SUMMARY STATEMENT 

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 11/22/2021 

Revised Date: 

Principal Investigators (Listed Alphabetically): 

Application Number: 1 R21 DC019832-01A1 

Formerly: 1 R21 DC019832-01 

GAN, LIN 

MCCLUSKEY, LYNNETTE MARIE (Contact) 

Applicant Organization: 

Review Group: NIC 

Neuroscience of lnteroception and Chemosensation Study Section 

Meeting Date: 10/28/2021 

Council: JAN 2022 

Requested Start: 04/01/2022 

RFAIPA: PA20-195 

PCC: CS04 

Project Title: Ace2 in the healthy and inflamed taste system 

SRG Action: Impact Score:17 Percentile:3 + 

Next Steps: Visit https://grants.nih.gov/grants/next_steps.htm 

Human Subjects: 10-No human subjects involved 

Animal Subjects: 30-Vertebrate animals involved - no SRG concerns noted 

Project 

Year 

1 

2 

TOTAL 

Direct Costs 

Requested 

Estimated 

Total Cost 

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been adjusted 

to reflect any recommendations made by reviewers. If an award is planned, the costs will be calculated by 

Institute grants management staff based on the recommendations outlined below in the COMMITTEE 

BUDGET RECOMMENDATIONS section. 

1
1 R21 DC019832-01A1 2 NIC 

MCCLUSKEY, L 

RESUME AND SUMMARY OF DISCUSSION: The proposed exploratory research project is designed 

to test the hypothesis that taste buds are SARS-CoV-2 targets, that 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. The reviewers agreed 

that insight into mechanisms responsible for taste dysfunction due to SARS- CoV-2 are likely. All of the 

needed expertise and infrastructure are in place and additional collaborators have been recruited to the 

project to help advise portions of the study that require analysis of neuroanatomy of taste-related areas 

of the PNS and CNS. The questions are timely and important, novel transgenic mice will be utilized and 

the response to the prior review was thorough, including an additional transgenic mouse line. Rigor of 

the prior research was well-addressed, including discussion of gaps in the current literature, the 

experiments are well-designed and controlled, and both male and female mice will be studied. All in all, 

the possibility of attaining a better mechanistic understanding of how SARS-CoV-2 interacts with the 

taste system is strong, which is highly significant. 

DESCRIPTION (provided by applicant): Taste deficits are prevalent in people infected with SARSCoV-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 SARSCoV-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. 

PUBLIC HEALTH RELEVANCE: Taste loss is a common symptom of infection by SARS-CoV-2, the 

virus which causes COVID- 19 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 SARS- CoV2. 

CRITIQUE 1 

Significance: 1 

Investigator(s): 1 

Innovation: 1 

Approach: 2 

Environment: 1 
1 R21 DC019832-01A1 3 NIC 

MCCLUSKEY, L 

Overall Impact: This is a revision of an R21 multi-PI proposal from Lynette McCluskey, an investigator 

with established expertise is taste-immune interactions. She has established a collaboration Dr Lin Gin, 

a developmental biologist with expertise in manipulating the mouse genome to develop three novel 

models appropriate to address critical questions about interactions of the SARS-COV-2 virus with the 

taste system. The experiments will (1) probe expression of ACE2 and TMPRSS2 throughout the 

gustatory neuraxis, (2) explore the function of endogenous murine ACE2 in peripheral taste 

responsiveness and (3) Use a humanized ACE2 knock-in model to assess if the SARS-CoV-2 spike 

protein interferes with normal taste function. The questions are timely and important. The mouse lines 

avoid problems presented by currently available models and have been revised in line with the previous 

reviews. Two of the three have already been developed. The team and environment are excellent, and 

the revision brings in extra consultants for analyzing the CNS data, as suggested by previous 

reviewers. Additional preliminary data partially allays previous concerns regarding the specificity of 

immunostaining. Enthusiasm is high. 

Rigor of the Prior Research: The literature indicates considerable uncertainty with regard to the 

prevalence of ACE2 and TMPRSS expression in taste buds; these studies are cited by the PI and 

represent a gap to be filled by the proposed research. The PI cites a study by Doyle et al., as evidence 

for ACE2 expression in human taste buds. That study is not very convincing. 

Level of Experimental Rigor in the proposed research project: Each experiment uses appropriate 

controls, e.g., including blinded analyses of anatomical data. The previous reviews questioned the 

specificity of the immunohistochemistry in the preliminary data and noted that there were no negative 

controls omitting the primary antibody. These images have been added and show a lack of staining. 

Nevertheless, the high level of staining and its apparent ubiquitous distribution in the bud remains 

surprising. However, the proposed studies should resolve remaining questions, at least for ACE2 

staining. 

Sex as a Biological Variable: Both male and female mice will be used in all studies and the possibility 

of differences based upon clinical observations is discussed. 

1. Significance: 

Strengths 

• Covid-19 has been associated with chemosensory deficits. Olfactory deficits are the best 

documented but taste deficits are also possible, perhaps likely. These experiments will perform 

critical experiments for establishing a cellular basis for these deficits. 

Weaknesses 

• None noted. 

2. Investigator(s): 

Strengths 

• Dr. Lynette McCluskey is a productive, established investigator. She is one of a small cohort of 

scientists who have focused on taste-immune interactions. 

• Dr Lin Gan is a developmental biologist who has made several significant discoveries in the 

molecular basis of retinal and inner ear development. He has long-standing expertise in 

manipulating the mouse genome and is director of the Transgenic and Genome Editing Core at 

Augusta University, Medical College of Georgia. 
1 R21 DC019832-01A1 4 NIC 

MCCLUSKEY, L 

• The other members of the team are likewise excellent, provided needed expertise in all aspects 

of the project: CT nerve recording (G. Dong), confocal imaging/immunoassays (S. Kogan), and 

statistical support (D. Linder). 

• In response to previous concerns that the team did not have expertise in CNS anatomy, 

additional members have been added to the team: Two individuals with established expertise in 

neuroanatomy, Camille King & Michael King will consult on the gustatory CNS mapping of ACE 

and Xin-Lun Yu will provide complementary expertise for central regions outside the gustatory 

system. 

Weaknesses 

• None 

3. Innovation: 

Strengths 

• Novel mouse models relevant to Covid-19. 

Weaknesses 

• None 

4. Approach: 

Strengths 

• The proposed mouse models study the expression and the effects of deletion and humanization 

of the ACE2 receptor under control of the endogenous promoter. 

• Two of the three mouse models have been developed and validated with PCR. 

• Elucidating the expression of ACE2 throughout the gustatory neuraxis using a conditional 

tdTomato reporter under the control of the endogenous promoter will greatly clarify the location 

of this receptor. These experiments have potential to extend beyond the gustatory system. 

• Recording experiments in the mouse that express the humanized ACE2 receptor to probe the 

effects of the Spike protein should be particularly revealing. 

• Sex as a biological variable is well-considered as are standard considerations for rigor. 

Weaknesses 

• Minor: Although the addition of negative control data for immunostaining is an improvement, the 

strong and ubiquitous staining for both the ACE2 and TMPRSS antibodies still raises some 

doubts. It is appreciated that the reporter mouse for ACE2 will help to resolve this question. 

However, the location of TMPRSS may still be in question and additional controls to check the 

antibody specificity would be appropriate. Overall, more skepticism about these data would 

have been reassuring. 

5. Environment: 

Strengths 

• Excellent facilities and a supportive environment. 

Weaknesses 
1 R21 DC019832-01A1 5 NIC 

MCCLUSKEY, L 

• None 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

YES, all criteria addressed 

• All the criteria are carefully & comprehensively addressed. 

Biohazards: 

Not Applicable (No Biohazards) 

Resubmission: 

• Addressed above. 

Resource Sharing Plans: 

Acceptable 

Authentication of Key Biological and/or Chemical Resources: 

Acceptable 

Budget and Period of Support: 

Recommend as Requested 

CRITIQUE 2 

Significance: 1 

Investigator(s): 1 

Innovation: 2 

Approach: 2 

Environment: 1 

Overall Impact: This revised R21 by PIs McCluskey and Gan proposes to study the role of ACE2 in 

the peripheral taste system and whether ACE2 when bound to SARS2-spike protein has a detrimental 

effect on TRCs and taste function. This is a very timely and important study given the world-wide 

impact of COVID and potential lingering taste loss after acute disease recovery. This proposal uses 

newly engineered mouse knock-in strains to label Ace2 expressing cells and also to introduce 

humanized Ace2 into the endogenous mouse locus to better understand the effects of SARS2 spike 

protein on the taste system. A floxed conditional knock-out strain will also be used to study the 

contribution of Ace2 in normal and inflamed taste tissues. The investigators have all of the expertise 

required to perform these studies, and these new strains, once characterized fully could be of great 
1 R21 DC019832-01A1 6 NIC 

MCCLUSKEY, L 

utility to the field (although there is already a humanized KI strain available). PIs have been very 

responsive to prior reviewer comments, changing the strategy to use a V5 epitope tag rather than a 

GFP for co-labeling (a nice improvement). The PIs have also recruited other collaborators to assist in 

the anatomical mapping of ACE2 expression throughout from the periphery to the CNS. 

Rigor of the Prior Research: Early studies of ACE2 expression within taste cells was not very vigorous, 

but now this is well established in both human and mouse taste tissues. 

Level of Experimental Rigor in the proposed research project: High 

Sex as a Biological Variable: Both sexes included, and sex will be used as a biological variable since 

COVID affects males more than females. ACE2 is located on the X chromosome – but this was not 

mentioned in the research strategy, which may play a role in sex differences in the engineered mice. 

1. Significance: 

Strengths 

• The widespread and long-term effects of COVID is a significant problem facing the world today. 

Understanding how SARS-coV2 infection affects the taste system may reveal ways to combat 

taste loss in patients. 

• Understanding the normal role of ACE2 in the taste system is an important goal itself. 

Weaknesses 

• None noted. 

2. Investigator(s): 

Strengths 

• PI McCluskey is perfectly suited to lead these investigations having a distinguished record of 

studying the interactions of inflammation and immunology in the taste system. 

• PI Gan is the director of the transgenic core at Augusta and has all of the expertise necessary to 

engineer the transgenic mouse lines for these experiments. 

• Collaborators have been recruited to the project to help advise portions of the study that require 

analysis of neuroanatomy of taste-related areas of the PNS and CNS. 

Weaknesses 

• None noted. 

3. Innovation: 

Strengths 

• The transgenic mouse lines developed here are elegantly designed and would be very useful for 

others in the field. 

Weaknesses 

• One of the lines – the humanized knockin already has been developed and is available at JAX. 

Is this a duplication of effort, or is there a rationale for developing this line to overcome some 

weakness of the currently available strain? 

4. Approach: 
1 R21 DC019832-01A1 7 NIC 

MCCLUSKEY, L 

Strengths 

• Each aim has a straightforward approach and a well-justified rationale. 

Weaknesses 

• All of the aims depend on the success of the transgenic lines. Given the skill and expertise of 

the PIs, I am confident that these lines will indeed be successfully generated and will allow the 

experiments to be completed as planned. 

5. Environment: 

Strengths 

• Excellent environment to successfully complete this work. 

Weaknesses 

• None noted. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

YES, all criteria addressed 

• Acceptable 

Biohazards: 

Not Applicable (No Biohazards) 

Resubmission: 

• PIs have been very responsive to previous critiques. 

Resource Sharing Plans: 

Acceptable 

• Acceptable 

Authentication of Key Biological and/or Chemical Resources: 

Acceptable 

Budget and Period of Support: 

Recommend as Requested 

CRITIQUE 3 
1 R21 DC019832-01A1 8 NIC 

MCCLUSKEY, L 

Significance: 2 

Investigator(s): 2 

Innovation: 3 

Approach: 3 

Environment: 2 

Overall Impact: This is an exciting resubmission from a highly productive (even in pandemic times) 

researcher from Augusta University, who proposes to study the role of ACE2 receptor in taste 

responsiveness. This effort has both basic and translational arms, in that ACE2-R is involved in COVID19 infection, and the investigator will examine how the presence of the spike protein impacts peripheral 

taste function. Thus, this research holds the promise of both aiding in our understanding of peripheral 

taste function and potentially providing the beginnings of an explanation of COVID-related perturbations 

of that function. Furthermore, the investigator has been satisfyingly responsive to the prior critiques, 

making major changes and bolstering questionable aspects of the proposal. An eminently worthy 

proposal. 

Rigor of the Prior Research: Extremely high 

Level of Experimental Rigor in the proposed research project: Great, and even better in the 

resubmission. 

Sex as a Biological Variable: handled. 

THE FOLLOWING SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO 

SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE, OR REVIEWERS' 

WRITTEN CRITIQUES, ON THE FOLLOWING ISSUES: 

VERTEBRATE ANIMALS: ACCEPTABLE 

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. 

Footnotes for 1 R21 DC019832-01A1; PI Name: MCCLUSKEY, LYNNETTE Marie 

+ Derived from the range of percentile values calculated for the study section that reviewed 

this application. 

NIH has modified its policy regarding the receipt of resubmissions (amended applications).See 

Guide Notice NOT-OD-18-197 at https://grants.nih.gov/grants/guide/notice-files/NOT-OD-18-

197.html. The impact/priority score is calculated after discussion of an application by 

averaging the overall scores (1-9) given by all voting reviewers on the committee and 

multiplying by 10. The criterion scores are submitted prior to the meeting by the individual 

reviewers assigned to an application, and are not discussed specifically at the review meeting 

or calculated into the overall impact score. Some applications also receive a percentile 

ranking. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring. 
MEETING ROSTER 

Neuroscience of lnteroception and Chemosensation Study Section 

Integrative, Functional and Cognitive Neuroscience Integrated Review Group 

CENTER FOR SCIENTIFIC REVIEW 

NIC 

10/28/2021 - 10/29/2021 

Notice of NIH Policy to All Applicants: Meeting rosters are provided for information purposes only. Applicant 

investigators and institutional officials must not communicate directly with study section members about an 

application before or after the review. Failure to observe this policy will create a serious breach of integrity 

in the peer review process, and may lead to actions outlined in NOT-OD-14-073 at 

https://grants.nih.gov/grants/guide/notice-files/NOT-OD-14-073.html, NOT-OD-15-106 at 

https://grants.nih.gov/grants/guide/notice-files/NOT-OD-15-106.html, and NOT-OD-18-115 at 

https://grants.nih.gov/grants/guide/notice-files/NOT-OD-18-115.html, including removal of the application from 

immediate review.