Neurodegeneration associated-proteins in human olfactory epithelium: immunocytochemical and biomolecular study in healthy subjects and patients with synucleinopathies

Olfactory impairment is considered an initial disturbance of several neurodegenerative diseases (NDs), including Parkinson\u2019s disease (PD) and Alzheimer\u2019s disease (AD). In addition, smell impairment precedes a decade, or even longer, the onset of motor or cognitive symptoms. Olfactory signals are detected by olfactory receptor proteins (ORPs) expressed in the cilia of olfactory receptor neurons (ONs). ONs are the distinctive cellular components of the peripheral olfactory epithelium (OE) and lie in the nasal vault. ONs axons pass the cribriform plate and reach the olfactory bulb (OB) where the olfactory stimuli are processed and sent to the superior nuclei of the CNS. Previous studies in AD and other neurodegenerative disorders have shown the presence of \u3b2-amyloid deposits in the OB, neurofibrillary tangles, as well as Lewy body pathology. OB represents the brain area earlier involved in the neuropathological process, decades before the development of clinical symptoms. Therefore, OB can be considered a target in the study of neurodegenerative diseases in their early molecular processes. Moreover, the OB of healthy subjects presents deposits of aggregated proteins confirming that these aggregates are deposited in a prodromal disease stage. Since the OB is an early accumulation site of aggregated proteins and the synapses derive from the ONs, it is possible that the first event of protein aggregation occurs in OE. ONs are directly exposed to the external environment including chemical/physical toxic injuries and such micro-environment predisposes to abnormal protein processing and folding (Sammeta and McClintock 2010). In addition, ONs and all other mature cell components have a half-life of three months and programmed apoptosis. The neural activity is maintained by a constant cellular turn-over, which is sustained by the basal stem cells. This regeneration process is persistent during the whole life of an individual, albeit with a decreasing rate with aging. Extensive scientific literature indicates the neuronal damage as the consequence of exposure to toxic injuries leading to neurodegeneration and ONs are a natural model of this noxious process (Lema Tom\ue9, Tyson et al. 2013). The hypothesis of this pathological pathway is supported by several studies, in which aggregated forms of \u3b1-synuclein, tau and \u3b2-amyloid are detected in olfactory mucosa (OM) biopsies as well as in autoptic samples of patients with Parkinson\u2019s disease (PD), Lewy body dementia (LBD), Frontotemporal dementia (FTD) and Alzheimer disease (AD) (Funabe, Takao et al. 2013) (Saito, Shioya et al. 2016) (Tabaton, Cammarata et al. 1991) (Talamo, Rudel et al. 1989) (Crino, Greenberg et al. 1995) (Arnold, Lee et al. 2010). In this study, we investigated for the first-time primary ONs sampled ex vivo using olfactory brushing (OBg) in normal subjects and patients with different neurodegenerative disorders. Because of its convenient location, OE is easily accessible and can be sampled to obtain the ONs in the tissue outer layer. This sampling method is harmless and non-invasive, bypassing potential artifacts due to post mortem specimens as well as avoiding the invasiveness of biopsy procedures. Recently, we showed that OBg procedure in Creutzfeldt-Jakob Disease (CJD) patients allows efficient OM sampling for the Real-Time Quaking-Induced Conversion (RT-QuIC) assay. We specifically amplified pathological prion protein (PrPSc) providing a diagnostic intra vitam test with sensitivity and specificity nearly to 100% (Orr\ufa, Bongianni et al. 2014). For the purpose of our study, we firstly defined the phenotypic characterization of the human olfactory cells sampled with OBg from healthy subjects. Distinct antibodies were selected to analyze the olfactory epithelium cells: olfactory marker protein (OMP), neuron-specific class III \u3b2-tubulin (TUJ-1), protein gene product 9.5 (PGP 9.5), Pan-Cytokeratin (PCK). Secondly, we aimed to determine the expression patterns of the major misfolded proteins involved in the main neurodegenerative diseases. In particular, the selected proteins were: \u3b1-synuclein, APP/beta-amyloid, tau, and TDP-43. The identification of the expression patterns of these proteins in the ONs might provide information to understand the abnormal molecular mechanisms in the initial misfolding species involved in the pathological process. Moreover, in this study, we speculated on the subcellular locale where the protein aggregation may occur. Furthermore, by demonstrating the constitutive expression of the native NDs-associated proteins in the OE, we could assume that they may represent a potential template for triggering the aggregation process. Based on the immunocytochemistry analysis, we investigated the \u3b1-synuclein expression in patients affected by different synucleinopathies. In fact, \u3b1-synuclein misfolding and aggregation mechanisms are involved in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (LBD) and multiple system atrophy (MSA), which are all characterized by \u3b1-synuclein fibrils deposition (Spillantini, Schmidt et al. 1997). Finally, we analyzed the immunocytochemistry results in OM samples tested by \u3b1-synuclein RT-QuIC (\u3b1-syn RT-QuIC)

Neurodegeneration-associated proteins in human olfactory neurons collected by nasal brushing

The olfactory neuroepithelium is located in the upper vault of the nasal cavity, lying on the olfactory cleft and projecting into the dorsal portion of the superior and middle turbinates beyond the mid-portion of the nasal septum. It is composed of a variety of cell types including olfactory sensory neurons, supporting glial-like cells, microvillar cells, and basal stem cells. The cells of the neuroepithelium are often intermingled with respiratory and metaplastic epithelial cells. Olfactory neurons undergo a constant self-renewal in the timespan of 2\u20133 months; they are directly exposed to the external environment, and thus they are vulnerable to physical and chemical injuries. The latter might induce metabolic perturbations and ultimately be the cause of cell death. However, the lifespan of olfactory neurons is biologically programmed, and for this reason, these cells have an accelerated metabolic cycle leading to an irreversible apoptosis. These characteristics make these cells suitable for research related to nerve cell degeneration and aging. Recent studies have shown that a non-invasive and painless olfactory brushing procedure allows an efficient sampling from the olfactory neuroepithelium. This approach allows to detect the pathologic prion protein in patients with sporadic Creutzfeldt\u2013Jakob disease, using the real-time quaking-induced conversion assay. Investigating the expression of all the proteins associated to neurodegeneration in the cells of the olfactory mucosa is a novel approach toward understanding the pathogenesis of human neurodegenerative diseases. Our aim was to investigate the expression of \u3b1-synuclein, \u3b2-amyloid, tau, and TDP-43 in the olfactory neurons of normal subjects. We showed that these proteins that are involved in neurodegenerative diseases are expressed in olfactory neurons. These findings raise the question on whether a relationship exists between the mechanisms of protein aggregation that occur in the olfactory bulb during the early stage of the neurodegenerative process and the protein misfolding occurring in the olfactory neuroepithelium

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

Observation of gravitational waves from the coalescence of a 2.5–4.5 M ⊙ compact object and a neutron star

We report the observation of a coalescing compact binary with component masses 2.5–4.5 M ⊙ and 1.2–2.0 M ⊙ (all measurements quoted at the 90% credible level). The gravitational-wave signal GW230529_181500 was observed during the fourth observing run of the LIGO–Virgo–KAGRA detector network on 2023 May 29 by the LIGO Livingston observatory. The primary component of the source has a mass less than 5 M ⊙ at 99% credibility. We cannot definitively determine from gravitational-wave data alone whether either component of the source is a neutron star or a black hole. However, given existing estimates of the maximum neutron star mass, we find the most probable interpretation of the source to be the coalescence of a neutron star with a black hole that has a mass between the most massive neutron stars and the least massive black holes observed in the Galaxy. We provisionally estimate a merger rate density of 55−47+127Gpc−3yr−1 for compact binary coalescences with properties similar to the source of GW230529_181500; assuming that the source is a neutron star–black hole merger, GW230529_181500-like sources may make up the majority of neutron star–black hole coalescences. The discovery of this system implies an increase in the expected rate of neutron star–black hole mergers with electromagnetic counterparts and provides further evidence for compact objects existing within the purported lower mass gap

Persistent chemosensory dysfunction in a young patient with mild COVID-19 with partial recovery 15 months after the onset

Objective: It is reported that recovery from COVID-19 chemosensory deficit generally occurs in a few weeks, although olfactory dysfunction may persist longer. Here, we provide a detailed follow-up clinical investigation in a very young female patient (17-year-old) with a long-lasting anosmia after a mild infection, with partial recovery 15 months after the onset. Methods: Neuroimaging and neurophysiologic assessments as well as olfactory mucosa swabbing for microbiological and immunocytochemical analyses were performed. Olfactory and gustatory evaluations were conducted through validated tests. Results: Chemosensory evaluations were consistent with anosmia associated with parosmia phenomena and gustatory impairment, the latter less persistent. Brain MRI (3.0 T) showed no microvascular injury in olfactory bulbs and brain albeit we cannot rule out slight structural abnormalities during the acute phase, and a high-density EEG was negative. Immunocytochemistry of olfactory mucosa swabs showed high expression of ACE2 in sustentacular cells and lower dot-like cytoplasmic positivity in neuronal-shaped cells. Discussion: The occurrence of long-term persistent olfactory deficit in spite of the absence of structural brain and olfactory bulb involvement supports the view of a possible persistent dysfunction of both sustentacular cells and olfactory neurons. The gustatory dysfunction even if less persisting for the described features could be related to a primary gustatory system involvement. Future longitudinal studies are needed to investigate the persistence of chemosensory impairment, which could have a relevant impact on the daily life

RNAseq analysis of olfactory neuroepithelium cytological samples in individuals with Down syndrome compared to euploid controls: a pilot study

Down syndrome is a common genetic disorder caused by partial or complete triplication of chromosome 21. This syndrome shows an overall and progressive impairment of olfactory function, detected early in adulthood. The olfactory neuronal cells are located in the nasal olfactory mucosa and represent the first sensory neurons of the olfactory pathway. Herein, we applied the olfactory swabbing procedure to allow a gentle collection of olfactory epithelial cells in seven individuals with Down syndrome and in ten euploid controls. The aim of this research was to investigate the peripheral gene expression pattern in olfactory epithelial cells through RNAseq analysis. Validated tests (Sniffin' Sticks Extended test) were used to assess olfactory function. Olfactory scores were correlated with RNAseq results and cognitive scores (Vineland II and Leiter scales). All Down syndrome individuals showed both olfactory deficit and intellectual disability. Down syndrome individuals and euploid controls exhibited clear expression differences in genes located in and outside the chromosome 21. In addition, a significant correlation was found between olfactory test scores and gene expression, while a non-significant correlation emerged between olfactory and cognitive scores. This first preliminary step gives new insights into the Down syndrome olfactory system research, starting from the olfactory neuroepithelium, the first cellular step on the olfactory way

Alpha-synuclein seeds in olfactory mucosa of patients with isolated REM sleep behaviour disorder

Isolated REM sleep behaviour disorder (RBD) is an early-stage alpha-synucleinopathy in most, if not all, affected subjects. Detection of pathological alpha-synuclein in peripheral tissues of patients with isolated RBD may identify those progressing to Parkinson's disease, dementia with Lewy bodies or multiple system atrophy, with the ultimate goal of testing preventive therapies. Real-time quaking-induced conversion (RT-QuIC) provided evidence of alpha-synuclein seeding activity in CSF and olfactory mucosa of patients with alpha-synucleinopathies. The aim of this study was to explore RT-QuIC detection of alpha-synuclein aggregates in olfactory mucosa of a large cohort of subjects with isolated RBD compared to patients with Parkinson's disease and control subjects. This cross-sectional case-control study was performed at the Medical University of Innsbruck, Austria, the Hospital Clinic de Barcelona, Spain, and the University of Verona, Italy. Olfactory mucosa samples obtained by nasal swab in 63 patients with isolated RBD, 41 matched Parkinson's disease patients and 59 matched control subjects were analysed by alpha-synuclein RT-QuIC in a blinded fashion at the University of Verona, Italy. Median age of patients with isolated RBD was 70 years, 85.7% were male. All participants were tested for smell, autonomic, cognitive and motor functions. Olfactory mucosa was alpha-synuclein RT-QuIC positive in 44.4% isolated RBD patients, 46.3% Parkinson's disease patients and 10.2% control subjects. While the sensitivity for isolated RBD plus Parkinson's disease versus controls was 45.2%, specificity was high (89.8%). Among isolated RBD patients with positive alpha-synuclein RT-QuIC, 78.6% had olfactory dysfunction compared to 21.4% with negative alpha-synuclein RT-QuIC (P<0.001). The extent of olfactory dysfunction was more severe in isolated RBD patients positive than negative for olfactory mucosa a-synuclein RT-QuIC (P<0.001). We provide evidence that the alpha-synuclein RT-QuIC assay enables the molecular detection of neuronal alpha-synuclein aggregates in olfactory mucosa of patients with isolated RBD and Parkinson's disease. Although the overall sensitivity was moderate in this study, nasal swabbing is attractive as a simple, non-invasive test and might be useful as part of a screening battery to identify subjects in the prodromal stages of alpha-synucleinopathies. Further studies are needed to enhance sensitivity, and better understand the temporal dynamics of alpha-synuclein seeding in the olfactory mucosa and spreading to other brain areas during the progression from isolated RBD to overt alpha-synucleinopathy, as well the impact of timing, disease subgroups and sampling technique on the overall sensitivity