Pathological expression of PHFtau, amyloid-β, and α-synuclein have been described in the OE of older subjects with and without neurodegenerative diseases (for review, see42
) but the frequency, extent, and disease specificity of these lesions have not been established. We found that amyloid-β and PHFtau pathological lesions are significantly more frequent and more abundant in subjects with AD than in normal elderly controls or subjects with other non-AD neurodegenerative diseases.
Pathological amyloid-β was observed in the OE principally as intracellular, cytoplasmic punctate, vesicular, and amorphous aggregates. These inclusions were immunolabeled similarly by both NAB228, an antibody directed against the N-terminus of amyloid-β, as well as NAB61, an antibody that recognizes pathological dimers, soluble oligomers, and higher order species of amyloid-β. Earlier work from our center examined expression of amyloid-β and its flanking sequences of amyloid precursor protein as well as thioflavin-S in the OE in smaller numbers of subjects.28
More diffuse labeling was noted, particularly in the basal third of the OE and was more common in AD (8/9 cases) than normal controls (2/10), but was also common in an assortment of other neurodegenerative diseases (10/12).
A number of small studies of OE obtained in biopsies of living people with AD also have investigated neurodenerative disease lesions and other phenomena that may be associated with neurodegeneration. Several early reports indicated frequent tau, amyloid, and ubiquitin immunoreactive neuritic pathologies in the OE biopsies of AD subjects,25, 26, 43
but subsequent studies did not support these.44, 45
Perry et al.46
examined OE biospy sections from 8 subjects with probable AD and 3 controls and reported greater expression of oxidative damage markers in AD. Rawson et al.47
used freshly dissociated olfactory receptor neurons obtained by biopsy in 3 AD, 1 vascular dementia, and 14 healthy elderly subjects OE biopsies to measure intracellular calcium flux response to chemical odorant exposure. While this study importantly demonstrated the feasibility of conducting neurophysiological experiments in OE biopsy, no diagnosis-related differences were discerned in this very small sample. Finally, in a recent study of OE biopsies in 7 subjects with Parkinson’s disease, Witt et al.48
reported no major histochemical differences (including α-synuclein immunolabeling) in the Parkinson’s group compared to control cases.
Over the past decade, better antibodies, such as NAB228 or NAB61, and better antigen retrieval methods have been developed and we surmise that the increased sensitivity and specificity offered by these advances allowed recognition of the extensive intracellular amyloid-β deposition in OE that we report here.
The presence and nature of intracellular amyloid-β in AD has been controversial.49
Amyloid-β is predominantly described in extracellular neuritic plaques in the cerebral cortex in AD and as diffuse plaques in AD and commonly in normal cognitive aging as well.19, 50
Some of the contention about whether intracellular amyloid-β is present in the brains of people with AD may be due to technical issues including sensitivity of stains and antibodies, antigen retrieval methods, and possibly time at which pathological examination occurs in the course of the illness. Nonetheless, many investigations employing a variety of approaches provide compelling evidence for the existence and potential importance of intracellular localization amyloid-β,49
in familial APP mutation AD,51
Down syndrome AD,52
and in transgenic models of AD.54, 55
The question also arises as to whether the amyloid-β we observed was fibrillar. A previous smaller study from our group that included electron microscopy failed to find fibrillar amyloid-β in OE.28
We attempted to discern fibrillar amyloid here with Thioflavin S fluorescent labeling in select cases with abundant amyloid-β pathology (data not shown). When present, thioflavin S staining was generally similar in distribution pattern to our immunohistochemical labeling, but given the inferior sensitivity of this staining method and the degree of autofluorescence and other artifacts associated with it, the extent of pathology was lower and its nature was difficult to interpret.
PHFtau also distinguished AD from normal control and other neurodegenerative disease cases, though not as much as did amyloid-β in this series. As in previous work of ours and others’, PHFtau was especially evident in dystrophic neurites coursing through the lamina propria and in lower portions of the OE. Here, we also observed PHFtau expression in neuron somata with a morphological appearance typical of neurofibrillary tangles. While these were less frequent than dystrophic neurites, their presence is highly noteworthy as, to our knowledge, they have not heretofore been reported.
In the normal adult, the OE consists of clusters of sensory neurons interspersed among patches of metaplastic respiratory epithelium. The number of sensory neurons decreases across the lifespan, with the greatest decrease occurring after the age of 65.56
Furthermore, the proportion of respiratory epithelia increases as the olfactory neuroepithelium atrophies with the cumulative effects of environmental insult, inflammation, or disease. In this study, we did not systematically determine whether the amyloid-β, PHFtau, or α-synuclein lesions that we observed were confined to neuronal versus non-neuronal extents of OE. In several cases in which we conducted double immunofluorescence histochemistry to examine co-localization of the neuron-specific marker β-tubulin-III with amyloid-β (), we found amyloid-β expression in the vicinity of β-tubulin-III immunoreactive neurons, but also in areas in which there were no β-tubulin-III neurons. We speculate that pathological expression of amyloid-β contributes to olfactory neuron loss in AD and remains in areas of the OE now devoid of olfactory receptor neurons.
Impairments in olfactory functioning in AD and other neurodegenerative diseases are likely due to degenerative changes at multiple levels of the olfactory system and the relative contributions of changes in OE, olfactory bulb, and different olfaction-related cortices are difficult to determine. Consistent with pathology in multiple levels of the olfactory system in AD, a meta-analysis found extremely large effect sizes or impairments in AD across tests of odor identification and detection threshold sensitivity.57
In AD, accumulations of PHFtau neuropil threads and neurofibrillary tangles in the olfactory bulb and nerve have been found in all cases of definite AD, and many cases with probable AD as well as MCI and cognitively normal aging (though these may represent cases at risk for subsequent progression to AD).18, 58
It is also well-established that neurofibrillary pathology is early and severe in entorhinal, perirhinal, and piriform cortices in AD,19, 59, 60
all areas that play roles in odor identification and memory.61, 62
It is notable that the presence of peripheral OE amyloid-β and PHFtau correlated with higher levels of amyloid-β plaque and PHF tau pathology in the cortex, with the strength of this relationship being stronger for amyloid-β. These data suggest that measurement of these markers in OE may have some relevance to prediction of similar pathology in brain in living patients.
Identification and validation of biochemical and neuroimaging biomarkers of AD brain pathology is of major importance for early diagnosis and monitoring of disease-modifying treatment response. Significant strides have been made in characterizing the sensitivity and specificity of cerebrospinal fluid Aβ and tau levels and amyloid-β-ligand imaging with PET for AD and MCI.63, 64
Given the early and predictive functional olfactory changes that occur in people with and at risk for AD,2
the pathological findings for AD in the current study, and the relatively easy accessibility of olfactory epithelium for biopsy,65, 66
we suggest greater attention be paid to investigating the utility of OE as a biomarker source for studies of AD.