Impact Objectives• Delve deeper into the range of environmental factors affecting the sense of smell,
including age, inflammation and cigarette smoke• Understand the molecular mechanism of the pathology and the impact of upper
respiratory tract inflammation as a result of environmental factors such as smoking• Eventually devise methods to treat olfactory impairment through the use of easierand cheaper methods such as nose drop (collunarium) and oral supplements

Effects of smoking andageing on olfaction andolfactory neurogenesis
Assistant Professor Rumi Ueha from the University of Tokyo, Japan, and her colleagues Professors Satoshi
Ueha, Toshihiro Ito and Tatsuya Yamasoba, discuss their project to investigate the effects of smoking andageing on olfaction and olfactory neurogenesis

Assistant Professor
Rumi Ueha

Professor Satoshi Ueha

Professor Toshihiro ItoProfessor Tatsuya
Yamasoba

Could you expand on your work focusing on
ageing on the olfactory cell line?

Could you explain the processes involved inyour project?

We demonstrated a decline in the division anddifferentiation of neuro-progenitor cells anda decline in the number of mature olfactory
receptor neurons (ORNs) associated with thedecline in olfactory function related to ageing.
ORNs are complex ligand-gate channels
that, as a result of a significant number ofreceptor variants, enable the differentiationof different odours by the depolarisation ofreceptors on contact with different molecularcompositions. The mechanism underlying thisdecline in ORNs involves the upregulation ofinflammatory cytokines and a decrease in theexpression of Igf-1 and tissue structure-relatedgenes in the nasal mucosa. Considering ourresults, inflammatory cytokine inhibitors,
insulin-like growth factor-1 (IGF-1) promotersand supplements, and activators of IGF-1
signal transduction, may be important clinicaltargets for preventing the decline in olfactory
function associated with ageing.

In the research we used a comprehensive
microarray-based analysis of gene expression
in the nasal mucosa to investigate themolecular mechanisms underlying ageingassociated reduction of ORNs. The method of
research uses three distinct stages; the first is
the production of the animal model and tissue
collection followed by the verification of anumber of cells and preparation of the coronal
section of the olfactory epithelium. Following
the measurement of the number of cells by
immunohistochemical staining, the final
stage of the research is to assess olfactory
cell behaviour and the reaction to odoursby an olfactory habituation/dishabituation
test, and by measuring levels of a number of
inflammatory cytokines by extraction of mRNAand DNA analysis and the level of IL-1β andIL-6 expression by QRT-PCR.
What results are you able to discuss?

Firstly, the results showed that cigarettesmoke solution (CSS) impaired the olfactory
progenitor cells and induced olfactory
dysfunction in mice. Secondly, the results
showed that the intranasal administration ofCSS suppressed the recovery of ORNs andolfaction following methimazole-induced
olfactory epithelial (OE) injury. Cigarettesmoke solution administration decreases
Igf1 gene and IGF-1 protein expression in
the injured-OE, and recombinant human
IGF-I administration reverses CSS-induced
suppression of ORN recovery. The results
found that ORN recovery was not linked
to changes in SOX2+ ORN progenitor
cells in the basal layer of the OE, but waslinked to the impaired dividing of ORN
progenitors and impaired increase in thenumber of GAP43+ immature ORNs. At
the molecular level, the data showed thatin the nasal mucosa, mRNA expression
levels of neurotrophic factors such as brainderived neurotrophic factor, neurotrophin-3,neurotrophin-5, glial cell-derived
neurotrophic factor and insulin-like growth
factor-1 (IGF-1) were increased following OE
injury. In addition, in the aged mice, it wasalso demonstrated that smoking reduced
the number of mature ORNs and olfactory
dysfunction by increasing ORN death inthe OE, which eventually overwhelms theregenerative capacity of the epithelium. lAgeing and olfactory
disordersThe ‘Effects of smoking and ageing on olfaction and olfactory neurogenesis’ project is working to develop agreater understanding of how smoking in Japan influences olfactory disorders and how they could be treatedO

ur sense of smell is directly connected
to survival and wellbeing. In Japan,
the number of patients with olfactory
disorder has increased, but prevention
and treatment protocols remain limited.
Research by Assistant Professor Rumi
Ueha takes into consideration a range ofenvironmental factors affecting the senseof smell, including age, inflammation andcigarette smoke. She highlights: ‘Significant
oxidation occurs to human beings exposed
to daily life-creating stress and chronicrhinitis in the nasal region, and smoking
is involved in the onset and deteriorationof sinusitis.’ Risk factors for the onset ofchronic nasal and secondary nasal disorders
affect sense of smell by changing the flowof air that exacerbates mucous membrane
inflammation of the cavity and makes thepassage of air more difficult, resulting inboth breathing and olfactory disorders.
Olfactory disorders can occur as a result ofa variety of factors ranging from ageing to
toxic chemical exposure, airway allergies,
upper-airway viral infections, head trauma
or development of neurodegenerative
diseases, and in most cases loss of olfactory
senses is a characteristic symptom. With anageing population and increasing incidencesof respiratory infections and asthma, as well
as allergy related illnesses such as rhinitis,
it is becoming increasingly important tounderstand the molecular mechanism ofthe pathology and understand the impact
of upper respiratory tract infections as
a result of environmental factors suchas smoking. Ueha outlines three key
objectives to her research: ‘The objectives
are to understand at the molecular level
the effect of cigarette smoke on olfactorymechanisms, to assess how smoking caninfluence olfactory epithelium homeostasis
and epithelial disorder recovery process,and determine how the age of patients caninfluence inflammation and recovery.’ The
analysis will focus on histological analysis ofchanges to cells at the molecular level.
MOLECULAR ANALYSIS OF CIGARETTE
SMOKE IMPACT
In order to understand the impact ofcigarette smoke at both the molecular andbehavioural level, cigarette smoke solution(CSS) was administered periodicallyto mice prior to tissue extraction andmolecular analysis were monitored inbehavioural assessments. Ueha outlines:‘We administered CSS to mice over 24days and then examined ORN (olfactory
receptor neurons) existence, cell survival,
inflammatory cytokine levels in theolfactory epithelium (OE), and olfaction
using histological analyses, gene analyses
and olfactory habituation/dishabituation
testing.’ In terms of molecular assessment,
Ueha details: ‘Olfactory marker proteinpositive (OMP+) ORN levels were thenquantified and the number of SOX2+ ORN
progenitors, GAP43+ immature ORNs,
Ki67+ cells, and cleaved Cas3+ apoptotic
cells per mm of basal layer length weremanually counted using digital imaginganalysis.’ This molecular analysis wasselected as olfactory marker proteins areexpressed specifically in mature ORNs andKi67 proteins are also a known cellularmarker linked to cell proliferation and canbe detected throughout the depth of theolfactory epithelium, mainly in the basallayer. In addition, Ueha says: ‘Caspases
are also crucial mediators of programmed
cell death (apoptosis), and Cas3 is a major
death protease activated during apoptotic
processes, catalysing the specific cleavageof many key cellular proteins. As a result, it
is important to determine rates of decline

in olfactory receptors following exposure
to cigarette smoke.’ The research also
recorded the time sensitivity of potential
treatment intervention as the length ofexposure to CSS is critical to the numberof mature ORNs that do not recoverafter cessation of treatment resulting inpersistent olfactory dysfunction only inthe aged mice. As Ueha explains: ‘We firstexamined the effect of continuous intranasal
administration of CSS on ORNs in theyoung mice. We then examined the effects
in the aged mice. In the young mice, thereduction in mature ORN numbers became
more severe on day seven after the final
intranasal administration of CSS, but thengradually recovered during days 14 to 28. Inaddition, olfaction also recovered within 14days after the final intranasal administrationof CSS. But, in the aged mice, the number ofmature ORNs and olfaction did not recoverwith time.’
IGF1 REGULATION AND ROLE IN
OLFACTORY DISORDERS
One of the key findings of the researchis related to the first demonstration of alow-dose rhIGF-1 administration increasing
the number of OMP+ mature ORNs in theaged OE, although in comparison, a higher
rhIGF-1 dose did not correspondingly havepositive effects on the aged OE. Ueha
comments: ‘These dose-dependent effectsof rhIGF-1 on the aged OE may be importantfor developing treatments for ageing-related
olfactory dysfunction as IGF-1 is a growth
factor that exerts trophic effects on neuronaldevelopment and regeneration.’ The increasein the number of OMP+ mature ORNs
after IGF-1 administration can be partly
explained by the overwhelmingly higher cellproliferation of olfactory progenitors andimmature ORNs compared to the increase inI think nutritional supplements, vitamin-rich
supplements and neurotrophic factors are also
thought of as possible therapeutic measures
Cas3+ apoptotic cells. Ueha also recognises
that: ‘Our results indicate that the numberof olfactory progenitors and immatureand mature ORNs rises only by low-doserhIGF-1 treatment and that high-dose rhIGF-1
administration did not increase the numberof ORN progenitors and only promoted
both proliferation and apoptosis of ORN
progenitors/immature ORNs in the aged
OE.’ As it is the low-dose (not high-dose)rhIGF-1 administration that increases thenumber of OMP+ mature ORNs in the aged
OE, this will affect dose levels and intervals
for IGF administration, which is a key factor
in treatments for ageing-related olfactory
dysfunction and as Ueha acknowledges,
requires further research. She says: ‘The
reduction in mature ORN numbers is
associated with olfactory impairment inolder populations and IGF-1 administered at
an appropriate dose could prevent ageinginduced negative effects on ORNs. However,Ueha admits: ‘Because our results were only
obtained from mouse studies, work needs tofocus on developing IGF-1 application for thetreatment of human olfactory impairment.’
PREVENTING DYSOSMIA IN THE FUTURE
The results of this study into the impactof olfactory disorders has recognised thelong-term impact of smoking. As Ueha
explains: ‘Not only the mature olfactory cells
are impaired, but the olfactory epithelium
progenitor cells are also impaired at thebeginning.’ She adds: ‘Olfactory progenitor
cells are impaired by the effect on olfactory
cells by continuous exposure to smoke andthere is an increase in the effects of theolfactory epithelium changes in olfactory
disorders and the inflammatory response.’
The research demonstrates how neuronalcells did not recover and cell death isincreased and there is a strong differentiationin the ability of olfactory cells to regenerate
and illness persists. ‘In particular, the abilityof epithelial cells to recover and regenerate
reduces after prolonged exposure totobacco smoke and in older people thiscan determine the severity of subsequentolfactory disorders,’ Ueha explains. Ageingis associated with increasing levels of
inflammatory activity and proinflammatory
circulation of cytokines such as IL-1 andTNF, and as a result, impacts are increasedin older people as the regenerative capacityof the olfactory epithelium is overwhelmed.
Ueha underlines: ‘In younger people,
olfactory cell inflammation response isaddressed the same way as any other cell
inflammation and as a result, cell condition
and response limits the short-term impact on
health.’ She adds: ‘The research clarifies thatdelays to the recovery mechanism is a resultof the division and differentiation processof olfactory progenitor cells and a failure toinhibit differentiation into mature olfactory
cells is thought to determine the type andlevel of the olfactory disorder sustained ascell death is increased.’ Ueha concludes:‘Whilst smoking should be deterred, thereis the potential to develop mucosal receptor
targeted treatment to reduce the rate of cell
apoptosis and persistent olfactory
disorders.’ lFURTHER READING
For more information please visit the website: https://researchmap.jp/ruu/RELEVANT PAPERS
1. Damage to Olfactory Progenitor Cells Is Involved in Cigarette Smoke-Induced Olfactory
Dysfunction in Mice. The American Journal of Pathology, doi: 10.1016/j.ajpath.2015.11.009
2. Cigarette Smoke Delays Regeneration of the Olfactory Epithelium in Mice Neurotoxicity
research, doi: 10.1007/s12640-016-9617-5
3. Reduction of Proliferating Olfactory Cells and Low Expression of Extracellular Matrix
Genes Are Hallmarks of the Aged Olfactory Mucosa. Frontiers in Aging Neuroscience,
doi: 10.3389/fnagi.2018.00086
4. Cigarette Smoke-Induced Cell Death Causes Persistent Olfactory Dysfunction in AgedMice. Frontiers in Aging Neuroscience, doi: 10.3389/fnagi.2018.00183
5. Dose-Dependent Effects of Insulin-Like Growth Factor 1 in the Aged Olfactory
Epithelium. Frontiers in Aging Neuroscience, doi: 10.3389/fnagi.2018.00385

Project Insights
FUNDING
Grant 1: Smoking Research Foundation• Grant 2: A Japan Society for thePromotion of Science Grant-in-Aid for
Scientific Research: KAKENHI [grantnumbers 24791749, 16KT0190].
COLLABORATORS AND ADVISORS• Prof Tatsuya Yamasoba MD, PhD,
Chairman and Professor (tyamasobatky@umin.ac.jp) – Department ofOtolaryngology Head and Neck Surgery,
Faculty of Medicine, University of Tokyo,
Japan & Executive Board Member ofIFOS and Asia Oceania Associationof
ORL Societies• Prof Steven L. Kunkel PhD, Endowed
Professor of Pathology Research, Headof the Immunology Program, and Senior
Associate Dean Research (slkunkel@med.umich.edu) –University of Michigan
Medical School, US• Prof Toshihiro Ito MD, PhD, Professor(toshi-ito@naramed-u.ac.jp) –Department of Immunology, Nara
Medical University, Japan• Prof Satoshi Ueha PhD, Associate
Professor (ueha@rs.tus.ac.jp) – Divisionof Molecular Regulation of Inflammatory
and Immune Diseases, ResearchInstitute for Biomedical Sciences, Tokyo
University of Science, Japan
CONTACTRumi Ueha
Project Coordinator
T: +81 358008665
E: UEHAR-OTO@h.u-tokyo.ac.jp
W: https://researchmap.jp/ruu/PROJECT COORDINATOR BIO
Professor Rumi Ueha graduated fromNara Medical University, Faculty ofMedicine, Japan, in 2003, and thenstudied at the University of Michigan,
US, in 2012 and the University ofCalifornia Davis, US, in 2018. She
currently works as an AssistantProfessor at the Department ofOtorhinolaryngology, Head and Neck
Surgery, University of Tokyo, Japan.
Swallowing disorder and olfactory
impairment are her specialist fields. She
is absorbed in many surgeries and basic
researches.