Tau seeding activity in skin biopsy differentiates tauopathies from synucleinopathies.

Most neurodegenerative diseases lack definitive diagnostic tests, and the identification of easily accessible and reliable biomarkers remains a critical unmet need. Since tau protein is highly expressed in skin of tauopathies patients, we aimed to exploit the ultrasensitive seeding activity assay (SAA) to assess tau seeding activity in skin of patients with tauopathies. In this multicentric, case-control study, patients with tauopathies and synucleinopathies were consecutively recruited and sex-matched to healthy controls (HC). Subjects underwent a double 3 mm skin biopsy in cervical area and ankle. Skin tau-SAA, using TauK18 and TauK19 as reaction substrates for 4R and 3R isoforms, seeding score, clinical scales, biochemical and morphological characterization of SAA end-products were evaluated. We analyzed 58 subjects: 24 tauopathies (18 progressive supranuclear palsy, PSP, and 6 corticobasal degeneration, CBD), 20 synucleinopathies (14 Parkinson's disease, PD, and 6 multiple system atrophy, MSA), and 14 HC. PSP and CBD showed higher tau seeding activity at both anatomical sites. A greater sensitivity of 4R-SAA than 3R-SAA was observed. 4R tau-SAA identified tauopathies with 71% sensitivity and 93% specificity. Accuracy was higher for PSP than CBD: PSP vs HC / PD (AUC 0.825), while CBD vs HC / PD (AUC 0.797), and PSP vs MSA (AU 0.778). SAA end-products showed differences in biochemical and morphological characterization according to the anatomical site. Skin tau-SAA identifies tauopathies with good accuracy and can be used to implement the in-vivo clinical diagnosis of patients with neurodegenerative diseases. Further characterization of peripheral tau seed in skin may elucidate the structure of tau deposits in brain.

Amyloid detection and typing yield of skin biopsy in systemic amyloidosis and polyneuropathy.

OBJECTIVE: Disease-modifying therapies are available for amyloidosis but are ineffective if end-organ damage is severe. As small fiber neuropathy is an early and common feature of amyloidosis, we assessed detection and typing yield of skin biopsy for amyloid in patients with confirmed systemic amyloidosis and neuropathic symptoms. METHODS: In this case-control study, patients with transthyretin and light chain amyloidosis (ATTRv, ATTRwt, and AL) were consecutively recruited. They were sex and age-matched to three control groups (1) non-neuropathic controls (NNC), (2) monoclonal gammopathy of undetermined significance (MGUS), and (3) other neuropathic disease controls (ONC). Patients underwent a double 3 mm skin biopsy in proximal and distal leg. Amyloid index and burden, protein typing by immuno-electron microscopy, intraepidermal nerve fiber density, electroneuromyography, and clinical characteristics were analyzed. RESULTS: We studied 15 subjects with confirmed systemic amyloidosis, 20 NNC, 18 MGUS, and 20 ONC. Amyloid was detected in 100% of patients with amyloidosis (87% in ankle and 73% in thigh). It was not detected in any of the control groups. A small fiber neuropathy was encountered in 100% of amyloidosis patients, in 80% of MGUS, and in 78% of ONC. Amyloid burden was higher in ATTRv, followed by AL and ATTRwt. The ultrastructural examination allowed the identification of the precursor protein by immunotyping in most of the cases. INTERPRETATION: Skin biopsy is a minimally invasive test with optimal sensitivity for amyloid. It allows amyloid typing by electron microscope to identify the precursor protein. The diagnostic work up of systemic amyloidosis should include a skin biopsy.

Prevalence of amyloid in ligamentum flavum of patients with lumbar spinal stenosis.

BACKGROUND: Transthyretin (ATTR) amyloidosis is often diagnosed in an advanced stage, when irreversible cardiac damage has occurred. Lumbar spinal stenosis (LSS) may precede cardiac ATTR amyloidosis by many years, offering the opportunity to detect ATTR already at the time of LSS surgery. We prospectively assessed the prevalence of ATTR in the ligamentum flavum by tissue biopsy in patients aged >50 years undergoing surgery for LSS. METHODS: Ligamentum flavum thickness was assessed pre-operatively on axial T2 magnetic resonance imaging (MRI) slices. Tissue samples from ligamentum flavum were screened centrally by Congo red staining and immunohistochemistry (IHC). RESULTS: Amyloid in the ligamentum flavum was detected in 74/94 patients (78.7%). IHC revealed ATTR in 61 (64.9%), whereas amyloid subtyping was inconclusive in 13 (13.8%). Mean thickness of ligamentum flavum was significantly higher at all levels in patients with amyloid (p < .05). Patients with amyloid deposits were older (73.1 ± 9.2 vs. 64.6 ± 10.1 years, p = .01). No differences in sex, comorbidities, previous surgery for carpal tunnel syndrome or LSS were observed. CONCLUSIONS: Amyloid, mostly of the ATTR subtype, was found in four out of five patients with LSS and is associated with age and ligamentum flavum thickness. Histopathological work-up of ligamentum flavum might inform future decision making.

Dorsal Root Ganglion Stimulation in Chronic Painful Polyneuropathy: A Potential Modulator for Small Nerve Fiber Regeneration.

OBJECTIVES: Neuromodulatory treatments like spinal cord stimulation and dorsal root ganglion stimulation (DRGS) have emerged as effective treatments to relieve pain in painful polyneuropathy. Animal studies have demonstrated that neurostimulation can enhance nerve regeneration. This study aimed to investigate if DRGS may impact intraepidermal nerve fiber regeneration and sensory nerve function. MATERIALS AND METHODS: Nine patients with chronic, intractable painful polyneuropathy were recruited. Intraepidermal nerve fiber density (IENFD) quantification in 3 mm punch skin biopsy was performed 1 month before DRGS (placed at the level of the L5 and S1 dorsal root ganglion) and after 12- and 24-month follow-up. Quantitative sensory testing, nerve conduction studies, and a clinical scale score were also performed at the same time points. RESULTS: In 7 of 9 patients, DRGS was successful (defined as a reduction of ≥ 50% in daytime and/or night-time pain intensity), allowing a definitive implantable pulse generator implantation. The median baseline IENFD among these 7 patients was 1.6 fibers/mm (first and third quartile: 1.2; 4.3) and increased to 2.6 fibers/mm (2.5; 2.9) and 1.9 fibers/mm (1.6; 2.4) at 1- and 2-years follow-up, respectively. These changes were not statistically significant (p = 1.000 and 0.375). Sensory nerve tests did not show substantial changes. CONCLUSIONS: Although not significant, the results of this study showed that in most of the patients with implants, there was a slight increase of the IENFD at the 1- and 2-year follow-up. Larger-scale clinical trials are warranted to explore the possible role of DRGS in reversing the progressive neurodegeneration over time. CLINICAL TRIAL REGISTRATION: The Clinicaltrials.gov registration number for the study is NCT02435004; Swiss National Clinical Trials Portal: SNCTP000001376.

Risk stratification of patients with SARS-CoV-2 by tissue factor expression in circulating extracellular vesicles.

Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressing tissue factor (CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n = 60) and an independent validation cohort including unselected consecutive enrolled patients (n = 201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC = 0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC = 0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest as risk stratification tool to support clinical decision making in COVID-19.

Tau protein quantification in skin biopsies differentiates tauopathies from alpha-synucleinopathies.

Abnormal accumulation of microtubule-associated protein tau (τ) is a characteristic feature of atypical parkinsonisms with tauopathies, such as progressive supranuclear palsy and corticobasal degeneration. However, pathological τ has also been observed in α-synucleinopathies like Parkinson's disease and multiple system atrophy. Based on the involvement of the peripheral nervous system in several neurodegenerative diseases, we characterized and compared τ expression in skin biopsies of patients clinically diagnosed with Parkinson's disease, multiple system atrophy, progressive supranuclear palsy and corticobasal degeneration and in healthy control subjects. In all groups, τ protein was detected along both somatosensory and autonomic nerve fibres in the epidermis and dermis by immunofluorescence. We found by western blot the presence of mainly two different bands at 55 and 70 kDa, co-migrating with 0N4R/1N3R and 2N4R isoforms, respectively. At the RNA level, the main transcript variants were 2N and 4R, and both were more expressed in progressive supranuclear palsy/corticobasal degeneration by real-time PCR. Enzyme-linked immunosorbent assay demonstrated significantly higher levels of total τ protein in skin lysates of progressive supranuclear palsy/corticobasal degeneration compared to the other groups. Multivariate regression analysis and receiver operating characteristics curve analysis of τ amount at both sites showed a clinical association with tauopathies diagnosis and high diagnostic value for progressive supranuclear palsy/corticobasal degeneration versus Parkinson's disease (sensitivity 90%, specificity 69%) and progressive supranuclear palsy/corticobasal degeneration versus multiple system atrophy (sensitivity 90%, specificity 86%). τ protein increase correlated with cognitive impairment in progressive supranuclear palsy/corticobasal degeneration. This study is a comprehensive characterization of τ in the human cutaneous peripheral nervous system in physiological and pathological conditions. The differential expression of τ, both at transcript and protein levels, suggests that skin biopsy, an easily accessible and minimally invasive exam, can help in discriminating among different neurodegenerative diseases.

Supervised and unsupervised learning to define the cardiovascular risk of patients according to an extracellular vesicle molecular signature.

Cardiovascular (CV) disease represents the most common cause of death in developed countries. Risk assessment is highly relevant to intervene at individual level and implement prevention strategies. Circulating extracellular vesicles (EVs) are involved in the development and progression of CV diseases and are considered promising biomarkers. We aimed at identifying an EV signature to improve the stratification of patients according to CV risk and likelihood to develop fatal CV events. EVs were characterized by nanoparticle tracking analysis and flow cytometry for a standardized panel of 37 surface antigens in a cross-sectional multicenter cohort (n = 486). CV profile was defined by presence of different indicators (age, sex, body mass index, hypertension, hyperlipidemia, diabetes, coronary artery disease, cardiac heart failure, chronic kidney disease, smoking habit, organ damage) and according to the 10-year risk of fatal CV events estimated using SCORE charts of European Society of Cardiology. By combining expression levels of EV antigens using unsupervised learning, patients were classified into 3 clusters: Cluster-I (n = 288), Cluster-II (n = 83), Cluster-III (n = 30). A separate analysis was conducted on patients displaying acute CV events (n = 82). Prevalence of hypertension, diabetes, chronic heart failure, and organ damage (defined as left ventricular hypertrophy and/or microalbuminuria) increased progressively from Cluster-I to Cluster-III. Several EV antigens, including markers for platelets (CD41b-CD42a-CD62P), leukocytes (CD1c-CD2-CD3-CD4-CD8-CD14-CD19-CD20-CD25-CD40-CD45-CD69-CD86), and endothelium (CD31-CD105) were independently associated with CV risk indicators and correlated to age, blood pressure, glucometabolic profile, renal function, and SCORE risk. EV profiling, obtained from minimally invasive blood sampling, allows accurate patient stratification according to CV risk profile.

Alpha-synuclein oligomers and small nerve fiber pathology in skin are potential biomarkers of Parkinson's disease.

The proximity ligation assay (PLA) is a specific and sensitive technique for the detection of αSyn oligomers (αSyn-PLA), early and toxic species implicated in the pathogenesis of PD. We aimed to evaluate by skin biopsy the diagnostic and prognostic capacity of αSyn-PLA and small nerve fiber reduction in PD in a longitudinal study. αSyn-PLA was performed in the ankle and cervical skin biopsies of PD (n = 30), atypical parkinsonisms (AP, n = 23) including multiple system atrophy (MSA, n = 12) and tauopathies (AP-Tau, n = 11), and healthy controls (HC, n = 22). Skin biopsy was also analyzed for phosphorylated αSyn (P-αSyn) and 5G4 (αSyn-5G4), a conformation-specific antibody to aggregated αSyn. Intraepidermal nerve fiber density (IENFD) was assessed as a measure of small fiber neuropathy. αSyn-PLA signal was more expressed in PD and MSA compared to controls and AP-Tau. αSyn-PLA showed the highest diagnostic accuracy (PD vs. HC sensitivity 80%, specificity 77%; PD vs. AP-Tau sensitivity 80%, specificity 82%), however, P-αSyn and 5G4, possible markers of later phases, performed better when considering the ankle site alone. A small fiber neuropathy was detected in PD and MSA. A progression of denervation not of pathological αSyn was detected at follow-up and a lower IENFD at baseline was associated with a greater cognitive and motor decline in PD. A skin biopsy-derived compound marker, resulting from a linear discrimination analysis model of αSyn-PLA, P-αSyn, αSyn-5G4, and IENFD, stratified patients with accuracy (77.8%), including the discrimination between PD and MSA (84.6%). In conclusion, the choice of pathological αSyn marker and anatomical site influences the diagnostic performance of skin biopsy and can help in understanding the temporal dynamics of αSyn spreading in the peripheral nervous system during the disease. Skin denervation, not pathological αSyn is a potential progression marker for PD.

Extracellular Vesicle Surface Markers as a Diagnostic Tool in Transient Ischemic Attacks.

Background and Purpose: Extracellular vesicles (EVs) are promising biomarkers for cerebral ischemic diseases, but not systematically tested in patients with transient ischemic attacks (TIAs). We aimed at (1) investigating the profile of EV-surface antigens in patients with symptoms suspicious for TIA; (2) developing and validating a predictive model for TIA diagnosis based on a specific EV-surface antigen profile. Methods: We analyzed 40 subjects with symptoms suspicious for TIA and 20 healthy controls from a training cohort. An independent cohort of 28 subjects served as external validation. Patients were stratified according to likelihood of having a real ischemic event using the Precise Diagnostic Score, defined as: unlikely (score 0­1), possible-probable (score 2­3), or very likely (score 4­8). Serum vesicles were quantified by nanoparticle tracking analysis and EV-surface antigen profile characterized by multiplex flow cytometry. Results: EV concentration increased in patients with very likely or possible-probable TIA (P<0.05) compared with controls. Nanoparticle concentration was directly correlated with the Precise Diagnostic score (R=0.712; P<0.001). After EV immuno-capturing, CD8, CD2, CD62P, melanoma-associated chondroitin sulfate proteoglycan, CD42a, CD44, CD326, CD142, CD31, and CD14 were identified as discriminants between groups. Receiver operating characteristic curve analysis confirmed a reliable diagnostic performance for each of these markers taken individually and for a compound marker derived from their linear combinations (area under the curve, 0.851). Finally, a random forest model combining the expression levels of selected markers achieved an accuracy of 96% and 78.9% for discriminating patients with a very likely TIA, in the training and external validation cohort, respectively. Conclusions: The EV-surface antigen profile appears to be different in patients with transient symptoms adjudicated to be very likely caused by brain ischemia compared with patients whose symptoms were less likely to due to brain ischemia. We propose an algorithm based on an EV-surface-antigen specific signature that might aid in the recognition of TIA.

Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection.

BACKGROUND: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents. METHODS: We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serum-derived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-α levels were measured by ELISA. FINDINGS: Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-α serum levels. INTERPRETATION: In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity. FUNDING: Cardiocentro Ticino Institute, Ente ospedaliero Cantonale, Lugano-Switzerland.

Profiling Inflammatory Extracellular Vesicles in Plasma and Cerebrospinal Fluid: An Optimized Diagnostic Model for Parkinson's Disease.

Extracellular vesicles (EVs) play a central role in intercellular communication, which is relevant for inflammatory and immune processes implicated in neurodegenerative disorders, such as Parkinson's Disease (PD). We characterized and compared distinctive cerebrospinal fluid (CSF)-derived EVs in PD and atypical parkinsonisms (AP), aiming to integrate a diagnostic model based on immune profiling of plasma-derived EVs via artificial intelligence. Plasma- and CSF-derived EVs were isolated from patients with PD, multiple system atrophy (MSA), AP with tauopathies (AP-Tau), and healthy controls. Expression levels of 37 EV surface markers were measured by a flow cytometric bead-based platform and a diagnostic model based on expression of EV surface markers was built by supervised learning algorithms. The PD group showed higher amount of CSF-derived EVs than other groups. Among the 17 EV surface markers differentially expressed in plasma, eight were expressed also in CSF of a subgroup of PD, 10 in MSA, and 6 in AP-Tau. A two-level random forest model was built using EV markers co-expressed in plasma and CSF. The model discriminated PD from non-PD patients with high sensitivity (96.6%) and accuracy (92.6%). EV surface marker characterization bolsters the relevance of inflammation in PD and it underscores the role of EVs as pathways/biomarkers for protein aggregation-related neurodegenerative diseases.

Dorsal Root Ganglion Stimulation for the Management of Intractable Painful Polyneuropathy: A Prospective Pilot Study.

OBJECTIVES: Dorsal root ganglion stimulation (DRGS) is a promising neurostimulation modality in the treatment of painful polyneuropathy. The aim of this prospective pilot study was to investigate the effect of DRGS on pain intensity in patients with intractable painful polyneuropathy. MATERIALS AND METHODS: Nine patients with chronic, intractable painful polyneuropathy in the lower limbs were recruited. In each subject, between two and four DRGS leads were placed at the level of the L5 and S1 dorsal root ganglion. If trial stimulation was successful, a definitive implantable pulse generator (IPG) was implanted. Pain intensity was scored using an 11-point numeric rating scale (NRS) and reported as median and interquartile range (IQR), and compared to baseline values using the Wilcoxon signed-rank test. Additionally, patients' global impression of change (PGIC), pain extent, presence of neuropathic pain, physical functioning, quality of life, and mood were assessed. RESULTS: Eight out of nine patients had a successful trial phase, of which seven received an IPG. Daytime pain decreased from a median (IQR) NRS score of 7.0 (5.9-8.3) to 2.0 (1.0-3.5) and 3.0 (1.6-4.9) in the first week and at six months after implantation, respectively. Similar effects were observed for night time and peak pain scores. CONCLUSIONS: The results of this study suggest that DRGS significantly reduces both pain intensity and PGIC in patients with intractable painful polyneuropathy in the lower extremities. Large-scale clinical trials are needed to prove the efficacy of DRGS in intractable painful polyneuropathy.

Immune profiling of plasma-derived extracellular vesicles identifies Parkinson disease.

OBJECTIVE: To develop a diagnostic model based on plasma-derived extracellular vesicle (EV) subpopulations in Parkinson disease (PD) and atypical parkinsonism (AP), we applied an innovative flow cytometric multiplex bead-based platform. METHODS: Plasma-derived EVs were isolated from PD, matched healthy controls, multiple system atrophy (MSA), and AP with tauopathies (AP-Tau). The expression levels of 37 EV surface markers were measured by flow cytometry and correlated with clinical scales. A diagnostic model based on EV surface markers expression was built via supervised machine learning algorithms and validated in an external cohort. RESULTS: Distinctive pools of EV surface markers related to inflammatory and immune cells stratified patients according to the clinical diagnosis. PD and MSA displayed a greater pool of overexpressed immune markers, suggesting a different immune dysregulation in PD and MSA vs AP-Tau. The receiver operating characteristic curve analysis of a compound EV marker showed optimal diagnostic performance for PD (area under the curve [AUC] 0.908; sensitivity 96.3%, specificity 78.9%) and MSA (AUC 0.974; sensitivity 100%, specificity 94.7%) and good accuracy for AP-Tau (AUC 0.718; sensitivity 77.8%, specificity 89.5%). A diagnostic model based on EV marker expression correctly classified 88.9% of patients with reliable diagnostic performance after internal and external validations. CONCLUSIONS: Immune profiling of plasmatic EVs represents a crucial step toward the identification of biomarkers of disease for PD and AP.

Tau and Alpha Synuclein Synergistic Effect in Neurodegenerative Diseases: When the Periphery Is the Core.

In neuronal cells, tau is a microtubule-associated protein placed in axons and alpha synuclein is enriched at presynaptic terminals. They display a propensity to form pathologic aggregates, which are considered the underlying cause of Alzheimer's and Parkinson's diseases. Their functional impairment induces loss of axonal transport, synaptic and mitochondrial disarray, leading to a "dying back" pattern of degeneration, which starts at the periphery of cells. In addition, pathologic spreading of alpha-synuclein from the peripheral nervous system to the brain through anatomical connectivity has been demonstrated for Parkinson's disease. Thus, examination of the extent and types of tau and alpha-synuclein in peripheral tissues and their relation to brain neurodegenerative diseases is of relevance since it may provide insights into patterns of protein aggregation and neurodegeneration. Moreover, peripheral nervous tissues are easily accessible in-vivo and can play a relevant role in the early diagnosis of these conditions. Up-to-date investigations of tau species in peripheral tissues are scant and have mainly been restricted to rodents, whereas, more evidence is available on alpha synuclein in peripheral tissues. Here we aim to review the literature on the functional role of tau and alpha synuclein in physiological conditions and disease at the axonal level, their distribution in peripheral tissues, and discuss possible commonalities/diversities as well as their interaction in proteinopathies.

Neurologic complications of acute hepatitis E virus infection.

OBJECTIVE: To assess the prevalence and clinical features of neurologic involvement in patients with acute hepatitis E virus (HEV) infection in Southern Switzerland. METHODS: Among 1,940 consecutive patients investigated for acute hepatitis E, we identified 141 cases of acute of HEV infection (anti-HEV immunoglobulin M and immunoglobulin G both reactive and/or HEV RNA positive) between June 2014 and September 2017. Neurologic cases were followed up for 6 months. We compared patients with and without neurologic symptoms. RESULTS: Neurologic symptoms occurred in 43 acute HEV cases (30.4%) and consisted of neuralgic amyotrophy (NA, n = 15, 10.6%) and myalgia (n = 28, 19.8%). All NA cases were immunocompetent. Men had higher odds (OR = 5.2, CI 1.12-24.0, p = 0.03) of developing NA after infection with HEV, and in 3 couples simultaneously infected with HEV, only men developed NA. Bilateral involvement of NA was predominant (2:1) and occurred only in men. Seven NA cases were viremic (all genotype 3), but HEV was undetectable in their CSF. In the acute phase of NA, 9 patients were treated with intravenous immunoglobulin and 4 with prednisone, reporting no side effects and improvement in pain and strength. Myalgia occurred both without (n = 16) or with (n = 12) concomitant elevated serum creatinine kinase. Seven cases with myalgia in the shoulder girdle did not have muscle weakness ("forme fruste" of NA). CONCLUSIONS: Neurologic symptoms occurred in one-third of acute HEV infections and consisted of NA and myalgia. NA seems to occur more frequently in men infected by HEV and has a predominant (but not exclusive) bilateral involvement.

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson's disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (αSyn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of αSyn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting αSyn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark αSyn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Cervical skin denervation associates with alpha-synuclein aggregates in Parkinson disease.

OBJECTIVE: Autonomic nervous system is involved at the onset of Parkinson disease (PD), and alpha-synuclein (α-Syn) and its phosphorylated form (p-αSyn) have been detected in dermal autonomic nerve fibers of PD. We assessed disease specific conformation variant of α-Syn immunoreactivity in cutaneous nerves and characterized skin denervation patterns in PD and atypical parkinsonism (AP). METHODS: We enrolled 49 subjects, 19 with PD, 17 age-matched healthy controls, and 13 with AP. The manifestations of disease were rated on clinical scales. Skin biopsies from ankle, thigh, and neck were analyzed by immunofluorescence for p-αSyn, 5G4 as a conformation specific antibody to pathogenic α-Syn and PGP9.5 as axonal marker. Intraepidermal nerve fiber density was measured in all anatomical sites as marker of neurodegeneration. Thirteen of the 19 PD underwent a 1 year follow-up visit plus skin biopsies. RESULTS: PD subjects displayed more severe cervical skin denervation (P < 0.03), which correlated to disease duration and worsened between initial and follow-up examination (P < 0.001). p-αSyn and 5G4 were equally sensitive and specific for the diagnosis of PD (area under the ROC was 0.839 for p-αSyn and 0.886 for 5G4). PD and AP with possible alpha-synucleinopathies share the features of marked cervical denervation and the presence of 5G4. In contrast AP with possible tauopathies were normal. INTERPRETATION: Conformational specific forms of α-Syn are detectable in skin biopsy by immunofluorescence in PD, with a promising diagnostic efficiency similar to p-αSyn. Cervical cutaneous denervation correlates with disease duration and increases over time standing out as a potential biomarker of PD progression.

Clinical phenotype and outcome of hepatitis E virus-associated neuralgic amyotrophy.

OBJECTIVE: To determine the clinical phenotype and outcome in hepatitis E virus-associated neuralgic amyotrophy (HEV-NA). METHODS: Cases of NA were identified in 11 centers from 7 European countries, with retrospective analysis of demographics, clinical/laboratory findings, and treatment and outcome. Cases of HEV-NA were compared with NA cases without evidence of HEV infection. RESULTS: Fifty-seven cases of HEV-NA and 61 NA cases without HEV were studied. Fifty-six of 57 HEV-NA cases were anti-HEV IgM positive; 53/57 were IgG positive. In 38 cases, HEV RNA was recovered from the serum and in 1 from the CSF (all genotype 3). Fifty-one of 57 HEV-NA cases were anicteric; median alanine aminotransferase 259 IU/L (range 12-2,961 IU/L); in 6 cases, liver function tests were normal. HEV-NA cases were more likely to have bilateral involvement (80.0% vs 8.6%, p < 0.001), damage outside the brachial plexus (58.5% vs 10.5%, p < 0.01), including phrenic nerve and lumbosacral plexus injury (25.0% vs 3.5%, p = 0.01, and 26.4% vs 7.0%, p = 0.001), reduced reflexes (p = 0.03), sensory symptoms (p = 0.04) with more extensive damage to the brachial plexus. There was no difference in outcome between the 2 groups at 12 months. CONCLUSIONS: Patients with HEV-NA are usually anicteric and have a distinct clinical phenotype, with predominately bilateral asymmetrical involvement of, and more extensive damage to, the brachial plexus. Involvement outside the brachial plexus is more common in HEV-NA. The relationship between HEV and NA is likely to be causal, but is easily overlooked. Patients presenting with NA should be tested for HEV, irrespective of liver function test results. Prospective treatment/outcome studies of HEV-NA are warranted.

Stimulation of the neurotrophin receptor TrkB on astrocytes drives nitric oxide production and neurodegeneration.

Neurotrophin growth factors support neuronal survival and function. In this study, we show that the expression of the neurotrophin receptor TrkB is induced on astrocytes in white matter lesions in multiple sclerosis (MS) patients and mice with experimental autoimmune encephalomyelitis (EAE). Surprisingly, mice lacking TrkB specifically in astrocytes were protected from EAE-induced neurodegeneration. In an in vitro assay, astrocytes stimulated with the TrkB agonist brain-derived neurotrophic factor (BDNF) released nitric oxide (NO), and conditioned medium from activated astrocytes had detrimental effects on the morphology and survival of neurons. This neurodegenerative process was amplified by NO produced by neurons. NO synthesis in the central nervous system during EAE depended on astrocyte TrkB. Together, these findings suggest that TrkB expression on astrocytes may represent a new target for neuroprotective therapies in MS.