie, X., Liang, G., Huang, L., Hu, F., Kiernan, A., and Gan, L. 

(2014). LMO4 functions as a negative regulator of sensory organ formation in the mammalian cochlea. 

J. Neurosci. 34, 10072-7. PMCID: PMC4107398. (Selected for F1000Prime) 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 26 
d. Xu, J., Yu, D., Dong, X., Xie, X., Xu, M., Guo, L., Huang, L., Tang, Q., and Gan, L. (2021). GATA3 

maintains the quiescent state of cochlear supporting cells by regulating p27kip1. Sci. Rep. 11:15779. 

4. Other contributions to the neuroscience field 

While my major research focus has always been in the development of retina and inner ear, my study using 

mouse genetic models often leads to observations of great significance in the development and disease of other 

nervous systems. I have been able to recognize the importance of these findings and to elucidate the underlying 

mechanisms by myself as well as in collaboration with experts in the related field. Often, these studies are 

published in high-impact journals and demonstrate my commitment to broad scientific collaborations and our 

ability to initiate and carry to fruition research at high level in research fields other than my specialty in retina and 

inner ear. 

a. Elshatory, Y. and Gan, L. (2008). The LIM-homeobox gene Islet-1 is required for the development of 

restricted forebrain cholinergic neurons. J. Neurosci. 28:3291–3297. PMCID: PMC2786914. 

b. Joshi, P.S., Molyneaux, B.J., Feng, L., Xie, X., Macklis, J., and Gan, L. (2008). Bhlhb5 Regulates the 

Post-Mitotic Acquisition of Area Identities in Layers II-V of the Developing Neocortex. Neuron 60, 258–

272. PMCID: PMC2643370. 

c. Ding, Q. Joshi, P.S., Xie, Z., Xiang, M., and Gan, L. (2012). BARHL2 transcription factor regulates the 

ipsilateral/contralateral subtype divergence in postmitotic dI1 neurons of the developing spinal cord. 

Proc. Natl. Sci. Acad., USA. 109: 1566-1571. PMCID: PMC3277160. 

d. Luo, X., Li, M., Huang, L., Steinberg, S., Mattheisen, M., Donohoe, G., Shi, Y., Chen, C., Yue, W., Alkelai, 

A., Lerer, B., Li, Z., Yi, Q., Rietschel, M., Cichon, S., Collier, D.A., Tosato, S., Suvisaari, J., Rujescu, D., 

Golimbet, V., Silagadze, T., Durmishi, N., Milovancevic, M.P., Stefansson, H., Schulze, T.G., Nöthen, 

M.M., Chen, C., Lyne, R., Morris, D.W., Gill, M., Corvin, A., Zhang, D., Dong, Q., Moyzis, R.K., 

Sigurdsson, E., Stefansson, K., Hu, F., MooDS SCZ Consortium, Su, B., and Gan, L. (2014). Convergent 

lines of evidence support CAMKK2 as a schizophrenia susceptibility gene. Molecular Psychiatry. 19, 

774-83. PMID: 23958956. (Selected for F1000Prime). 

Complete List of 110 Published Work in MyBibliography: 

https://www.ncbi.nlm.nih.gov/sites/myncbi/lin.gan.1/bibliography/40475286/public/?sortby=pubDate&sdirection 

=descending 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 27 
OMB No. 0925-0001 and 0925-0002 (Rev. 03/2020 Approved Through 02/28/2023) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Linder, Daniel Frederick 

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Assistant Professor of Biostatistics and Data Science 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) 

INSTITUTION AND LOCATION 

DEGREE 

(if 

applicable) 

Completion 

Date 

MM/YYYY 

FIELD OF STUDY 

Georgia Southern University, Statesboro, Georgia BS 07/2006 Mathematics 

Georgia Southern University, Statesboro, Georgia MS 05/2008 Mathematics 

AU (Medical College of Georgia), Augusta, Georgia PhD 07/2013 Biostatistics 

A. Personal Statement 

My background is in mathematics and biostatistics, and I have specific research interests in developing 

Bayesian statistical methods for stochastic reaction networks and more generally in developing Bayesian 

machine learning methods for high-dimensional problems, key research areas for this application. I have been 

involved in several funded projects that involved developing new statistical methodology for reaction networks, 

big biomedical data, longitudinal data, and cluster analysis, all of which were eventually published in peer 

reviewed journals. In May of 2016, I joined the faculty in the Department of Population Health Sciences in the 

Medical College of Georgia at Augusta University. As a faculty member I have directed a doctoral dissertation 

on Bayesian modeling using global-local shrinkage rules for inferring gene regulatory networks, and I have also 

directed another PhD dissertation on the topic of statistical methods in stochastic reaction networks. Further, I 

am currently directing a PhD dissertation in which my student has proposed efficient Bayesian modeling 

strategies that link patient phenotypes to genotypes. As part of an Early Career Award from the Mathematical 

Biosciences Institute (MBI) at Ohio State University, I have worked with colleagues on developing survival 

dynamical models to model the spread of infectious diseases on networks. Since joining the department, I 

have developed collaboration with several clinical and basic sciences researchers at Augusta, and I am 

currently a collaborator on Dr. Lynnette McCluskey’s R01. Part of this work is to understand the mechanisms of

taste bud regeneration and the role that cytokine networks and genes play in the pro- and anti-regenerative 

process, with a goal of translating this work to help those with tongue injury repair taste. We currently have two 

manuscripts in preparation related to this work. For the current project, I will work closely with Dr. McCluskey 

and her team to investigate the role of the SARS-CoV-2 receptor, ACE2, in the taste system. 

B. Positions and Honors 

Positions and Employment 

2012-2013 Visiting Assistant Professor of Biostatistics, Georgia Southern University (GSU), Statesboro, 

Georgia 

2013-2016 Assistant Professor of Biostatistics, GSU 

2016-Present Assistant Professor of Biostatistics and Data Science, Medical College of Georgia, Augusta 

University 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 28 
Other Experience and Professional Memberships 

2013-Present Eastern North American Region International Biometric Society, ENAR 

2016-Present American Statistical Association 

Honors 

2006 Mathematical Association of America, 4 th place in math jeopardy, Knoxville, TN 

2006 Outstanding mathematics student award, GSU 

2012 Travel award by Mathematical Biosciences Institute, CAMBAM-NIMBIOS Summer graduate program: 

Stochastics applied to biological systems, Ohio State University, Columbus, OH 

2014 Modified Bayesian Lasso with L1 Loss, 1st place in applied statistics, APHA, New Orleans, LA 

2015 Mathematical Biosciences Institute travel award: Treatment, Clinical Trials, Resistance Workshop 

2016 Mathematical Biosciences Institute travel award: Topological, Geometric, and Statistical Techniques in 

Biological Data Analysis. 

C. Contributions to Science 

1. My main area of statistical research is at the cross-section of Bayesian statistics and computational 

biology. A large part of my work has focused on balancing model accuracy with computational 

feasibility. Some of the approaches I have developed have been based on both empirical and 

hierarchical Bayesian modeling. Specifically, my colleagues and I have developed highly tractable and 

efficient Bayesian techniques for inference in complex stochastic dynamical systems with a wide range 

of applications from systems biology to political science. My work on these mathematical models and 

their connection to Bayesian model selection methodology for high-dimensional data provides the 

requisite experience necessary to discover novel findings. 

1. Linder, D.F. & Rempala, G.A. (2020). Synthetic likelihood method for reaction network 

inference. Mathematical Methods in the Applied Sciences. 

2. Linder, D.F., & Rempala, G.A. (2013). Algebraic statistical model for biochemical network 

dynamics inference. Journal of Coupled Systems and Multiscale Dynamics, 1(4),468–475. 

3. Panchal, V. & Linder, D.F. (2020). Reverse engineering gene networks using global-local 

shrinkage rules. Interface Focus. 

4. Volkening, A., Linder, D.F., Porter, M.A., & Rempala, G.A. (2020). Forecasting elections using 

compartmental models of infections. In press at SIAM Review. 

2. I have also worked on a series of publications developing efficient sampling strategies to maximize 

power in clinical studies. Efficient experimental design has become pertinent with the growing 

availability of high-throughput technologies, like next generation sequencing, where researchers are 

confronted with a need to collect measurements that are often costly and under constrained budgets. 

Part of my research activity has been on how to maximize the information content of a study by using 

carefully designed sampling strategies. The methodology developed in the publications below leverage 

the additional structure of order statistics to impart additional information into the study samples that are 

collected and measured. Thus, the collected samples will contain considerably more information than a 

corresponding simple random sample of the same size. Through my expertise in efficient sampling 

design, I will work with clinicians to design highly efficient and powerful clinical studies that have high 

power of success, while at the same time preserving valuable resources. 

1. Linder, D.F., Samawi, H.M., Yu, L., Chatterjee, A., Huang, Y., & Vogel, R. (2015). On stratified 

bivariate ranked set sampling for regression estimators. Journal of Applied Statistics, 42(12), 

15–23. 

2. Linder, D.F., Yin, J., Rochani, H., Samawi, H., & Sethi, S. (2017). Increased Fisher’s 

information for parameters of association in count regression via extreme ranks. 

Communications in Statistics (Theory and Methods). DOI: 10.1080/03610926.2017.1316859 

3. Samawi, H.M., Cai, J., Linder, D.F., Rochani, H., Yin, J. (2018). A simpler approach for 

mediation anaylsis for dichotomous mediators in logistic regression. Journal of Statistical 

Computation and Simulation. DOI: 10.1080/00949655.2018.1426762 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 29 
4. Samawi, H.M., Rochani, H., Yin, J., Linder, D.F., Vogel,R. (2018). Notes on kernel-based mode 

estimation using more efficient sampling designs. Computational Statistics. DOI: 

10.1007/s00180-017-0787-2 

3. Further, I have collaborated with clinical and public health researchers on several projects ranging from 

the mining and analysis of large national databases to analyzing data from clinical trials, of which my 

role was as the principal biostatistician. 

1. Tarasenko, Y., Linder, D.F., & Miller, E.E. (2018). Physical activity and mortality among 3+ year 

cancer survivors in the U.S. Cancer Causes and Control. 

2. Majeed, B., Linder, D.F., Eissenberg, T., Tarasenko, Y.N., Smith, D., & Ashley, D.A. (2020). 

Cluster analysis of urinary biomarkers among US adults: Population Assessment of Tobacco 

and Health (PATH). Preventive Medicine. 

3. Park, Y.D., Linder, D.F., Pope, J., Flamini, R.J., & Moretz, K., Diamond, M.P., Long, S.A. 

(2020). Long-term efficacy and safety of cannabidiol (CBD) in children with treatment-resistant 

epilepsy: Results from a state-based expanded access program. Epilepsy & Behavior. 

D. Additional Information: Research Support and/or Scholastic Performance 

Ongoing Research Support 

• NIH 1R01 DC 016668 McCluskey, Lynnette (PI) 01/2018-12/2023 

Cytokines in the Regenerating Taste System 

The goal of this study is to examine the role of cytokines in the regeneration of taste. 

Role: Biostatistician 

Completed Research Support 

• The Department of Defense Chen, Jie (PI) 08/2019-07/2020 

Machine Learning 

Developed machine learning course for the United States military 

Role: Co-Investigator 

• The Department of Defense Chen, Jie (PI) 08/2019-07/2020 

Theoretical Foundations of Machine Learning 

Developed a theoretical foundations course for the United States military 

Role: Co-Investigator 

• Georgia Cannabidiol Study Park, Yong (PI) 06/2018-05/2020 

The Georgia Cannabidiol Study is an Intermediate Size Expanded Access clinical trial of the drug 

Epidiolex in pediatric seizure patients. 

Role: Biostatistician 

• Early Career Award MBI/NSF Linder, Daniel (PI) 01/2018-12/2018 

The Ohio State University 

Role: Visiting research faculty 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 30 
OMB No. 0925-0001 and 0925-0002 (Rev. 03/2020 Approved Through 02/28/2023) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Camille Tesstiore King, Ph.D. 

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Chair & Professor of Psychology 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) 

INSTITUTION AND LOCATION 

DEGREE 

(if applicable) 

Completion 

Date 

MM/YYYY 

FIELD OF STUDY 

University of Virginia, Charlottesville, VA 

University of Virginia, Charlottesville, VA 

University of Virginia, Charlottesville, VA 

University of Virginia, Charlottesville, VA 

University of Michigan Ann Arbor, MA 

B.A. 

M.A. 

Ph.D. 

Post-doc 

Post-doc 

05/1985 

05/1988 

05/1990 

12/1991 

08/1993 

Psychology 

Biological Psychology 

Biological Psychology 

Central Taste Anatomy 

Central Taste Anatomy 

University of Florida, Gainesville, FL Post-doc 08/1998 Behavioral Neuroscience 

A. Personal Statement 

My entire research career has been centered on the anatomy of the central gustatory system, chiefly on its 

development or regeneration, with a second focus on taste behaviors in adults in response to central 

gustatory manipulations. I have examined how the central termination fields of the taste nerves (using 

HRP-labeling) and the structures of first-order neurons (using Golgi-staining) are altered during 

development as a consequence of maternal sodium deprivation. For many years, I examined patterns of 

central gustatory activity (via fos-immunohistochemistry) in several central taste structures (nucleus of the 

solitary tract, parabrachial nucleus, amygdala, and gustatory cortex) following gustatory nerve transection 

and regeneration. I have also spent years examining how ibotenic lesions of the gustatory cortex affect 

taste behaviors (taste reactivity, two bottle preference tests). 

B. Positions and Honors 

Professional Positions 

1994-1996 Adjunct Professor of Psychology, Stetson University & Daytona Beach Community College 

1996-1998 Visiting Assistant Professor of Psychology/Postdoc, University of Florida 

1998-1999 Research Assistant Professor, Stetson University 

1999-2000 Lecturer in Psychology, Stetson University 

2000- 2001 Visiting Assistant Professor, Psychology Department, Stetson University 

2001-2007 Assistant Professor, Psychology Department, Stetson University 

2007-2012 Associate Professor, Psychology Department, Stetson University 

2012-present Professor, Psychology Department, Stetson University 

2016-present Chair, Psychology Department, Stetson University 

Honors 

1988 First recipient of the Becky Boone Award for Teaching Excellence, University of Virginia 

2004 Recipient of the Hand Award for Excellence in Research, Stetson University 

2011 Recipient, William Hugh McEniry Award for Teaching Excellence, Stetson University 

2012 Invited keynote address speaker, 2nd Annual Florida Undergraduate Research Conference 

Other Professional Activities 

1988-present Associate for Chemoreception Sciences Member 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 31 
2002-2003 Educational Outreach Program, Association for Chemoreception Sciences 

2004-present Society for the Study of Ingestive Behaviors Member 

2004-present Served as reviewer for the following: Brain Research, Journal of Comparative Neurology, 

Chemical Senses, Psi Chi Journal of Undergraduate Research, NCUR Conference 

Proceeding Papers, a National Science Foundation Grant, Textbook Chapters for 

Houghton/Mifflin: Introductory Psychology, Biological Psychology; for Cengage: 

Introductory Psychology; for Oxford University Press: Behavioral Neuroscience 

2010 & 2012 Session Presider, International Conference for Learning and the Brain Conference 

2011 Association for Chemoreception Sciences, Election Committee Member 

2016-present Association for Heads of Departments of Psychology (AHDP) Member 

C. Contributions to Science

1. My dissertation work revealed for the first time how the developing central gustatory system is altered

as a consequence of maternal sodium chloride deprivation.

King CT and Hill DL (1991). Dietary sodium chloride deprivation throughout development selectively 

influences the terminal field organization of gustatory afferent fibers projecting to the rat nucleus of 

the solitary tract. Journal of Comparative Neurology, 303:159-

169. https://doi.org/10.1002/cne.903030114 

King CT and Hill DL (1993). Neuroanatomical alterations in the rat nucleus of the solitary tract 

following early maternal NaCl deprivation and subsequent NaCl repletion. Journal of Comparative 

Neurology, 333:531-42. 10.1002/cne.903330406 

2. My postdoctoral work and continued collaborations with Dr. Alan Spector while I have been at Stetson

demonstrated the regenerative capacity of the central gustatory system. (* denotes undergraduate)

King CT, Travers SP, Rowland NE, Garcia M, Spector AC (1999). Glossopharyngeal nerve 

transection eliminates quinine-stimulated fos-like immunoreactivity in the nucleus of the solitary tract: 

Implications for a functional topography of gustatory nerve input in rats. Journal of Neuroscience, 

19:3107-3121. https://doi.org/10.1523/JNEUROSCI.19-08-03107 

King CT, Garcea M and Spector AC (2000). Glossopharyngeal nerve regeneration is essential for 

the complete recovery of quinine-stimualted oromotor rejection behaviors and central patterns of 

neuronal activity in the nucleus of the solitary tract in the rat. Journal of Neuroscience, 20:8426-

8434. https://doi.org/10.1523/JNEUROSCI.20-22-08426 

King CT, *Deyrup LD, *Dodson SE, *Galvin KE, Garcea M, and Spector AC (2003). Effects of 

gustatory nerve transection on quinine-stimulated Fos-like immunoreactivity in the parabrachial 

nucleus of the rat. Journal of Comparative Neurology, 465:296-

308. https://doi.org/10.1002/cne.10851 

King CT, Garcea M, and Spector AC (2014). Restoration of quinine-stimulated Fos-immunoreactive 

neurons in the central nucleus of the amygdala and gustatory cortex following reinnervation or crossreinnervation of the lingual taste nerves in rats. Journal of Comparative Neurology, 522:2498-2517. 

DOI 10.1002/cne.23546 

3. My most recent work has focused on the role of the gustatory cortex in taste behaviors

King CT, Hashimoto, Garcia M, and Spector AC. (2015). Unconditioned oral-motor taste reactivity

elicited by sucrose and quinine is unaffected by extensive bilateral damage to the gustatory zone of the 

insular cortex in rats. Brain Research, 1599: 9-19. DOI: 10.1016/j.brainres.2014.12.035 

.D. Additional Information: Research Support and/or Scholastic Performance 

Completed Research Support (the last federally funded project): 

2010-2016, NIH R01DC009821, The Functional Organization of the Central Gustatory System, 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 32 
PI (primary grant): Alan C. Spector, Florida State University 

Co-I (Subcontract #R01335): Camille Tessitore King, Stetson University 

Total Funding (FSU subcontract with Stetson): 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 33 
OMB No. 0925-0001 and 0925-0002 (Rev. 09/17 Approved Through 03/31/2020) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Michael S. King 

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Professor and Chair of Biology 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) 

INSTITUTION AND LOCATION 

DEGREE 

(if 

applicable) 

Completion 

Date 

MM/YYYY 

FIELD OF STUDY 

Princeton University, Princeton, NJ B.A. 06/1986 Biology 

University of Virginia, Charlottesville, VA Ph.D. 01/1992 Neuroscience 

University of Michigan, Ann Arbor, MI Postdoc 07/1993 Central Taste 

Physiology 

A. Personal Statement 

Beginning with my post-doctoral training, my research has focused on the neuroanatomy and physiology of the 

central gustatory system as well as behavioral responses to taste input in rodents. Specifically, my work has 

examined several aspects of the taste system including the physiology of ganglion and nucleus of solitary tract 

(NST) neurons, details of the connectivity and neurotransmitters expressed by NST neurons that project to the 

parabrachial nucleus (PBN), and the role of CNS structures in the behavioral responses to taste input. 

Recently, I have been combining behavioral and neuroanatomical approaches to determine how the CNS 

controls taste reactivity responses to gustatory input. In this recent work, Fos-immunochemistry has been the 

main method by which I have studied role of central gustatory and related structures. I feel that this 

background will allow me to contribute to Dr. McCluskey’s work if needed.

B. Positions and Honors 

Professional Positions 

1993-1999 Assistant Professor of Biology, Stetson University 

1999-2005 Associate Professor of Biology, Stetson University 

2005-present Professor of Biology, Stetson University 

2001-2006 Chair, Dept. of Biology, Stetson University 

2021-present Chair, Dept. of Biology, Stetson University 

Honors 

1998 Hand Faculty Research Award, Stetson University 

2008 McEniry Award for Teaching Excellence, Stetson University 

Other Professional Activities 

1990-present Society for Neuroscience member 

1992-present Association for Chemoreception Sciences member 

1995-present National Association of Advisors for the Health Professions member 

1993-1998 Council on Undergraduate Research member 

2010-2016 Council on Undergraduate Research member 

2000 Project Kaleidoscope member 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 34 
1995-present Reviewer of ~25 manuscripts for scientific journals including: American Journal of Physiology, 

Brain Research, Brain Structure and Function, Chemical Senses, Journal of Autonomic 

Neuroscience, Journal of Neurophysiology, Neuroscience, Neuroscience Letters, Physiology and 

Behavior, Plos One 

1995-present Reviewed text book chapters for the Raven and Johnson (General Biology), Hadley 

(Endocrinology), Nordell and Valone (Animal Behavior) 

C. Contributions to Science 

1. My post-doctoral work and subsequent sabbatical research revealed some details about the physiological 

properties of central taste neurons. The following publications are reports of this work. 

King, M.S., Wang, L. and Bradley, R.M., 1993, Substance P excites neurons in the gustatory zone of the 

rat nucleus tractus solitarius. Brain Research 619:120-130. 

https://www.ncbi.nlm.nih.gov/pubmed/7690670 

King, M.S. and Bradley, R.M., 1994, Relationship between structure and function of neurons in the rat 

rostral nucleus tractus solitarii. J. Comparative Neurology 344:50-64. 

https://www.ncbi.nlm.nih.gov/pubmed/8063955 

Bradley, R.M., King, M.S., Wang, L. and Shu, X., 1996, Neurotransmitter and neuromodulatory activity in 

the gustatory zone of the nucleus tractus solitarius. Chemical Senses 21:377-385. 

https://www.ncbi.nlm.nih.gov/pubmed/8670717 

King, M.S. and Bradley, R.M., 2000, Biophysical properties and responses to glutamate receptor 

agonists of identified subpopulations of rat geniculate ganglion neurons, Brain Research 866: 237-246. 

https://www.ncbi.nlm.nih.gov/pubmed/10825499 

2. My initial work in my current position at Stetson University relied on fluorescent tract tracing and 

immunohistochemistry to investigate details of the connectivity and neurotransmitter expression of brainstem 

gustatory neurons. This work lead to the following publications. 

Williams, J.B., Murphy, D.M., Reynolds, K.E., Welch, S.J. and King, M.S., 1996, Demonstration of a 

bilateral projection from the rostral nucleus of the solitary tract to the medial parabrachial nucleus in rat. 

Brain Research 737:231-237. https://www.ncbi.nlm.nih.gov/pubmed/8930370 

Gill, C.F., Madden, J.M., Roberts, B.P., Evans, L.D. and King, M.S., 1999, A subpopulation of neurons 

in the rat rostral nucleus of the solitary tract that project to the parabrachial nucleus express glutamatelike immunoreactivity, Brain Research 821:251-262. https://www.ncbi.nlm.nih.gov/pubmed/10064811 

King, M.S., 2003, Distribution of immunoreactive GABA and glutamate receptors in the gustatory 

portion of the nucleus of the solitary tract in rat, Brain Research Bulletin 60:241-254. 

https://www.ncbi.nlm.nih.gov/pubmed/12754086 

Harrison, T.A., Hoover, D.B. and King, M.S., 2004, Distinct regional distributions of NK1 and NK3 

neurokinin receptor immunoreactivity in rat brainstem gustatory centers, Brain Research Bulletin 63:7- 

17. https://www.ncbi.nlm.nih.gov/pubmed/15121234 

3. Over the past twenty years or so my work has explored the role of central gustatory structures in behavioral 

responses to taste input. These studies required a combination of behavioral and anatomical techniques and 

led to the following publications. 

Galvin, K.E., King, C.T. and King, M.S., 2004, Stimulation of specific parabrachial subnuclei elicits 

ingestive oromotor behaviors in conscious rats, Behavioral Neuroscience 118:163-172. 

https://www.ncbi.nlm.nih.gov/pubmed/14979793 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 35 
Morganti, J.M., Odegard, A.K. and King, M.S., 2007, The number and location of Fos-like 

immunoreactive neurons in the central gustatory system following electrical stimulation of the 

parabrachial nucleus in conscious rats. Chemical Senses 32:543-555. 

https://www.ncbi.nlm.nih.gov/pubmed/17488743 

Riley, C.A. and King, M.S., 2013, Differential effects of electrical stimulation of the central amygdala 

and lateral hypothalamus on Fos-immunoreactive neurons in the gustatory brainstem and taste 

reactivity behaviors in conscious rats. Chemical Senses 38: 705-717. 

https://www.ncbi.nlm.nih.gov/pubmed/23978688 

King, M.S., 2018, Distribution of Fos-immunoreactive neurons in the gustatory cortex elicited by intraoral infusion of taste solutions in conscious rats. Brain Research 1683:67-77. 

https://www.ncbi.nlm.nih.gov/pubmed/29371098 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 36 
OMB No. 0925-0001 and 0925-0002 (Rev. 11/16 Approved Through 10/31/2018) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Xin-Yun Lu 

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Professor and Chair 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) 

INSTITUTION AND LOCATION 

DEGREE 

(if 

applicable) 

Completion 

Date 

MM/YYYY 

FIELD OF STUDY 

Washington State University, Pullman, WA Ph.D. 06/1998 Pharmacology 

University of Michigan, Ann Arbor, MI Postdoc. 1998-2001 Neuroscience 

A. Personal Statement 

The long-term goals of my research are to understand the molecular, cellular and circuit bases of 

neuropsychiatric and neurodegenerative disorders, especially for those with comorbid metabolic disorders. Type 

2 diabetes and other metabolic consequences of obesity are associated with higher rates of depression, anxiety 

and dementia. We have made significant contributions to our understanding of the roles of the adipose-brain 

axis in the regulation of mood, emotion and cognition. Adipose tissue, the primary site of energy storage, is now 

recognized as a highly active metabolic and endocrine organ, producing a number of secretory bioactive 

substances termed adipokines. Based upon our studies on two major adipokines (leptin and adiponectin), we 

have developed a novel theory of dysfunctional adipose-brain axis in the pathogenesis and treatment of 

depression, post-traumatic stress disorder (PTSD) and Alzheimer’s disease. We are particularly interested in

how adipokines interact with specific neural circuits orchestrating behavioral and cognitive responses to 

emotionally and motivationally relevant stimuli, and how they modulate neuroplasticity and tune adaption to 

experiences and environments in a sex-specific and age-dependent manner. 

a. Yu T, Guo M, Garza JC, Rendon S, Sun XL, Zhang W, Lu XY (2011). Cognitive and neural correlates 

of depression-like behavior in socially defeated mice: An animal model of depression with cognitive 

dysfunction. Int J Neuropsychopharmacol, 14:303-317. PMCID: PMC3432579 

b. Guo M, Lu Y, Garza JC, Li Y, Chua SC, Zhang W, Lu B, Lu XY (2012). Forebrain glutamatergic 

neurons mediate leptin action on depression-like behaviors and synaptic depression. Translational 

Psychiatry, 2: e83. PMCID: PMC3298113 

c. Zhang D, Wang X, Wang B, Garza JC, Fang X, Wang J, Scherer, PE, Brenner R, Lu XY (2017). 

Adiponectin regulates contextual fear extinction and intrinsic excitability of dentate gyrus neurons 

through AdipoR2 receptors. Molecular Psychiatry, 22(7):1044-1055. PMCID: PMC5491689 

d. Kim NS, Wen Z, Liu J, Zhou Y, Guo Z, Xu C, Lin YT, Yoon KJ, Park J, Cho M, Kim M, Wang X, Yu 

H, Salamuru S, Christian KM, Hsu KS, Xia M, Li W, Ross CA, Margolis RL, Lu XY# , Song H# , Ming 

GL#(2021). Pharmacological rescue in patient iPSC and mouse models with a rare DISC1 mutation. 

Nature Commun. 2021 Mar 3;12(1):1398. doi: 10.1038/s41467-021-21713-3. (

#

corresponding 

author) 

B. Positions and Honors 

Positions and Employment 

2001-2003 Research Investigator, Mental Health Research Institute, University of Michigan Medical School 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 37 
2003-2009 Assistant Professor, Department of Pharmacology, University of Texas Health Science Center 

2009-2013 Associate Professor, Department of Pharmacology, University of Texas Health Science Center 

2010-2017 Cross-appointed Faculty of Psychiatry, Department of Psychiatry, University of Texas Health 

Science Center 

2013-2017 Professor, Department of Pharmacology, University of Texas Health Science Center 

2017- Professor, Department of Neuroscience and Regenerative Medicine, Medical College of Georgia 

at Augusta University 

2019- Chair, Department of Neuroscience and Regenerative Medicine, Medical College of Georgia at 

Augusta University 

Honors, Awards, and Other Professional Activities 

2005 Scientist Development Award, American Heart Association 

2007 Presidential Distinguished Junior Research Scholar Award, University of Texas Health Science 

Center 

2009- Ad hoc reviewer of NIH study sections 

2010 Independent Investigator Award, National Alliance for Research on Schizophrenia and 

Depression 

2016-2020 Standing member, NIH/Neuroendocrinology, Neuroimmunology, Rhythms and Sleep Study 

Section 

2020-2022 Standing member, NIH/Behavioral Neuroendocrinology, Neuroimmunology, Rhythms, and 

Sleep (BNRS) 

2016- Member, American College of Neuropharmacology (ACNP) 

2017- Georgia Research Alliance Eminent Scholar Chair in Translational Neuroscience 

2018-2019 Member, External Scientific Advisory Board for the State University of New York 

Upstate Medical University 

C. Contribution to Science 

1. Glutamatergic neurons and mood-related behaviors. Leptin and its receptor LepRb are well known to 

play an important role in the pathogenesis of obesity. We identified novel functions of leptin in regulating 

different aspects of emotion-related behaviors. We found that leptin produces antidepressant-like effects 

through activating LepRb in forebrain glutamatergic neurons by reducing stress-evoked glutamate 

release/transmission at dentate gyrus (DG)-CA3 synapses. Ablation of LepRb in forebrain glutamatergic 

neurons causes pro-depressive behavior and abolishes leptin-s antidepressant-like effects. Moreover, we 

found that leptin treatment in intact female mice induces behavioral effects and stimulates hippocampal Akt 

phosphorylation in the proestrus phase but not in the diestrus phase of the estrous cycle. These effects in 

female mice can be abolished by ovariectomy and restored by pretreatment with 17β-estradiol in 

ovariectomized female mice. Our results suggest that leptin regulates mood-related behaviors in a sexspecific and estrous cycle-dependent manner. 

a. Guo M, Lu Y, Garza JC, Li Y, Chua SC, Zhang W, Lu B, Lu XY (2012). Forebrain glutamatergic 

neurons mediate leptin action on depression-like behaviors and synaptic depression. Translational 

Psychiatry, 2: e83. PMCID: PMC3298113 

b. Wang X, Zhang D, Lu XY (2015). Dentate gyrus-CA3 glutamate release/NMDA transmission 

mediates behavioral despair and antidepressant-like responses to leptin. Molecular Psychiatry, 

20(4):509-19. PMCID: PMC4362753 

c. Lei Y, Wang J, Wang D, Li C, Liu B, Fang X, You J, Guo M, Lu XY (2020). SIRT1 in forebrain 

excitatory glutamatergic neurons exerts sexually dimorphic effects on depression-related behaviors 

and modulates neuronal excitability and synaptic transmission in the medial prefrontal cortex. 

Molecular Psychiatry, 25:1094–1111. 

d. Li C, Meng F, Lei Y, Liu J, Liu J, Zhang J, Liu F, Liu C, Guo M, Lu XY (2020). Leptin regulates 

exon-specific transcription of the Bdnf gene via epigenetic modifications mediated by an AKT/p300 

HAT cascade. Molecular Psychiatry. 2020 Oct 26. doi: 10.1038/s41380-020-00922-0. Online ahead 

of print. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 38 
2. Leptin, dopamine neurons and emotional behaviors. Obesity has been associated with anxiety disorders. 

Current antidepressants are effective for the treatment of anxiety disorders after chronic administration. 

However, they can worsen anxiety in the initial phase of treatment. We found that both fluoxetine and leptin show 

antidepressant-like behavioral effects in rodent models, but they exhibit opposite effects on anxiety behavior. 

Acute administration of leptin produces anxiolytic effects, whereas acute fluoxetine elicits anxiogenic effects. 

Furthermore, we identified that dopamine neurons in the ventral tegmental area (VTA) mediate leptin’s effects

on anxiety through the Jak2/Stat3 signaling pathway. 

a. Liu J, Garza, JC, Bronner J, Kim CS, Zhang W, Lu XY (2010) Acute administration of leptin produces 

anxiolytic-like effects: A comparison with fluoxetine. Psychopharmacology, 207:535–545. PMCID: 

PMC4057895 

b. Liu J, Perez S, Zhang W, Lodge D, Lu XY (2011). Selective deletion of the leptin receptor in dopamine 

neurons produces anxiogenic-like behavior and increases dopaminergic activity in amygdala. 

Molecular Psychiatry, 16(10):1024-1038. PMCID: PMC3432580 

c. Liu J, Guo M, Lu XY (2015) Leptin/LepRb in the ventral tegmental area mediates anxiety-related 

behaviors. The International Journal of Neuropsychopharmacology, 19(2). pii: pyv115. PMCID: 

PMC4772826 

d. Carrier N, Wang X, Sun L, Lu XY (2015) Sex-specific and estrous cycle-dependent antidepressantlike effects and hippocampal Akt signaling of leptin. Endocrinology, 156(10):3695-3705. PMCID: 

PMC4588814 

3. Adiponectin, emotion-related behaviors and fear memories. Adiponectin is a metabolic hormone that is 

secreted exclusively by adipocytes and can cross the blood-brain barrier to exert effects on the central nervous 

system beyond metabolic control. We have made major contributions to general understanding how adiponectin 

mediates the crosstalk between adipocytes and neurons and how adiponectin expression in adipose tissue is 

regulated by stress and how adiponectin in the brain modulates the negative feedback action 

of glucocorticoid hormones, neuronal excitability, emotional behaviors and fear memories. We found that 

adipose PPARγ and adiponectin expression levels are downregulated after exposure to chronic stress and 

correlate with changes in behavioral responses. Central infusion of adiponectin induces antidepressant and 

anxiolytic effects. By contrast, adiponectin insufficiency increases susceptibility for stress-induced depressiveand anxiety-like behaviors. Moreover, we found that adiponectin in the ventral tegmental area inhibits dopamine 

neuron (DA) population activity and can reverse stress-induced increase in DA neuron firing and loss of AdipoR1 

specically in DA neurons induces anxiogenic behavior and enhances DA neuron responses to stress. In the 

hippocampus, adiponectin suppresses neuronal excitability of hippocampal granule neurons and modulates 

extinction of contextual fear memories in a mouse model of post-traumatic stress disorder. Adiponectin 

deficiency impairs fear extinction, whereas intra-hippocampal infusions of adiponectin facilitate extinction of 

contextual fear. 

a. Liu J, Guo M, Zhang D, Cheng SY, Liu M, Ding J, Scherer PE, Liu F, Lu XY (2012). Adiponectin is 

critical in determining susceptibility to depressive behaviors and has antidepressant-like activity. Proc 

Natl Acad Sci U S A, 109(30):12248-12253. PMCID: PMC3409774 

b. Guo M, Li C, Lei Y, Xu S, Zhao D, Lu XY (2017). Role of the adipose PPARγ/adiponectin axis in

susceptibility to stress and emotion-related disorders. Molecular Psychiatry, 22(7):1056-1068. 

PMCID: PMC5468488 

c. Zhang D, Wang X, Wang B, Garza JC, Fang X, Wang J, Scherer, PE, Brenner R, Lu XY (2017). 

Adiponectin regulates contextual fear extinction and intrinsic excitability of dentate gyrus neurons 

through AdipoR2 receptors. Molecular Psychiatry, 22(7):1044-1055. PMCID: PMC5491689 

d. Sun F, Lei Y, You J, Li C, Sun L, Garza J, Zhang D, Guo M, Scherer P, Lodge D, Lu XY (2019). 

Adiponectin modulates ventral tegmental area dopamine neuron activity and anxiety-related 

behavior through AdipoR1. Molecular Psychiatry, 24:126–144. PMID: 29988086 

2. Adipokines, neurogenesis and neuroplasticity. Remodeling of dendritic spines and synapses of existing 

neurons and adult neurogenesis in the hippocampus are critical for memory and emotional processing. Using in 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 39 
vitro and in vivo models, we found that leptin and adiponectin, two major adipokines, promote adult neurogenesis 

in the hippocampus via different intracellular signaling mechanisms. Moreover, we observed that adiponectin 

elicits neurotrophic effects on dendritic growth, arborization and spinogenesis. Our studies indicate that 

adipokines modulate two forms of structural plasticity in the hippocampus, i.e. morphological modifications of 

dendritic spines/synapses of existing neurons and generation of new neurons in adulthood. 

a. Garza JC, Guo M, Zhang W, Lu XY (2008). Leptin increases adult hippocampal neurogenesis in vivo 

and in vitro. The Journal of Biological Chemistry, 283(26):18238-47. PMCID: PMC2440628 

b. Zhang D, Guo M, Zhang W, Lu XY (2011). Adiponectin stimulates proliferation of adult hippocampal 

neural stem/progenitor cells through activation of p38MAPK/GSK-3β/β-catenin signaling cascade. 

The Journal of Biological Chemistry, 286(52):44913-20. PMCID: PMC3247954 

c. Garza JC, Guo M, Zhang W, Lu XY (2012). Leptin restores adult hippocampal neurogenesis in a 

chronic unpredictable stress model of depression and reverses glucocorticoid-induced inhibition of 

GSK-3β/β-catenin signaling. Molecular Psychiatry, 17(8):790-80. PMCID: PMC3368076 

d. Zhang D, Wang X, Lu XY (2016). Adiponectin exerts neurotrophic effects on dendritic arborization, 

spinogenesis and neurogenesis of the dentate gyrus of male mice. Endocrinology, 157(7):2853-2869. 

PMCID: PMC4929553 

5. Central melanocortin signaling, HPA and stress responses. The central melanocortin system has been 

extensively investigated for its role in the control of feeding behavior and body weight. Our anatomical studies 

show that POMC and AgRP neurons, the two major components of the melanocortin system, interact with the 

stress system. We found that corticotrophin releasing hormone (CRH) neurons in the paraventricular nucleus 

relay melanocortin signaling to influence the hypothalamo-pituitry-adrenal (HPA) axis activity and food intake. We 

demonstrated that melanocortins induces stress-like responses, such as anorexia, anxiety and HPA activation. 

Using emotional stressors, we compared the responsiveness of POMC and AgRP neurons and discovered that 

these neurons in the arcuate nucleus are rapidly but differentially recruited by emotional stress. This body of 

work emphasizes the important role of melanocortinergic signaling in mediating endocrine, emotional and 

feeding responses to acute and chronic stress. 

a. Liu J, Garza JC, Truong VTH, Henschel J, Zhang W, Lu XY (2007). The melanocortinergic 

pathway is rapidly recruited by acute emotional stress and contributes to stress-induced anorexia 

and anxiety-like behavior. Endocrinology, 148(11): 5531-5540. PMCID: PMC3708592 

b. Liu J, Garza JC, Li W, Lu XY (2013). Melanocortin-4 receptor in the medial amygdala regulates 

emotional stress-induced anxiety-like behaviour, anorexia and corticosterone secretion. The 

International Journal of Neuropsychopharmacology, 16(1):105-120. PMCID: PMC3708461 

c. Fang X, Jiang S, Wang J, Bai Y, Kim CS, Blake D, Wentraub NL, Lei Y, Lu XY (2021). Chronic 

unpredictable stress induces depression-related behaviors by suppressing AgRP neuron activity. 

Molecular Psychiatry, 2021 Jan 11;10.1038/s41380-020-01004-x. doi: 10.1038/s41380-020-01004-

x. Online ahead of print. 

d. Fang X, Chen Y, Wang J, Zhang Z, Bai Y, Denney K, Gan L, Guo M, Weintraub NL, Lei Y, Lu XY. 

Activation of hypothalamic POMC neurons is sufficient and necessary to induce depression-related 

behaviors. Molecular Psychiatry. Under revision. 

Complete List of Published Work in MyBibliography: 

https://www.ncbi.nlm.nih.gov/myncbi/1rAyIBH9C4ikN/bibliography/public/ 

D. Research Support 

1 RF1 AG062166-01 (Lu) 02/15/2019 – 01/31/2024 

NIH/NIA 

“The adipocyte PPARgama/adiponectin axis and Alzheimer's disease”

Major Goal(s): To examine the role of the adipocyte-brain PPARγ/adiponectin/AdipoR axis in the onset and 

progression of Alzheimer’s disease.

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 40 
Role: Principal Investigator 

1R56 MH119456-01 (Lu) 04/01/2019 – 02/28/2022 (NCE) 

NIH/NIMH 

“Distinct roles of POMC and AgRP neurons in chronic stress-induced depressive behaviors”

Major Goal(s): To study how hypothalamic POMC and AgRP neurons modulate chronic stress-induced 

depressive behaviors. 

Role: Principal Investigator 

1R56AG064895-01 (Lu/Weintraub) 09/15/2019 – 06/30/2022 (NCE) 

NIH/NIA 

“HDAC9, Aging and Alzheimer's Disease”

Major Goal(s): This application aims at identifying the molecular bases underlying the effects of aging on ADrelated neuropathology and cognitive deficits. We expect that the results will provide insights into therapeutic 

interventions targeting specific subtypes of HDACs and tissue-selective therapy. 

Role: Co-Principal Investigator 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Biosketches Page 41 
PHS 398 Cover Page Supplement OMB Number: 0925-0001

Expiration Date: 02/28/2023

1. Vertebrate Animals Section

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Cumulative Budget Information

1. Total Costs, Entire Project Period

Section A, Total Direct Cost less Consortium Indirect (F&A) for Entire Project Period ($)

Section A, Total Consortium Indirect (F&A) for Entire Project Period ($)

Section A, Total Direct Costs for Entire Project Period ($)

Section B, Total Indirect (F&A) Costs for Entire Project Period ($)

Section C, Total Direct and Indirect (F&A) Costs (A+B) for Entire Project Period ($)

2. Budget Justifications

Personnel Justification McCluskey_Personnel_Just2.pdf

Consortium Justification

Additional Narrative Justification

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195. Received Date: 2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 46 
PERSONNEL JUSTIFICATION 

Name Role Person Months on Project 

Lynnette McCluskey, Ph.D. PI 2.04 

Lin Gan, Ph.D. PI 0.96 

Daniel Linder, Ph.D. Collaborator 0.3 

Camille Tessitore King, PhD Consultant 0 

Michael King, PhD Consultant 0 

Xin-Yun Lu, MD/PhD Consultant 0 

Guangkuo Dong, MS Research Associate 6.0 

Schuyler Kogan, BS Research Assistant 10.2 

Lynnette McCluskey (PI) is a project PI. She will oversee and be responsible for all 

experiments. Dr. McCluskey will analyze results, perform imaging experiments, and 

write and edit manuscripts detailing results. 

Lin Gan (PI) is a project PI. He will oversee and be responsible for all mouse breeding 

and genotyping. Dr. Gan will analyze results, perform experiments, and edit 

manuscripts detailing results. 

Daniel Linder (Co-I) will perform mixed-model statistical analyses for the project. 

Camille Tessitore King (Consultant) will provide guidance on mapping Ace2+ cells in 

central taste structures in Aim 1. 

Michael King (Consultant) will provide guidance on mapping Ace2+ cells in central 

taste structures in Aim 1. We anticipate that undergraduate students in his laboratory 

will analyze images uploaded to a shared account. 

Xin-Yun Lu (Consultant) will advise on brain regions outside the taste system that may 

contain novel populations of Ace2+ cells. We will first focus on regions near taste 

structures using conventional anatomical techniques but expect that laboratory 

personnel will be trained on a new light-sheet microscope by early 2022. 

Guangkuo Dong (Research Associate) will perform experiments (qPCR, ELISAs, and 

neurophysiology). He is highly skilled at recording chorda tympani responses in mice. 

Schuyler Kogan (Research Assistant) will perform immunoassays, confocal imaging 

and image analyses for Aims 1 and 2. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Personnel Justification Page 47 
PHS 398 Research Plan OMB Number: 0925-0001

Expiration Date: 02/28/2023

Introduction

1. Introduction to Application

(for Resubmission and Revision applications) McCluskey_Introduction_FINAL.pdf

Research Plan Section

2. Specific Aims McCluskey_Aims2.pdf

3. Research Strategy* McCluskey_Research_FINAL.pdf

4. Progress Report Publication List

Other Research Plan Section

5. Vertebrate Animals McCluskey_Animals2.pdf

6. Select Agent Research

7. Multiple PD/PI Leadership Plan McCluskey_LeadershipPlan.pdf

8. Consortium/Contractual Arrangements

9. Letters of Support Compiled_Letters.pdf

10. Resource Sharing Plan(s) RESOURCE_SHARING_PLAN2.pdf

11. Authentication of Key Biological and/or

Chemical Resources McCluskey_Authentication2.pdf

Appendix

12. Appendix

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195. Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 48 
INTRODUCTION 

We thank the reviewers for their helpful comments on our previous R21 submission. The reviewers agreed 

that the proposed studies to investigate the role of ACE2 in the taste system of novel mouse models are 

“highly significant” given our meager understanding of taste loss due to SARS-CoV-2 infection. Reviewers 

also noted that the “PI, her colleagues, and the research environment were judged outstanding, fully capable of

bringing the project to fruition as designed”. We respond to reviewers’ comments and summarize revisions to 

the current application below. 

ACE-2 expression in taste buds. 

• Two reviewers commented that ACE2 staining in taste buds may be non-specific. We now include negative 

control images for ACE2 (Fig. 1) and TMPRSS (Fig. 2) immunofluorescence demonstrating antibody 

specificity. 

• When the previous application was submitted the premise that taste cells express ACE2 was more 

controversial. Ace2 expression was reported in a just a small subset of mouse type III taste cells by RNA-seq 

(Wang et al., 2020) although preliminary transcriptional profiling of public databases indicated more 

widespread expression in subsets of type II and III taste cells (Cooper et al., 2020). Ace2 expression in 

mouse taste buds was also demonstrated by PCR though only mentioned briefly since Ace1 was the focus of 

that study (see Fig. 1A by Shigemura et al., 2019). In our preliminary results, ACE2 immunoreactivity was 

widely detected in anterior and posterior mouse taste buds. Since the previous submission, ACE2 expression 

in type II human taste cells was shown by immunostaining and RNAscope in situ hybridization (Doyle et al., 

2021). In Aim 1 we propose to use a novel reporter mouse strain to map Ace2 expression with high 

sensitivity, resolving discrepancies between detection methods. 

Transgenic approaches 

• One reviewer was positive about mapping Ace2 expression in the taste system using the proposed reporter 

mice. Another reviewer commented that co-staining with cellular markers will be overly complicated in Ace2 

knock-in mice expressing both TdTomato and GFP. Therefore we have revised our design to replace GFP 

with the V5 epitope. We have recently generated Ace2-P2A-V5-CreERT2 (Ace2CE) knock-in founder mice 

using CRISPR/Cas9 method (Fig. 4) and will breed this new Ace2CE line with Ai14 Rosa26-tdTomato reporter 

mice. This strategy allows us to trace the lineage of Ace2+ cells with high sensitivity and straightforward 

immunolabeling with cell-specific markers. 

• The keratin (K14) promoter allows lingual epithelium-specific but not taste cell-specific Ace2 knockout. This 

approach is widely used in the field but has implications for interpreting results as now addressed (Aim 2a). 

• As suggested we have added collaborators with anatomical expertise in central taste regions (Dr. Michael 

King and Dr. Camille Tessitore King) and brain regions outside taste structures (Dr. Xin-Yun Lu). The Dept. 

of Neuroscience & Regenerative Medicine has also added a new workstation with NeuroInfo software which 

outlines brain regions on images using the Allen Mouse Brain Atlas. Thus we have the mouse models, 

equipment and collaborative expertise to map Ace2 expression in the central and peripheral taste system for 

Aim 1. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Introduction to Application Page 49 
SPECIFIC AIMS 

The virally-mediated disease known as COVID-19 has infected and killed millions worldwide. COVID19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by the 

loss of taste, smell and chemesthesis sometimes in the absence of other common symptoms such as 

respiratory distress, fatigue, cough, and fever. Taste deficits are prevalent and appear to be independent from 

anosmia. However, the biological basis of taste loss is largely unknown, including the potential for SARS-CoV2 infection and ensuing inflammation in taste receptor cells and ascending neural pathways. A fundamental 

question is whether central and peripheral taste structures are targets of SARS-Cov-2. Insight to host viral 

entry pathways and their normal function in taste buds is needed before SARS-Cov-2 induced taste deficits 

can be treated. While most patients regain taste sensation after several weeks, hypoguesia is longer lasting in 

a subset of patients leading to prolonged negative effects on nutrition and quality of life. 

The angiotensin-converting enzyme (ACE) 2 receptor is a negative regulator of the renin-angiotensin 

system (RAS) that mediates fluid balance and blood pressure. RAS hormones angiotensin II and aldosterone 

also modulate taste function, behavior, and immune responses to taste nerve injury. A member of the RAS, 

ACE2, binds the SARS-CoV-2 spike protein. Prior to host cell entry the spike protein must be cleaved by 

proteolytic enzymes such as furin, cathepsins, and most prominently, TMPRSS2. ACE2 is downregulated as 

receptor bound virus is internalized by host cells, driving the RAS to proinflammatory, vasoconstrictive, and 

fibrotic actions that contribute to organ damage. Thus ACE2 is essential for SARS-CoV-2 infection but also 

mediates protective responses through the RAS. Our preliminary results demonstrate robust ACE2 and 

TMPRSS2 expression in anterior and posterior mouse taste buds. 

Mouse models were highly useful in understanding the related SARS-CoV tropism and pathogenesis 

responsible for the severe acute respiratory syndrome (SARS) epidemic of 2002-2004. Yet the murine ACE2 

receptor binds SARS-CoV-2 ineffectively compared to human ACE2. This R21 Exploratory/Developmental 

Research application addresses the urgent need to understand the role of ACE2 in the taste system using 

suitable animal models. We have developed novel genetic mouse strains to address this gap in knowledge. 

We hypothesize that ACE2 is expressed in multiple taste cell types, modulates taste nerve responsivity, and 

has an anti-inflammatory, protective influence on the peripheral taste system. We also test whether ACE2 

bound to SARS-CoV-2 spike protein has detrimental effects on taste receptor cells and taste function which 

may contribute to taste deficits in COVID-19 patients. We test these hypotheses in two aims: 

Aim 1. Identify ACE2-expressing cells in peripheral and central taste pathways as potential targets of 

SARS-CoV-2. We will map ACE2 expression using standard cell markers in a novel Ace2-P2A-CreERT2 

knock-in (Ace2CE) strain of mice which express inducible CreERT2 at the endogenous Ace2 locus combined 

with Rosa26-tdTomato reporter mice. We will use this strain combined with antibodies to identify ACE2+ taste 

cell types, neurons, glia and blood vessels. 

Aim 2. Determine the contribution of lingual ACE2 to taste function and taste receptor cell dynamics in 

the healthy and inflamed peripheral taste system and potential modulation by SARS-CoV-2 spike 

protein. 

a. We will record responses to taste and tactile stimuli from a primary taste afferent nerve, the chorda tympani 

(CT), and quantify taste receptor cell number and turnover in lingual epithelium-specific Ace2 knockout 

mice using a new floxed Ace2 strain. Conditional Ace2 knockout and control mice will also be treated with 

systemic lipopolysaccharide (LPS) to test taste function during lingual inflammation. 

b. We will record neural responses and measure taste cell dynamics in humanized Ace2 knock in mice 

(hAce2-KI) which recapitulate endogenous ACE2 expression. Mice will be challenged with LPS and/or a 

human SARS-CoV-2 spike-Fc fusion protein to determine the impact on taste function. 

We expect taste changes in the absence of ACE2 reflecting the dysregulation of the RAS. Inflammation (Aim 

2a) and/or downregulation of ACE2 by spike-Fc (Aim 2b) are expected to exacerbate taste alterations based 

on a similar approach in a mouse lung injury model. 

These studies address the compelling need to understand ACE2 function in the taste system. If taste buds 

and associated pathways are indeed potential SARS-CoV-2 targets, we will test the effects of pseudovirus 

typed with human SARS-CoV-2 spike protein on taste function and behavior in hACE2-KI mice in future 

studies. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Specific Aims Page 50 
RESEARCH STRATEGY 

1. Significance 

The ongoing global pandemic caused by the novel coronavirus SARS-CoV-2 has resulted in millions of 

infections and deaths. Smell and taste deficits are major neurological symptoms of COVID-19 occurring in up 

to 80% of those infected often in the absence of respiratory ailments and fever1-9 . Chemosensory symptoms 

are highly predictive of SARS-CoV-2 infection and hospitalizations10-12 . Chemesthesis, mediated by sensory 

neurons in the lingual epithelium, also contributes to flavor perception. Recent reports indicate that loss of 

taste and chemesthesis can occur independently from anosmia2,3,6 . This is consistent with prior studies 

demonstrating taste deficits after upper respiratory viral infection and inflammation13-15 . Most patients regain 

taste sensation after recovery from viral infection but taste deficits persist in some people16-19 . Taste is 

important for nutrition and quality of life lending urgency to the search for mechanisms responsible for 

hypoguesia in COVID-19 patients20-25 . There is currently little known about the SARS-CoV-2 infection and 

associated inflammation in the taste system. This R21 application responds to the Notice of Special 

Interest (NOT-DC-20-008) from NIDCD focused on “the molecular mechanisms underlying chemosensory 

dysfunction due to SARS-CoV-2 infection”.

Angiotensin-converting enzyme (ACE)2 in the renin-angiotensin system (RAS) and SARS-CoV-2 

infection. 26Coronaviruses are composed of a sphere of structural proteins coated with a “corona” of viral

spike protein27 . The receptor for the SARS-CoV-2 spike protein is ACE228,29 , a negative regulator of the reninangiotensin-system (RAS) which controls extracellular fluid volume and blood pressure30 . In one wing of the 

RAS, ACE converts Ang I to Ang II which binds the AT1 receptor leading to vascular constriction, fibrosis, 

oxidative stress, and inflammation (Fig. 1). ACE2 counteracts these effects by converting Ang II to Ang (1-7) 

inducing vascular dilation, anti-fibrosis and anti-inflammatory effects30,31 . Thus ACE2 is protective in the context 

of the RAS. Conversely, ACE2 enables SARS-CoV-2 infection, and the widespread expression of ACE2 

throughout tissues is thought to underlie multi-organ damage in patients with severe COVID-1931,32 . Proteases 

are also needed to activate the ACE2-spike protein enabling viral fusion to the host cell membrane and 

internalization28,29 (Fig. 1). Host transmembrane protease, serine 2 (TMPRSS2) appears to be the most 

prominent spike activating protease though cathepsins28 , furin33 , and neuropilin-1 (NRP-1) are also cofactors34 . In Aim 1 we map endogenous Ace2 expression in the taste system of Ace2CE reporter mice to 

identify potential SARS-CoV-2 infection sites underlying taste deficits. 

RAS modulation of taste and inflammation. In the classic RAS feedback loop Ang II stimulates the adrenal 

cortex to produce aldosterone which causes Na+ reabsorption via epithelial Na+ channels (ENaCs)35 . ENaC 

also transduces Na+ in taste buds36-39 . Acute aldosterone treatment increases 

ENaC expression40 and function40 in taste receptor cells and elevates amiloridesensitive CT responses to Na+ 41 . Chronic aldosterone treatment in combination 

with contralateral CT nerve injury42 or with Na+ in drinking water43 inhibited CT 

responses to Na+ . Early macrophage responses to CT nerve sectioning were 

also reduced by aldosterone in contrast to the hormone’s well-established 

proinflammatory role in chronic cardiovascular injury44 . The tissue 

microenvironment, injury model, and length of aldosterone treatment may 

account for different effects on salt taste responsivity and inflammation44-46 . 

Ang II, upstream from aldosterone, suppressed amiloride-sensitive CT 

responses to Na+ but enhanced sweet responses. The authors suggest that Ang 

II increases Na+ ingestion by rapidly decreasing neural Na+ sensitivity before 

aldosterone restores homeostasis by elevating amiloride-sensitive Na+ 

responses47 . The Ang II receptor, AT1, is co-expressed with αENaC or the 

sweet taste receptor subunit, T1r3, in taste receptor cells demonstrating another 

role of RAS in the peripheral taste system47 . ACE2’s role in taste cell function is 

unknown48 . We will gain insight to the contribution of ACE2 to peripheral 

taste function under baseline and inflammatory conditions using lingual 

epithelial-specific Ace2 (LE-Ace2) knockout mice. 

ACE2 and lung injury. ACE2 is protective in mouse models of severe lung injury induced by LPS, sepsis and 

acid aspiration49-53 . ACE2 knockout worsened lung function in injury models while exogenous ACE2 acting via 

Fig. 1 ACE2 in the peripheral taste system.

We propose hat taste cells express ACE2 

(Aim 1) as a substrate for SARS-CoV-2 spike 

protein binding and entry. In Aim 2a we test 

whether taste nerve responses and taste cell 

dynamics are altered in the absence of lingual 

epithelium-specific Ace2, a negative regulator 

of Angiotensin (Ang) II, which drives the reninangiotensin system (RAS) to a destructive 

phenotype in lung. We will test taste function 

in humanized Ace2 mice in Aim 2b. 

Lipopolysaccharide (LPS) induced 

inflammation and SARS-CoV-2 spike-Fc 

protein are expected to further worsen taste 

outcomes. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Research Strategy Page 51 
the AT2 receptor protects against lung damage50 . Mice with acid-induced lung injury were given systemic 

SARS-CoV spike protein to establish a model that mimics SARS-CoV infection52 . This model was critical in 

demonstrating viral tropism and pathogenesis in the lung49,51,52 . The major limitation in following the same 

blueprint for the current pandemic is the ineffective binding of SARS-CoV-2 to the murine ACE2 receptor. We 

have addressed this problem by generating novel mouse models including humanized Ace2 knock in mice 

(hAce2-KI) which recapitulate endogenous Ace2 expression. In an approach used in lung, in Aim 2b we will 

determine taste effects of delivering a chimeric human spike protein to humanized ACE2 mice in combination 

with systemic LPS, which stimulates inflammation and alters taste cell dynamics and taste responses54,55 . 

Expression of ACE2 and associated SARS-CoV-2 entry molecules in taste buds. ACE2 expression in the 

human tongue and oral cavity has been reported56-60 most recently in type II taste cells bearing GPCRs for 

sweet, bitter and umami stimuli and near taste buds in 

post-mortem circumvallate papillae and fungiform 

biopsies61 . In the latter study, replicating SARS-CoV-2 

was detected in type II taste cells in fungiform papillae and 

taste stem cell proliferation was decreased by viral 

infection in patient biopsies. This study, while small, 

suggests that direct infection of type II taste cells could 

explain deficits in sweet, bitter and umami taste stimuli 

while indirect mechanisms could impact salt and sour 

taste perception61 . ACE2, TMPRSS2 and furin expression 

in human taste cell cultures have also been reported56 . 

Whether rodent taste buds express ACE2 is 

currently less clear. Ace2 was only detected in a small 

subset of mouse type III taste cells and lingual epithelial 

cells using RNA-Seq62 . Preliminary data mining from 

public databases indicated more widespread Ace2 

expression in subsets of mouse type II, III and LGR-5+ 

taste stem cells63 . A type I cell database was not 

available63 . Though a minor part of the publication, Ace2 

was also demonstrated in mouse taste buds and non-taste 

epithelium64 . ACE2 and TMPRSS immunoreactivity was 

reported in rat taste buds though not localized to specific 

types65 . Tmprss2 was not detected in type II or III taste 

cells in other analyses63 . 

Preliminary results. We demonstrate robust immunoreactivity for 

ACE2 (Fig. 2) and TMPRSS2 (Fig. 3) in K8+ taste cells from C57BL/6J 

mice. ACE2 and TMPRSS2 staining largely overlaps with K8, 

expressed by mature taste cells66 , indicating widespread expression in 

anterior and posterior lingual taste buds. We verified our results using 

lung as a positive control for ACE2 (Fig. 2C) and TMPRSS2 (Fig. 

3C)67,68 . Minimal staining was observed in negative control sections in 

which primary antibody was omitted (Fig. 2 and 3). Compared to lung, 

Ace2 mRNA expression levels are elevated in the CV papillae and 

anterior lingual epithelium containing fungiform taste buds (Fig. 2D). 

Tmprss2 expression levels are similar in lung, CV and anterior lingual 

epithelium (Fig. 3D). The reason for the difference between 

preliminary results and RNA-seq studies62,63 is unclear. In Aim 1 we 

resolve this discrepancy with a new reporter mouse to identify even 

weakly-positive and transiently-expressing Ace2+ cells. 

To facilitate the identification of Ace2-expressing cells and the 

functional study of ACE2 in the taste tissue, we have been creating in the 

C57BL/6J background the following three novel Ace2 mouse models 

Fig. 3 TMPRSS2 expression in mouse taste buds. 

TMPRSS2 is expressed in K8+ taste buds in (A) CV and 

(B) FG papilla andTMPRSS2-omitted negative control. 

Positive and negative staining is also shown in (C) lung. 

Results are representative of n=3-4 mice in assays 

performed 3x. (E) Tmprss2 mRNA expression levels in 

the CV and anterior lingual epithelium were similar to 

those in lung which was set as 1 (n=3 mice / tissue). 

Fig. 2 Robust ACE2 expression in mouse taste buds. ACE2 expression 

(green) in (A) circumvallate (CV) and (B) fungiform (FG) taste buds labeled with 

the taste cell marker keratin (K)8 (red). CV taste buds in white squares are 

shown at higher magnification on the right. ACE2 antibody was omitted in 

negative control sections and laser settings matched to within-assay positive 

tissues. (C) Lung positive tissue control and nega ive staining control. Results 

are representative of n=3-4 mice in assays performed >3x. (D) Ace2 mRNA 

expression in CV papillae, anterior lingual epithelium containing fungiform taste 

buds and lingual mucosa. Transcript levels are expressed relative to lung which 

was set as 1 (n=3 mice/tissue). 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Research Strategy Page 52 
Contact PD/Pl: MCCLUSKEY, LYNNETTE Marie 

(Fig. 4 and Table 1 ): (1) Ace2-P2A-CreERT2 (Ace2CE) knockin mice: a tamoxifen-inducible CreERT2 (CE) fusion protein is 

inserted in-frame to the C-terminus of endogenous ACE2, an 

approach that we have recently used to create Gfi1GcE mice69. 

The self-cleaving P2A peptide allows the co-expression 

of CE from the endogenous Ace2 locus without disrupting 

ACE2 expression and function. In Aim 1, we will use 

this Ace2cE strain to activate reporter gene expression 

in Ace2-expressing cells and determine the taste cell types 

and primary taste structures potentially targeted by SARSCoV-2. (2) Ace2 conditional knockout (Ace2'0 xP) mice: Exon 4 

is flexed by loxP and can be deleted by Cre recombinase to 

cause a shift in reading-frame and the inactivation of Ace2. 

This strain allows study of Ace2 function in tissue/cell typespecific fashion; and (3) Humanized Ace2 knock-in (hAce2-KI) 

mice: Human Ace2 and SV4O polyadenylation sequences are 

knocked in the first Ace2 coding exon (exon 2) and in-frame 

with the translation initiation Met codon at mouse Ace2 locus. 

Thus, hACE2 expression is driven by the mouse endogenous 

Ace2 promoter and should recapitulate the expression of 

endogenous ACE2. AcezoxP and hAce2-KI mice will be used 

A - Cas9 - PCR4(wll 

Ace2 allele ~ ~ 

repalru,mplall! :::;;:;:¢1\fi~ 

n homologous 

vrecombination ➔ ... PCR4 (KJ) 

... PCR1 (5' •• mal) 

..... PCRS(intemll) ➔ • P(:,R% (3' ex:temaf) 

hACE2 Kl allele ~ PCRi PCR2Pat!PCR< 

B 5' PCR - - ➔ - 3'PCR 

Ace2 allele ~ 

X X repair template+# 

IO)(P loxP 

n homologous V recombination S'PCR ➔ ... ◄ ... 3'PCR = 

Ace2 ""P allele ~ 

IC)XP k»cP 

~ Cre recombinase - - ..a - --...-

Ace2-null allele -m-e-El-------t ~ 

lo,P 5'PCR 3'PCR 

Fig. 4 Generation of novel Ace2 mice. (A) hAce2-KI targeting strategy and PCR genotyping confinnation of hAce2-KI heterozygotes. (B) Ace'2"'1' targeting strategy and PCR genotyping confinnation of 

Ace;!>•P mice. (C) Ace;!JE targeting strategy and lineage tracing of Ace2-expressing cells by tamoxifen-inducible CE and Rosa26-

for neurophysiological studies in Aim 2 Currently we have tdTomato reporter gene. ArroWheads and arrows indicate WT and 

• ' targeted Ace2 PCR fragments. respectively. successfully generated germline-transmitted 

hA 2 Kl d A ,..JoxP • II A ,-,cE Table 1: Novel mouse strains used in proposal Ce use - an Ce, mice as We as Ce,- Strain Description 1----------=---------------------1 (see PCR genotyping confirmation of A 2-P2A- Expresses CreERT2 at the endogenous . . . ce . ce2 locus combined with the tdTomato m 1: Map Ace2 expression in the heterozygous mice m Fig. 4) and expect to CreE!J_J2 knoek-tn rom Ai1 4 Rosa26-STOP-tdTomato reporter peripheral and central taste system 

b • A ,-,CE 1

. . d (Acee-) 0 tam ce,- germ me transm1tte _____ 1 ..._m_ou_se_ m_e ________________ -----1 

. Bred to K14-cre mice (JAX #018964) to . . . heterozygous mice by September' 2021. ce2 conditional enerate taste receptor cell and lingual m 2a. Role of LE-Ace2 m taSt

e knockout mice ith 1

• ifi k k ut t . LE A 2 nct,on, dynamics, and response Scientific premise. ACE2 expression in (Ace'2"'1') KO eium-spec IC noc O s ram - ce O LPS challenge 

taste buds is supported by our preliminary Humanized Ace2 Expresses he human Ace2 gene under the m 2b_= Test taste func ion, 

It t t d. · h ctynam,cs. and response to human reSU S, recen S U leS USlng Uman knock-in mice control of the enoogeous Ace2 regulatory spike-Fe protein +/-LPS-induced 

samples56-61, and some64•

65 ( or partially in (Mce2-K/) uence ste inflamma ion 

agreement with63) but not all62 reports using rodent samples. Experiments proposed in Aim 1 using the novel 

Ace~E mouse strain will resolve inconsistencies. The premise that SARS-CoV-2 causes taste loss is widely 

accepted driving the need for biological insight to underlying mechanisms2•

63•

70. Strong evidence confirms 

SARS-CoV-2 entry mechanisms through ACE2 and TMPRSS similar to SARS-CoV28•

71 . Additional proteases 

activate SARS-CoV-2 in other systems72•

73, though which mechanisms prevail in taste buds is unknown63. 

Previous studies demonstrate the role of ACE2 in SARS-CoV pathogenesis in the lung49-51•

53. This work 

provides a guide for developing a clinically-relevant mouse model to test taste changes caused by the human 

SARS-CoV-2 spike protein. The involvement of the RAS in taste buds is also supported by evidence from 

multiple laboratories over time4043A7. Whether ACE2 counteracts Ang II to modulate taste function and 

inflammatory responses is unknown (Fig. 1 ), leading to a major gap in our understanding of this fundamental 

homeostatic system addressed in the proposed studies. 

2. Innovation 

The rapid emergence and spread of SARS-CoV-2 created a lag between the recognition of taste deficits in 

COVID-19 patients and insight to biological underpinnings. Major limitations in discovering mechanisms 

underlying taste loss have included limited mouse models due to ineffective binding between the SARS-CoV-2 

spike protein and mouse ACE274, the lack of BSL3 facilities needed to work with the virus, and the shortage of 

investigators trained in both chemosensation and immunology. We resolve each limitation in this R21 

Exploratory /Developmental application: (1) Dr. Gan used CRISPR/Cas9 to develop new mouse models 

including a humanized hAce2-KI strain (Table 1 ); (2) human spike-Fe protein will be used in hAce2-KI mice to 

define inflammatory responses and taste loss in standard labs; and (3) Dr. McCluskey trained in prominent 

Research Strategy Page 53 

Contact PD/Pl: MCCLUSKEY, LYNNETTE Marie 

(Fig. 4 and Table 1 ): (1) Ace2-P2A-CreERT2 (Ace2CE) knockin mice: a tamoxifen-inducible CreERT2 (CE) fusion protein is 

inserted in-frame to the C-terminus of endogenous ACE2, an 

approach that we have recently used to create Gfi1GcE mice69. 

The self-cleaving P2A peptide allows the co-expression 

of CE from the endogenous Ace2 locus without disrupting 

ACE2 expression and function. In Aim 1, we will use 

this Ace2cE strain to activate reporter gene expression 

in Ace2-expressing cells and determine the taste cell types 

and primary taste structures potentially targeted by SARSCoV-2. (2) Ace2 conditional knockout (Ace2'0 xP) mice: Exon 4 

is flexed by loxP and can be deleted by Cre recombinase to 

cause a shift in reading-frame and the inactivation of Ace2. 

This strain allows study of Ace2 function in tissue/cell typespecific fashion; and (3) Humanized Ace2 knock-in (hAce2-KI) 

mice: Human Ace2 and SV4O polyadenylation sequences are 

knocked in the first Ace2 coding exon (exon 2) and in-frame 

with the translation initiation Met codon at mouse Ace2 locus. 

Thus, hACE2 expression is driven by the mouse endogenous 

Ace2 promoter and should recapitulate the expression of 

endogenous ACE2. AcezoxP and hAce2-KI mice will be used 

A - Cas9 - PCR4(wll 

Ace2 allele ~ ~ 

repalru,mplall! :::;;:;:¢1\fi~ 

n homologous 

vrecombination ➔ ... PCR4 (KJ) 

... PCR1 (5' •• mal) 

..... PCRS(intemll) ➔ • P(:,R% (3' ex:temaf) 

hACE2 Kl allele ~ PCRi PCR2Pat!PCR< 

B 5' PCR - - ➔ - 3'PCR 

Ace2 allele ~ 

X X repair template+# 

IO)(P loxP 

n homologous V recombination S'PCR ➔ ... ◄ ... 3'PCR = 

Ace2 ""P allele ~ 

IC)XP k»cP 

~ Cre recombinase - - ..a - --...-

Ace2-null allele -m-e-El-------t ~ 

lo,P 5'PCR 3'PCR 

Fig. 4 Generation of novel Ace2 mice. (A) hAce2-KI targeting strategy and PCR genotyping confinnation of hAce2-KI heterozygotes. (B) Ace'2"'1' targeting strategy and PCR genotyping confinnation of 

Ace;!>•P mice. (C) Ace;!JE targeting strategy and lineage tracing of Ace2-expressing cells by tamoxifen-inducible CE and Rosa26-

for neurophysiological studies in Aim 2 Currently we have tdTomato reporter gene. ArroWheads and arrows indicate WT and 

• ' targeted Ace2 PCR fragments. respectively. successfully generated germline-transmitted 

hA 2 Kl d A ,..JoxP • II A ,-,cE Table 1: Novel mouse strains used in proposal Ce use - an Ce, mice as We as Ce,- Strain Description 1----------=---------------------1 (see PCR genotyping confirmation of A 2-P2A- Expresses CreERT2 at the endogenous . . . ce . ce2 locus combined with the tdTomato m 1: Map Ace2 expression in the heterozygous mice m Fig. 4) and expect to CreE!J_J2 knoek-tn rom Ai1 4 Rosa26-STOP-tdTomato reporter peripheral and central taste system 

b • A ,-,CE 1

. . d (Acee-) 0 tam ce,- germ me transm1tte _____ 1 ..._m_ou_se_ m_e ________________ -----1 

. Bred to K14-cre mice (JAX #018964) to . . . heterozygous mice by September' 2021. ce2 conditional enerate taste receptor cell and lingual m 2a. Role of LE-Ace2 m taSt

e knockout mice ith 1

• ifi k k ut t . LE A 2 nct,on, dynamics, and response Scientific premise. ACE2 expression in (Ace'2"'1') KO eium-spec IC noc O s ram - ce O LPS challenge 

taste buds is supported by our preliminary Humanized Ace2 Expresses he human Ace2 gene under the m 2b_= Test taste func ion, 

It t t d. · h ctynam,cs. and response to human reSU S, recen S U leS USlng Uman knock-in mice control of the enoogeous Ace2 regulatory spike-Fe protein +/-LPS-induced 

samples56-61, and some64•

65 ( or partially in (Mce2-K/) uence ste inflamma ion 

agreement with63) but not all62 reports using rodent samples. Experiments proposed in Aim 1 using the novel 

Ace~E mouse strain will resolve inconsistencies. The premise that SARS-CoV-2 causes taste loss is widely 

accepted driving the need for biological insight to underlying mechanisms2•

63•

70. Strong evidence confirms 

SARS-CoV-2 entry mechanisms through ACE2 and TMPRSS similar to SARS-CoV28•

71 . Additional proteases 

activate SARS-CoV-2 in other systems72•

73, though which mechanisms prevail in taste buds is unknown63. 

Previous studies demonstrate the role of ACE2 in SARS-CoV pathogenesis in the lung49-51•

53. This work 

provides a guide for developing a clinically-relevant mouse model to test taste changes caused by the human 

SARS-CoV-2 spike protein. The involvement of the RAS in taste buds is also supported by evidence from 

multiple laboratories over time4043A7. Whether ACE2 counteracts Ang II to modulate taste function and 

inflammatory responses is unknown (Fig. 1 ), leading to a major gap in our understanding of this fundamental 

homeostatic system addressed in the proposed studies. 

2. Innovation 

The rapid emergence and spread of SARS-CoV-2 created a lag between the recognition of taste deficits in 

COVID-19 patients and insight to biological underpinnings. Major limitations in discovering mechanisms 

underlying taste loss have included limited mouse models due to ineffective binding between the SARS-CoV-2 

spike protein and mouse ACE274, the lack of BSL3 facilities needed to work with the virus, and the shortage of 

investigators trained in both chemosensation and immunology. We resolve each limitation in this R21 

Exploratory /Developmental application: (1) Dr. Gan used CRISPR/Cas9 to develop new mouse models 

including a humanized hAce2-KI strain (Table 1 ); (2) human spike-Fe protein will be used in hAce2-KI mice to 

define inflammatory responses and taste loss in standard labs; and (3) Dr. McCluskey trained in prominent 

Research Strategy Page 53 

Contact PD/Pl: MCCLUSKEY, LYNNETTE Marie 

(Fig. 4 and Table 1 ): (1) Ace2-P2A-CreERT2 (Ace2CE) knockin mice: a tamoxifen-inducible CreERT2 (CE) fusion protein is 

inserted in-frame to the C-terminus of endogenous ACE2, an 

approach that we have recently used to create Gfi1GcE mice69. 

The self-cleaving P2A peptide allows the co-expression 

of CE from the endogenous Ace2 locus without disrupting 

ACE2 expression and function. In Aim 1, we will use 

this Ace2cE strain to activate reporter gene expression 

in Ace2-expressing cells and determine the taste cell types 

and primary taste structures potentially targeted by SARSCoV-2. (2) Ace2 conditional knockout (Ace2'0 xP) mice: Exon 4 

is flexed by loxP and can be deleted by Cre recombinase to 

cause a shift in reading-frame and the inactivation of Ace2. 

This strain allows study of Ace2 function in tissue/cell typespecific fashion; and (3) Humanized Ace2 knock-in (hAce2-KI) 

mice: Human Ace2 and SV4O polyadenylation sequences are 

knocked in the first Ace2 coding exon (exon 2) and in-frame 

with the translation initiation Met codon at mouse Ace2 locus. 

Thus, hACE2 expression is driven by the mouse endogenous 

Ace2 promoter and should recapitulate the expression of 

endogenous ACE2. AcezoxP and hAce2-KI mice will be used 

A - Cas9 - PCR4(wll 

Ace2 allele ~ ~ 

repalru,mplall! :::;;:;:¢1\fi~ 

n homologous 

vrecombination ➔ ... PCR4 (KJ) 

... PCR1 (5' •• mal) 

..... PCRS(intemll) ➔ • P(:,R% (3' ex:temaf) 

hACE2 Kl allele ~ PCRi PCR2Pat!PCR< 

B 5' PCR - - ➔ - 3'PCR 

Ace2 allele ~ 

X X repair template+# 

IO)(P loxP 

n homologous V recombination S'PCR ➔ ... ◄ ... 3'PCR = 

Ace2 ""P allele ~ 

IC)XP k»cP 

~ Cre recombinase - - ..a - --...-

Ace2-null allele -m-e-El-------t ~ 

lo,P 5'PCR 3'PCR 

Fig. 4 Generation of novel Ace2 mice. (A) hAce2-KI targeting strategy and PCR genotyping confinnation of hAce2-KI heterozygotes. (B) Ace'2"'1' targeting strategy and PCR genotyping confinnation of 

Ace;!>•P mice. (C) Ace;!JE targeting strategy and lineage tracing of Ace2-expressing cells by tamoxifen-inducible CE and Rosa26-

for neurophysiological studies in Aim 2 Currently we have tdTomato reporter gene. ArroWheads and arrows indicate WT and 

• ' targeted Ace2 PCR fragments. respectively. successfully generated germline-transmitted 

hA 2 Kl d A ,..JoxP • II A ,-,cE Table 1: Novel mouse strains used in proposal Ce use - an Ce, mice as We as Ce,- Strain Description 1----------=---------------------1 (see PCR genotyping confirmation of A 2-P2A- Expresses CreERT2 at the endogenous . . . ce . ce2 locus combined with the tdTomato m 1: Map Ace2 expression in the heterozygous mice m Fig. 4) and expect to CreE!J_J2 knoek-tn rom Ai1 4 Rosa26-STOP-tdTomato reporter peripheral and central taste system 

b • A ,-,CE 1

. . d (Acee-) 0 tam ce,- germ me transm1tte _____ 1 ..._m_ou_se_ m_e ________________ -----1 

. Bred to K14-cre mice (JAX #018964) to . . . heterozygous mice by September' 2021. ce2 conditional enerate taste receptor cell and lingual m 2a. Role of LE-Ace2 m taSt

e knockout mice ith 1

• ifi k k ut t . LE A 2 nct,on, dynamics, and response Scientific premise. ACE2 expression in (Ace'2"'1') KO eium-spec IC noc O s ram - ce O LPS challenge 

taste buds is supported by our preliminary Humanized Ace2 Expresses he human Ace2 gene under the m 2b_= Test taste func ion, 

It t t d. · h ctynam,cs. and response to human reSU S, recen S U leS USlng Uman knock-in mice control of the enoogeous Ace2 regulatory spike-Fe protein +/-LPS-induced 

samples56-61, and some64•

65 ( or partially in (Mce2-K/) uence ste inflamma ion 

agreement with63) but not all62 reports using rodent samples. Experiments proposed in Aim 1 using the novel 

Ace~E mouse strain will resolve inconsistencies. The premise that SARS-CoV-2 causes taste loss is widely 

accepted driving the need for biological insight to underlying mechanisms2•

63•

70. Strong evidence confirms 

SARS-CoV-2 entry mechanisms through ACE2 and TMPRSS similar to SARS-CoV28•

71 . Additional proteases 

activate SARS-CoV-2 in other systems72•

73, though which mechanisms prevail in taste buds is unknown63. 

Previous studies demonstrate the role of ACE2 in SARS-CoV pathogenesis in the lung49-51•

53. This work 

provides a guide for developing a clinically-relevant mouse model to test taste changes caused by the human 

SARS-CoV-2 spike protein. The involvement of the RAS in taste buds is also supported by evidence from 

multiple laboratories over time4043A7. Whether ACE2 counteracts Ang II to modulate taste function and 

inflammatory responses is unknown (Fig. 1 ), leading to a major gap in our understanding of this fundamental 

homeostatic system addressed in the proposed studies. 

2. Innovation 

The rapid emergence and spread of SARS-CoV-2 created a lag between the recognition of taste deficits in 

COVID-19 patients and insight to biological underpinnings. Major limitations in discovering mechanisms 

underlying taste loss have included limited mouse models due to ineffective binding between the SARS-CoV-2 

spike protein and mouse ACE274, the lack of BSL3 facilities needed to work with the virus, and the shortage of 

investigators trained in both chemosensation and immunology. We resolve each limitation in this R21 

Exploratory /Developmental application: (1) Dr. Gan used CRISPR/Cas9 to develop new mouse models 

including a humanized hAce2-KI strain (Table 1 ); (2) human spike-Fe protein will be used in hAce2-KI mice to 

define inflammatory responses and taste loss in standard labs; and (3) Dr. McCluskey trained in prominent 

Research Strategy Page 53 

Contact PD/Pl: MCCLUSKEY, LYNNETTE Marie 

( Fig. 4 and Table 1 ): (1) Ace2-P2A-CreERT2 (Ace2cE) knockin mice: a tamoxifen-inducible CreERT2 (CE) fusion protein is 

inserted in-frame to the C-terminus of endogenous ACE2, an 

approach that we have recently used to create Gfi1GcE mice69. 

The self-cleaving P2A peptide allows the co-expression 

of CE from the endogenous Ace2 locus without disrupting 

ACE2 expression and function. In Aim 1, we will use 

this Ace2cE strain to activate reporter gene expression 

in Ace2-expressing cells and determine the taste cell types 

and primary taste structures potentially targeted by SARSCoV-2. (2) Ace2 conditional knockout (AcezaxP) mice: Exon 4 

is flexed by loxP and can be deleted by Cre recombinase to 

cause a shift in readi ng-frame and the inactivation of Ace2. 

This strai n allows study of Ace2 function in tissue/cell typespecific fashion; and (3) Humanized Ace2 knock-in (hAce2-KI) 

mice: Human Ace2 and SV4O polyadenylation sequences are 

knocked in the first Ace2 coding exon (exon 2) and i n-frame 

with the translation initiation Met codon at mouse Ace2 locus. 

Thus, hACE2 expression is driven by the mouse endogenous 

Ace2 promoter and should recapitulate the expression of 

endogenous ACE2. Ace2'0xP and hAce2-KI mice will be used 

for neurophysiological studies in Aim 2. Currently, we have 

successfully generated germline-transmitted 

hA 2 Kl d A rJoxP . II A -.CE Table 1: Novel mouse strains used in proposal Ce Use - an CeL mice as We as Ce..:::- 1--Strain -----+-----=------------,1--------------l Description 

(see PCR genotyping confirmation of A 2-P2A- ExpressesCreERT2atthe endogenous 

C ceERT2 kn ck-· ce2 locus combined with the tdTomato ·m 1: Map Ace2 expression in the heterozygous mice in Fig. 4) and expect to 0 m Ai1_

4 Rosa26-STOP-tdTomato reporter peripheral and central taste system 

obtain Ace2CE germline transmitted 1-------+---o

_u_se_h_ne__________________,1--------------l 

h · b S b 2021 ce2 conditional Bred 1° Kl4-cre mice (JAX #Ol B9G4) to ·m 2a: Role of LE-Ace2 in taste eterozygOUS mlCe y eptem er, . knockout m·ice enerate taste receptor cell and lingual nction. dynamics, and response Scientific premise. ACE2 expression in (Ace2""1') helium-specific knockout strain LE-Ace2 0 LPS challenge 

taste buds is supported by our preliminary Humanized Ace2 Expresses he human Ace2 gene under the a

:i ·

human results, recent studies using human knock-in mice trol of the endogeous Ace2 regulatory spike-Fe pr

otein +/-LPS-induced 

samples56-6 1 , and some64•

65 (or partially in (hAce2-K/) le inflamma ion 

agreement with63} but not all62 reports using rodent samples. Experiments proposed in Aim 1 using the novel 

Ace_2CE mouse strain will resolve inconsistencies. The premise that SARS-CoV-2 causes taste loss is widely 

accepted driving the need for biological insight to underlying mechanisms2•

63•

70. Strong evidence confirms 

SARS-CoV-2 entry mechanisms through ACE2 and TMPRSS similar to SARS-CoV28•

71 . Additional proteases 

activate SARS-CoV-2 in other systems72•

73 , though which mechanisms prevail in taste buds is unknown63. 

Previous studies demonstrate the role of ACE2 in SARS-CoV pathogenesis in the lung49-51 •

53. This work 

provides a guide for developing a clinically-relevant mouse model to test taste changes caused by the human 

SARS-CoV-2 spike protein. The involvement of the RAS in taste buds is also supported by evidence from 

multiple laboratories over time4D-43A7 _ Whether ACE2 counteracts Ang II to modulate taste function and 

inflammatory responses is unknown (Fig. 1 }, leading to a major gap in our understanding of this fundamental 

homeostatic system addressed in the proposed studies. 

2. Innovation 

The rapid emergence and spread of SARS-CoV-2 created a lag between the recognition of taste deficits in 

COVID-19 patients and insight to biological underpinnings. Major l imitations in discovering mechanisms 

underlying taste loss have included limited mouse models due to i neffective binding between the SARS-CoV-2 

spike protein and mouse ACE274, the lack of BSL3 facilities needed to work with the virus, and the shortage of 

investigators trained in both chemosensation and immunology. We resolve each limitation in this R21 

Exploratory /Developmental application: (1) Dr. Gan used CRISPR/Cas9 to develop new mouse models 

including a humanized hAce2-KI strain (Table 1 ); (2) human spike-Fe protein will be used in hAce2-KI mice to 

define inflammatory responses and taste loss in standard labs; and (3) Dr. McCluskey trained in prominent 

Research Strategy Page 53 
taste and neuroimmunology laboratories and has studied the taste-immune axis independently for >20 years. 

ACE2 in our humanized mice is expressed at the endogenous locus and is physiologically relevant. The use of 

the K18 promoter to drive ACE275,76 expression in commercially-available mice is of particular concern since a 

subset of rodent66,77,78 and human79 taste receptor cells express K18. One hAce2-KI strain, newly available 

from Jackson Laboratory80 , is available if needed for Aim 2b (Alternative outcomes). We have encountered 

unexpected hair cell and taste bud regeneration phenotypes in transgenic strains highlighting the need for 

multiple Ace2 mouse models. Together the novel mouse models and combined expertise of the team will 

enable fundamental insights to ACE2 and spike protein function in the taste system. 

3. Approach. 

In Aim 1 we map ACE2 expression in the taste system using novel Ace2CE and Rosa26-tdTomato reporter 

mice. In Aim 2 we use the conditional LE-Ace2 KO and hAce2-KI to test the role of ACE2 in taste function, 

inflammation, and in response to human SARS-CoV-2 spike protein. 

Scientific rigor, reproducibility, and sex as a biological variable. We determined group sizes by power 

analyses and chose statistical analyses in collaboration with Dr. Daniel Linder in the Dept. of Biostatistics & 

Epidemiology at the Medical College of Georgia. Analyses will be performed by personnel blinded to sample 

identity and treatment when possible. Each experiment will be and include multiple treatment groups. We will 

test both male and female mice and analyze gender as a variable since SARS-CoV-2 affects men and women 

differently. Men with COVID-10 die at a greater rate than female patients but it is currently unclear whether 

disparities in ACE2 expression, innate and acquired immunity or other factors are to blame81-87 . In contrast, 

chemosensory deficits appear to be more prevalent in SARS-CoV-2 positive females vs. males4,88 . Young adult 

mice (8-10 weeks old) will be used in the proposed studies since determining the role of ACE2 in taste across 

the lifespan is outside the scope of this 2-year project. 

Aim 1. Identify Ace2 expressing cells in peripheral and central taste pathways as potential targets of 

SARS-CoV-2. 

Rationale In this aim, we will generate Ace2CE tamoxifen-inducible CreERT2 knock-in mouse 

strain by crossing Ace2CE founders with wild type C57BL/6J mice. We will then cross Ace2CE/+ 

mice with conditional tdTomato reporter mice (Ai14 Rosa26-STOP-tdTomato, JAX #007908) 

to generate double heterozygous Ace2CE/+ , Rosa26-STOP-tdTomato mice. The expression of 

tdTomato in the Ace2-expressing cells will be activated by i.p. injection of tamoxifen (1-5 

consecutive daily dosage of 40 μg/g body weight) into adult Ace2CE/+ , Rosa26-STOPtdTomato mice. The high tdTomato expression level from Rosa26-CAG promoter allows easy 

detection of Ace2-expressing cells. We will co-immunolabel tdTomato and standard taste cell, 

neuronal, glial and vascular markers to determine endogenous Ace2 expression in 

peripheral and central taste pathways listed with abbreviations in Fig. 5: (1) Anterior, 

posterior and palatal taste buds; (2) Sensory afferent fibers from the CT (anterior), GL 

(posterior), GSP (palate) and lingual nerves (lingual epithelium); and (3) taste structures 

from taste ganglia through GC. We will also map SARS-CoV-2 protease activators which 

promote viral entry to host cells (Fig. 3). Together, these results will indicate potential 

mechanisms for SARS-CoV-2 induced taste deficits due to the loss of: (1) type I glial-like 

taste cells; (2) type II sweet, umami, and bitter-sensing cells; and (3) type III sour and saltsensing cells which also synapse with afferent nerve fibers89 . Infection of each taste cell 

population could affect taste qualities directly or through inflammatory bystander effects on 

intact taste cells. Ace2 expression in taste stem and progenitor cells suggests that taste cell replacement may 

also be dysregulated by viral infection61,63 . Together results from this aim provide insight to potential targets of 

SARS-CoV-2 in murine taste buds and central taste regions. 

Expected outcomes. We hypothesize that: (1) Ace2 and TMPRSS2 are expressed in type I, II, and III taste 

cells based on preliminary results (Fig. 2, 3); (2) stem cells in anterior and posterior taste buds express Ace2 

and TMPRSS261,63 and (3) taste afferent nerves and their cell bodies are Ace2 negative based on preliminary 

transcriptional profiling63 . ACE2 expression in the NTS is established90-92 but only preliminary in the PbN to 

date93 . To our knowledge endogenous vs. K18-driven76,94,95 Ace2 expression has not been determined in the 

mouse VPMpc or specifically in GC96-98 . Thus we will confirm Ace2 in the rostral NTS and determine its 

expression in taste projection areas because their potential for SARS-CoV-2 infection is currently 

Fig. 5 Quantification of Ace2 

expression in taste 

structures. Abbreviations: 

circumvallate (CV); greater 

superficial petrosal nerve 

(GSP); glossopharyngeal 

nerve (GL); chorda tympani 

nerve (CT); nucleus of the 

solitary tract (NTS); 

parabrachial nucleus (PbN); 

medial parvicellular region of 

the ventral posterior medial 

thalamus (VPMpc); gustatory 

cortex (GC). 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Research Strategy Page 54 
unresolved76,86,94,95,99-102 . PbN is not a major taste projection area in humans103 but its inclusion provides 

insight to potential RAS regulation of taste and cardiovascular function90,91,104 . Subsets of endothelial and/or 

glial cells may express Ace2+ though this remains contested and likely varies across brain regions105,106 . 

By colabeling with anti-V5 antibody, we can also determine cells that are expressing Ace2 presently 

(V5+ and tdTomato) or transiently (V5- and TdTomato+) in taste buds. Furthermore, the highly sensitive cell 

lineage tracing approach allows the identification of cells with transient and/or weak ACE2 expression, which 

may not be detected by immunolabeling with ACE2 antibody. These novel ACE2-expressing cells will be the 

focus of future neurophysiological and behavioral studies using pseudotyped virus. 

Immunofluorescence and qPCR. Tamoxifen-induced Ace2CE , Rosa26-TdTomato mice and control genotype 

littermates with and without tamoxifen (n=4/gender/group) will be perfused and taste tissues dissected and 

cryosectioned. Mouse taste buds will be co-labeled with anti-TdTomato to detect Ace2-expressing cells and 

antibodies to type I marker NTPdase2107-109 , type II marker PLCβ2110,111 , and type III marker carbonic 

anahydrase IV (CA4)111-113 . If type II and III taste cells are positive we will co-localize ACE2 with markers for 

functional subsets (e.g. T1r1, T1r2, T1r3, OTOP-1)89,114 . We will double label tdTomato with the stem cell 

markers Lgr6 in anterior taste buds115 , Lgr5 in posterior taste buds115-117 and the taste progenitor marker 

K14117,118 . CT fibers will be identified by P2X3119-121 , cell bodies with Phox2b122,123 , and GL, palatal and lingual 

nerve fibers with neurofilament124,125 . TdTomato-expressing Ace2+ neurons in the petrosal and trigeminal 

ganglia and central taste regions will be identified with βIII tubulin126 . Non-neuronal Ace2 expression will be colocalized with specific glial127 and vascular markers128 since these populations may be targets of SARS-CoV2129,130 . Negative control sections will be incubated in normal sera followed by secondary antibodies. 

Confocal images will be acquired with a Nikon A1R multiphoton/confocal system and analyzed with NIS 

Elements Version 4.3 (Nikon) and ImageJ (NIH). Double- and single-positive taste cells will be determined by 

cell counts or integrated density. Ganglia will be stained as whole mounts and double-positive and singlepositive cells counted as described122 . Ace2+ neurons, glia or vessels will be also be quantified in the NTS, 

PbN, and GC131-133 . The PIs’ department has a new automated analysis system with NeuroInfo software 

(Microbrightfield) which delineates brain regions on images using the Allen Mouse Brain Atlas134 . In Ace2-

TdTomato+ taste structures we will co-localize cell markers and SARS-CoV-2 activators TMPRSS2, furin, 

NRP-1, and Cathepsin B and Cathepsin L34,63,65,73,135-137. We will confirm Ace2 and viral entry co-factor gene 

expression by qPCR in the CV, FG punches, non-taste lingual epithelium and taste neurons in C57BL/6J mice 

as in Fig. 2-3138,139 . Single-positive cells, double-positive cells, and mRNA expression will be compared 

between groups with ANOVAs followed by Bonferroni posttests. 

Alternative outcomes and potential pitfalls. Dr. Gan is an expert in CRSPR/Cas9 technology and has 

extensive experience using Cre-activated reporter gene expression in cell lineage analysis69,140-143 . We do not 

expect problems in generating sufficient Ace2CE mice. We have validated ACE2 and TMPRSS antibodies in 

CV and FG taste buds (Fig. 2-3) in case of unforeseen circumstances. 

Aim 2a. Determine the contribution of lingual ACE2 to taste function and taste receptor cell dynamics 

in the healthy and inflamed peripheral taste system. We will record responses to taste and tactile stimuli 

from the chorda tympani (CT) nerve and quantify taste receptor cell number and turnover in LE-specific Ace2 

knockout mice using a new floxed Ace2 strain. Conditional Ace2 knockout and control mice will also be treated 

with systemic LPS to test taste function during lingual inflammation. 

Rationale. We will use conditional Ace2 knockout mice in this aim to probe the function of the SARS-CoV-2 

receptor in taste buds. Removing the ACE2 brake on Ang II exacerbates inflammatory responses to LPS and 

tissue injury in other tissues (Fig. 1). We hypothesize that lingual cytokine and leukocyte responses to LPS will 

also be elevated in conditional LE-Ace2 KO mice dampening neural taste responses to multiple stimuli. The 

unknown role of ACE2 in taste function is a barrier to understanding taste deficits caused by SARS-CoV-2. 

LPS treatment and neurophysiology. LE-Ace2 KO or heterogeneous littermates will receive LPS (5 mg/kg 

b.w.) or PBS i.p. We will record CT responses to salt (with and without 50 μm amiloride), sweet, bitter, acid, 

and sour tastants, 4oC water, and tactile stimulation at 6 and 24 hr after treatment according to standard 

methods (n=5 male and n=5 female mice/group/timepoint)138,139,144 . The timing is based on cytokine and 

neutrophil responses to LPS within hours and changes in taste cell dynamics at 24 hr54,145-149 . Terminal blood 

and tissues will be collected following recordings and used to measure circulating cytokine levels with 

multianalyte ELISAs. Cytokine levels in taste epithelium will also be analyzed by qPCR and local macrophage, 

T cell and neutrophil responses by immunofluorescence138,148-151 . We will count proliferating Ki67-positive, 

apoptotic cleaved caspase-3-positive, and type I, II, and III taste cells in fungiform papillae using confocal 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Research Strategy Page 55 
imaging as in Aim 1. The effects of genotype, LPS, gender and time will be identified with mixed-model 

analyses. 

Expected outcomes. Based on widespread ACE2 expression in taste buds (Fig. 2) we expect LPS treatment 

to suppress responses to multiple taste qualities in LE-Ace2 KO as taste cell turnover is dysregulated54,147 . In 

LPS-treated control littermates, we expect elevated Na+ responses, inflammatory cytokine levels, and 

leukocyte responses based on previous results54,145-149 . We expect LE-Ace2 KO to exacerbate inflammatory 

responses to LPS in as in other tissues31 . A common caveat in the field is that Ace2 will be deleted in taste 

cells and lingual keratinocytes because of their shared K14+ lineage152 . Thus, neural taste changes could be 

due to taste cell based or indirect mechanisms. 

Alternative outcomes and potential pitfalls. The techniques are standard to the field and our laboratories 

so no difficulties are anticipated. If CT responses to LPS are unaffected by LE-Ace2 at 6 or 24 hr we will 

extend survival time with s.c. fluid and record at day 2. However, taste bud levels of the cellular activity marker 

c-fos are suppressed at 6 hr after LPS injection so this is unexpected147 . ACE2 may play a fundamental role in 

salt taste in addition to its potential function as a SARS-CoV-2 receptor similar to its dual functions in the lung, 

kidney, and cardiovascular system30,31,153,154 . In that case amiloride-sensitive CT responses may be altered in 

vehicle-treated LE-Ace2 KO. 

Aim 2b. We will record neural responses and measure taste cell dynamics in hAce2-KI which recapitulate 

endogenous Ace2 expression. Mice will be challenged with LPS and/or a human SARS-CoV-2 spike-Fc fusion 

protein to determine the impact on taste function. 

Rationale. In this aim we determine the effect of human spike-Fc protein on inflammatory responses, taste 

bud composition, taste cell dynamics, and neural taste, tactile and temperature responses in mice expressing 

human Ace2. Fc-fusion proteins have improved stability and solubility in vivo155 , and this strategy has been 

successful in demonstrating ACE2-mediated mechanisms of lung injury during SARS-CoV infection49,51,52 . 

Results from this aim will contribute to our understanding of how SARS-CoV-2 causes taste deficits and 

strategies that might be used to treat long-term aguesia. 

Methods. CT recordings, LPS injections, analyses of inflammatory responses by ELISA, qPCR, 

immunofluorescence, taste cell confocal analyses and statistical analyses will be performed as described 

above. Groups of hAce2-KI mice and heterozygous littermates will be injected with LPS or PBS (i.p.). Human 

spike-Fc or control-Fc protein will be administered i.p. three times at 30 min before, 1 hr after, and 2 hr after 

LPS injections (n=5 mice/gender)52 . We will record from the CT and harvest samples at 6 hr after LPS 

injections since group differences emerged within hours in lung52 . Antibodies to the receptor binding domain of 

the human spike protein will be used to measure spike-Fc in Western blots on anterior lingual epithelium and 

lung lysates and in cryosections from anterior tongue co-labelled with type I, II, and III taste cell markers. 

Expected outcomes. We predict that spike-Fc injection will exacerbate inflammatory responses to LPS, 

reduce taste progenitor proliferation, increase taste cell death, alter the proportion of type I, II, and III taste 

cells, and decrease CT responses to multiple taste qualities more dramatically than LPS alone. 

Unexpected outcomes and potential pitfalls. If taste buds and CT responses are unaffected by LPS plus 

spike-Fc protein we will extend the survival period to 2 days post-LPS. LPS rapidly affects taste buds54,145-149 , 

however, and taste deficits appear to be early symptoms of SARS-CoV-2 infection156 . We could also deliver 

spike-Fc intranasally and/or i.v., though our goal is to test reactivity to the viral protein rather than immunize 

mice157 and human spike-Fc protein was administered systemically in lung models50,52 . If taste function is 

unaffected by treatment we would also test commercially-available hAce2-KI mice (JAX #035000). 

Timeline 

We are breeding mice and will have established breeding colonies by the start of year 1. We will begin Aim 1 

and neurophysiological studies for Aims 2a and 2b immediately. We expect to complete Aim 1 by the end of 

year 1 and CT recordings by the end of year 1.5. Immunological and immunofluorescent assays for Aims 2a 

and 2b will be ongoing as tissue is added. This allows time to complete all experiments before the end of year 

2 even if additional groups surviving 2 days are added. 

4. Future Studies 

We will test whether taste behavior is altered in LE-Ace2 KO and hAce2-KI mice receiving LPS and spike-Fc 

protein with collaborator Dr. David Pittman138 . A priority is to test whether exogenous ACE2 or the Ang II 

receptor antagonist, Losartan, ameliorates inflammation, structural changes in taste buds, and neural and 

behavioral taste deficits. We will also determine whether pseudotyped human SARS-CoV-2 virus, which can 

be used in BSL2 facilities63 , recapitulates mechanisms underlying taste loss in hAce2-KI mice. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Research Strategy Page 56 
PHS Human Subjects and Clinical Trials Information OMB Number: 0925-0001

Expiration Date: 02/28/2023

Use of Human Specimens and/or Data 

Does any of the proposed research in the 

application involve human specimens and/or data * ❍ Yes ● No 

Provide an explanation for any use of human 

specimens and/or data not considered to be 

human subjects research. 

Are Human Subjects Involved ❍ Yes ● No 

Is the Project Exempt from Federal regulations? ❍ Yes ❍ No 

Exemption Number ❏ 1 ❏ 2 ❏ 3 ❏ 4 ❏ 5 ❏ 6 ❏ 7 ❏ 8 

Other Requested Information 

Tracking Number: GRANT13439458 Funding Opportunity Number: PA-20-195 Received Date: 

2021-08-05T18:21:52.000-04:00 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Page 57 
VERTEBRATE ANIMALS 

1. Description of proposed use of animals. 

Mice. We propose to use n=192 8-10 week old male and female mice of the following strains: (1) Ace2-

P2A-V5-CreERT2 knock-in mice (Ace2CE); (2) Ace2 conditional knockout mice (Ace2loxP); (3) humanized 

Ace2 knock-in mice (hAce2-KI); (4) K14-Cre [#018964; B6N.Cg-Tg(Krt14-cre)1Amc/J; and (5) wild-type 

C57BL/6J mice (#000664). Novel Ace2 strains will be born and housed at the Medical College of Georgia 

at Augusta University and the remaining strains will be purchased from the Jackson Laboratory. 

The following animal procedures will be performed by trained personnel: 

a. Blood collection and i.p. injections. We will perform terminal bleeds for multianylate cytokine ELISAs. 

100 μl LPS or vehicle will be injected i.p. Some mice will also receive i.p. injections of human spike 

protein. 

b. Non-survival recording from the chorda tympani nerve. Mice will be deeply anesthetized with 

ketamine/xylazine, with supplemental injections given as necessary. Mice are tracheotomized, and the 

CT approached by a lateral dissection of the head. Sensory responses are recorded as taste stimuli are 

flowed over the tongue with a syringe. Body temperature is maintained with a water-circulating heating 

pad for the 1-2 hour recording. At the end of the recording, mice will be euthanized with isofluorane 

overdose followed by thoracotomy without awakening and tissues harvested for further analyses. 

2. Justification. Mice will be used for all aims because of the availability of transgenic strains generated by 

the Gan Laboratory to determine the expression and functional role of ACE2 in the taste system. The 

murine immune system is also well-studied and a wide variety of mouse reagents are available. The 

number of mice is minimized by using them for multiple experimental outcomes where possible. In all 

aims, mice used for neurophysiological recordings are euthanized then tissues harvested for 

immunofluorescence and qPCR. We will also work with Laboratory Animal Services to share tissues and 

animals with other PIs on campus when possible. Animal studies are needed because we are investigating 

complex processes in the taste-immune axis which cannot be adequately modeled in vitro at this time. 

3. Procedures to limit discomfort, distress, pain, and injury. Surgeries to dissect and record from the chorda 

tympani nerve will be performed under deep anesthesia to prevent pain and distress. Mice are euthanized 

following recordings before awakening. Dr. McCluskey has >20 years of experience recording sensory 

responses in rats and mice. All laboratory personnel are trained to recognize pain and distress in mice. 

4. Euthanasia. Euthanasia will be accomplished by methods consistent with the AVMA Guidelines for the 

Euthanasia of Animals. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Vertebrate Animals Page 58 
MULTIPLE PI LEADERSHIP PLAN 

Rationale for multiple PI approach. This application has two PIs: Dr. Lynnette McCluskey and Dr. Lin Gan, 

who are both faculty in the Department of Neuroscience & Regenerative Medicine at the Medical College of 

Georgia at Augusta University. Dr. McCluskey’s group has studied neural-immune interactions in the taste 

system for over 20 years. She has expertise in peripheral taste regulation by the immune system using 

molecular, cellular, immunological and neurophysiological methods. Dr. Gan’s lab studies sensory cell loss, 

neuronal differentiation, circuitry formation and neurodevelopment. He has extensive experience in mouse 

genetics and is the Director of the departmental Transgenic and Genome Editing Core Facility. The PIs have 

adjacent offices, laboratories on the same floor, and developed this R21 proposal after discussing newly 

generated ACE2 mouse models, sensory deficits, and the urgent need to determine mechanisms underlying 

SARS-CoV-2-induced taste loss. 

Governance, organizational structure, and responsibilities. Dr. McCluskey will oversee the administration of the 

grant and serve as the contact PI. She will manage her personnel and collaborators and oversee compliance 

(i.e. mouse IACUC and biosafety protocols) to complete studies in Aims 1 and 2. Dr. Gan will provide the Ace2 

mouse models and perform genotyping and histology. 

Plans for communication. The proximity of our adjacent offices and laboratories on the same floor ensures that 

communication will be frequent. Joint laboratory meetings with all laboratory personnel and Dr. Linder will be 

held monthly. Consultants will be briefed on results and assistance requested as needed. Data files will be 

shared, updated, and continually available to both PIs via Box software. 

Authorship and resolution of conflict. We do not anticipate any disagreement on scientific direction or 

authorship based on our collegial relationship since Dr. Gan’s arrival at the Medical College of Georgia. In the 

unlikely event that there is a significant disagreement PIs will submit to independent arbitration by a neutral 

party such as an AChemS officer. 

Authorship will be determined by the Society for Neuroscience (SFN) guidelines for authorship. We expect that 

Dr. McCluskey will be senior author on publications since the project is focused on the taste system which is 

her area of expertise, though in some cases senior authorship will be shared. The first author will be 

determined by the significance of contribution and may be a PI or member of either lab. All undergraduate 

students, graduate students and lab personnel at Augusta University or Stetson University making significant 

contributions to the research as defined by SFN will share co-authorship. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Multiple PD/PI Leadership Plan Page 59 
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Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 
RESOURCE SHARING PLAN 

We will adhere to the NIH Grants Policy on Availability of Research Results: Publications, Intellectual Property 

Rights, Sharing Biomedical Research Resources and Sharing Model Organisms for Biomedical Research. 

We will make available any protocols, reagents or requested designs. Specifically, sharing of research 

materials generated in this project will be made under terms no more restrictive than in the Simple Letter 

Agreement for the Transfer of Materials or the Uniform Biological Materials Transfer Agreement (UBMTA), and 

made without reach-through requirements. We will make it available to the research community any reagent or 

resource that is presented in a publication that is published or accepted for publication from our laboratories. 

We agree to release and share data/results/findings in a timely manner, with the date of release to be no later 

than acceptance for publication of our main findings from our final data set. All raw and processed data 

contributing to publications arising from this work will be packaged into well-documented datasets and 

distributed through public repositories. 

More specifically: 

A. Molecular tools 

Plasmid constructs generated by this project will be distributed freely or deposited into Addgene to 

make them available to broader research community, either before or immediately after publication. 

B. Model organisms: 

All new mouse lines generated in this study will be either deposited to The Jackson Laboratory or 

shared with individual investigators upon request after publication. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Resource Sharing Plan(s) Page 69 
AUTHENTICATION OF KEY BIOLOGICAL AND/OR CHEMICAL RESOURCES 

1. Antibodies. Each antibody will be purchased from reliable sources, and will have been cited in multiple 

publications and/or high-quality publications by colleagues. Antibodies used as taste cell and leukocyte 

markers have been used extensively in multiple publications from our laboratory and/or many others. 

Immunologist colleagues at the Medical College of Georgia are readily available for consultation and 

positive control samples as needed. Careful records of lot numbers and assay outcomes are kept in 

the laboratory. Our typical immunohistochemical controls include: omission of primary antibody, 

incubation in matched concentrations of preimmune sera and pre-adsorption with excess antigenic 

peptide and for newer antibodies Western blotting to test specificity (Shi et al., 2012). 

2. Chemicals and Biologicals. We will use fresh stocks of E. coli 0111:B4 LPS (5 mg/kg; Sigma) diluted in 

sterile, endotoxin-free water as in our previous work. Human spike-Fc fusion protein (R&D Systems) 

has been widely tested since the earlier SARS-CoV pandemic. Mice will be injected i.p. three times 

with spike-Fc or control-Fc (5.5 nmol/kg) diluted in sterile, endotoxin-free water (Kuba et al., 2005). 

Spike-Fc binding is validated with His-tagged human ACE2 in functional ELISAs and pull down assays. 

Elevated levels of spike-Fc will be confirmed in taste tissues and lung by Western blot and 

immunoassays to co-localize with taste cell markers. Taste solutions are freshly made weekly and 

stored at 4o C between recordings before warming to room temperature. Chemicals are purchased 

from Sigma. All other chemicals and biologicals have been verified in our groups’ prior research and in

publications. 

3. ELISAs. We have used multi-analyte and single-analyte ELISAs from Qiagen and R&D Systems for 

many years with good inter-assay and intra-assay reliability. Blood is collected at the same time of day 

to control for circadian influences in cytokine assays. All chromogenic assays are performed in 

duplicate or triplicate. Recombinant mouse cytokines that serve as kit positive controls have shown 

strong responses, while negligible optical density values are generated by negative wells. We also 

include sera, plasma, tissue lysates, or conditioned media from positive and negative controls. 

4. Primers. We will use primer pairs as published by multiple sources in the field and check specificity with 

Primer Blast. Primers that span an intron will be chosen when possible to avoid detecting 

contaminating genomic DNA. 

5. Animals. Health reports from Jackson and from the MCG-AU mouse facility are monitored to ensure 

that mice are free from specified pathogens. Humidity, temperature and light cycles are controlled 

within the barrier animal facility. 

Contact PD/PI: MCCLUSKEY, LYNNETTE Marie 

Authentication of Key Biological and/or Chemical Resources Page 70