Stochastic Optical Reconstruction Microscopy Imaging of Multiple System Atrophy Inclusions Suggests Stepwise α-Synuclein Aggregation.

BACKGROUND: The architecture and composition of glial (GCI) and neuronal (NCI) α-synuclein inclusions observed in multiple system atrophy (MSA) remain to be precisely defined to better understand the disease. METHODS: Here, we used stochastic optical reconstruction microscopy (STORM) to characterize the nanoscale organization of glial (GCI) and neuronal (NCI) α-synuclein inclusions in cryopreserved brain sections from MSA patients. RESULTS: STORM revealed a dense cross-linked internal structure of α-synuclein in all GCI and NCI. The internal architecture of hyperphosphorylated α-synuclein (p-αSyn) inclusions was similar in glial and neuronal cells, suggesting a common aggregation mechanism. A similar sequence of p-αSyn stepwise intracellular aggregation was defined in oligodendrocytes and neurons, starting from the perinuclear area and growing inside the cells. Consistent with this hypothesis, we found a higher mitochondrial density in GCI and NCI compared to oligodendrocytes and neurons from unaffected donors (P < 0.01), suggesting an active recruitment of the organelles during the aggregation process. CONCLUSIONS: These first STORM images of GCI and NCI suggest stepwise α-synuclein aggregation in MSA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Cancer in pathologically confirmed multiple system atrophy.

PURPOSE: The aim of this study was to assess whether cancer occurs with increased frequency in multiple system atrophy (MSA). The pathological hallmark of MSA is glial cytoplasmic inclusions containing aggregated α-synuclein, and the related protein γ-synuclein correlates with invasive cancer. We investigated whether these two disorders are associated clinically. METHODS: Medical records of 320 patients with pathologically confirmed MSA seen between 1998 and 2022 were reviewed. After excluding those with insufficient medical histories, the remaining 269 and an equal number of controls matched for age and sex were queried for personal and family histories of cancer recorded on standardized questionnaires and in clinical histories. Additionally, age-adjusted rates of breast cancer were compared with US population incidence data. RESULTS: Of 269 cases in each group, 37 with MSA versus 45 of controls had a personal history of cancer. Reported cases of cancer in parents were 97 versus 104 and in siblings 31 versus 44 for MSA and controls, respectively. Of 134 female cases in each group, 14 MSA versus 10 controls had a personal history of breast cancer. The age-adjusted rate of breast cancer in MSA was 0.83%, as compared with 0.67% in controls and 2.0% in the US population. All comparisons were nonsignificant. CONCLUSION: The evidence from this retrospective cohort found no significant clinical association of MSA with breast cancer or other cancers. These results do not exclude the possibility that knowledge about synuclein pathology at the molecular level in cancer may lead to future discoveries and potential therapeutic targets for MSA.

Activation of Pgk1 Results in Reduced Protein Aggregation in Diverse Neurodegenerative Conditions.

The prevention of protein condensates has emerged as a new drug target to treat diverse neurodegenerative disorders. We previously reported that terazosin (TZ), a prescribed antagonist of the α1 adrenergic receptor, is an activator of phosphoglycerate kinase 1 (Pgk1) and Hsp90. In this study, we aimed to determine whether TZ prevents the formation of diverse pathological condensates in cell cultures and animal disease models. In primary neuron culture, TZ treatment reduced both the protein density and abundance of fused in sarcoma (FUS)-P525L-GFP, a disease-associated mutant form of FUS. Regarding the mechanism, we found that increased intracellular ATP levels were critical for the reduction in protein aggregate density. In addition, Hsp90 activation by TZ enhanced Hsp90 interaction with ULK1, a master regulator of autophagy. Through in vivo studies, we examined neuron-specific overexpression of tau in Drosophila, mouse models of APP/PS1 Alzheimer's disease (AD), and a rat model of multiple system atrophy (MSA) via the viral expression of α-synuclein in the striatum. TZ prevented and reversed the formation of pathological protein condensates. Together, our results suggest that activation of Pgk1 in cytosol may dissolve pathological protein aggregates via increased ATP levels and degrade these proteins via autophagy; the FUS-P525L degradation pathway in nucleus is unclear.

Mitochondrial Dysfunction and Neurodegenerative Disorders: Role of Nutritional Supplementation.

Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, multisystem atrophy, and progressive supranuclear palsy. This article is concerned specifically with mitochondrial dysfunction as defined by reduced capacity for ATP production, the role of depleted levels of key nutritionally related metabolites, and the potential benefit of supplementation with specific nutrients of relevance to normal mitochondrial function in the above neurodegenerative disorders. The article provides a rationale for a combination of CoQ10, B-vitamins/NADH, L-carnitine, vitamin D, and alpha-lipoic acid for the treatment of the above neurodegenerative disorders.

Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models.

Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions. We also identify a KFERQ-like motif in the TPPP/p25A sequence that enables its effective degradation via chaperone-mediated autophagy (CMA) in an in vitro system of rat brain lysosomes. Furthermore, in a MSA-like setting established by addition of human recombinant SNCA pre-formed fibrils (PFFs) as seeds of pathological SNCA, we thoroughly characterize the contribution of CMA and macroautophagy in particular, in the removal of the exogenously added and the seeded oligodendroglial SNCA pathological assemblies. We also show that PFF treatment impairs autophagic flux and that TPPP/p25A exerts an inhibitory effect on macroautophagy, while at the same time CMA is upregulated to remove the pathological SNCA species formed within oligodendrocytes. Finally, augmentation of CMA or macroautophagy accelerates the removal of the engendered pathological SNCA conformations further suggesting that autophagy targeting may represent a successful approach for the clearance of pathological SNCA and/or TPPP/p25A in the context of MSA.Abbreviations: 3MA: 3-methyladenine; ACTB: actin, beta; ALP: autophagy-lysosome pathway; ATG5: autophagy related 5; AR7: atypical retinoid 7; CMA: chaperone-mediated autophagy; CMV: cytomegalovirus; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; Epox: epoxomicin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCIs: glial cytoplasmic inclusions; GFP: green fluorescent protein; HMW: high molecular weight; h: hours; HSPA8/HSC70: heat shock protein 8; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mcherry: monomeric cherry; MFI: mean fluorescence intensity; mRFP: monomeric red fluorescent protein; MSA: multiple system atrophy; OLN: oligodendrocytes; OPCs: oligodendroglial progenitor cells; PBS: phosphate-buffered saline; PC12: pheochromocytoma cell line; PD: Parkinson disease; PFFs: pre-formed fibrils; PIs: protease inhibitors; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; Rap: rapamycin; RFP: red fluorescent protein; Scr: scrambled; SDS: sodium dodecyl sulfate; SE: standard error; siRNAs: small interfering RNAs; SNCA: synuclein, alpha; SQSTM1: sequestosome 1; TPPP: tubulin polymerization promoting protein; TUBA: tubulin, alpha; UPS: ubiquitin-proteasome system; WT: wild type.

Therapeutic potential of iron modulating drugs in a mouse model of multiple system atrophy.

Multiple System Atrophy (MSA) is a rare neurodegenerative synucleinopathy which leads to severe disability followed by death within 6-9 years of symptom onset. There is compelling evidence suggesting that biological trace metals like iron and copper play an important role in synucleinopathies like Parkinson's disease and removing excess brain iron using chelators could slow down the disease progression. In human MSA, there is evidence of increased iron in affected brain regions, but role of iron and therapeutic efficacy of iron-lowering drugs in pre-clinical models of MSA have not been studied. We studied age-related changes in iron metabolism in different brain regions of the PLP-αsyn mice and tested whether iron-lowering drugs could alleviate disease phenotype in aged PLP-αsyn mice. Iron content, iron-ferritin association, ferritin protein levels and copper-ceruloplasmin association were measured in prefrontal cortex, putamen, substantia nigra and cerebellum of 3, 8, and 20-month-old PLP-αsyn and age-matched non-transgenic mice. Moreover, 12-month-old PLP-αsyn mice were administered deferiprone or ceruloplasmin or vehicle for 2 months. At the end of treatment period, motor testing and stereological analyses were performed. We found iron accumulation and perturbed iron-ferritin interaction in substantia nigra, putamen and cerebellum of aged PLP-αsyn mice. Furthermore, we found significant reduction in ceruloplasmin-bound copper in substantia nigra and cerebellum of the PLP-αsyn mice. Both deferiprone and ceruloplasmin prevented decline in motor performance in aged PLP-αsyn mice and were associated with higher neuronal survival and reduced density of α-synuclein aggregates in substantia nigra. This is the first study to report brain iron accumulation in a mouse model of MSA. Our results indicate that elevated iron in MSA mice may result from ceruloplasmin dysfunction and provide evidence that targeting iron in MSA could be a viable therapeutic option.

Phosphorylated α-synuclein and phosphorylated tau-protein in sural nerves may contribute to differentiate Parkinson's disease from multiple system atrophy and progressive supranuclear paralysis.

Differential diagnosis of Parkinson's disease (PD), multiple system atrophy (MSA) and progressive supranuclear paralysis (PSP) is challenging. This study aimed to investigate the expression of phosphorylated α-synuclein (p-α-syn) and phosphorylated tau-protein (p-tau) in sural nerves from patients with PD, MSA and PSP to find biomarkers for differential diagnosis. Clinical evaluations and sural nerve biopsies were performed on 8 PD patients, 8 MSA patients, 6 PSP patients and 8 controls (CTRs). Toluidine blue staining was used to observe morphological changes in sural nerves. The deposition of p-α-syn and p-tau was detected by immunohistochemistry with semiquantitative evaluation. Locations of p-α-syn and p-tau were identified by double immunofluorescent staining. In case groups, the density of nerve fibres decreased with swollen or fragmented Schwann cells (SCs). All cases (22/22) but no CTRs (0/8) presented p-α-syn immunoreactivity with gradually decreasing semiquantitative levels among the PD (6.00 ± 2.07), MSA (5.00 ± 2.33) and PSP (3.50 ± 1.52) groups. p-tau aggregates were found in 7/8 MSA (1.88 ± 1.46) and 6/6 PSP (1.67 ± 0.52) patients but not in PD patients or CTRs. There were different expression patterns of p-α-syn and p-tau in PD, MSA and PSP patients. These findings suggest that peripheral sensory nerve injury exists in PD, MSA and PSP patients. With a different expression pattern and level, p-α-syn and p-tau in sural nerves may serve as novel biomarkers for differential diagnosis of PD, MSA and PSP.

In Vivo Diagnosis of Synucleinopathies: A Comparative Study of Skin Biopsy and RT-QuIC.

OBJECTIVE: To determine whether (1) immunofluorescence is a reproducible technique in detecting misfolded α-synuclein in skin nerves and subsequently whether (2) immunofluorescence and real-time quaking-induced conversion (RT-QuIC) (both in skin and CSF) show a comparable in vivo diagnostic accuracy in distinguishing synucleinopathies from non-synucleinopathies in a large cohort of patients. METHODS: We prospectively recruited 90 patients fulfilling clinical and instrumental diagnostic criteria for all synucleinopathies variants and non-synucleinopathies (mainly including Alzheimer disease, tauopathies, and vascular parkinsonism or dementia). Twenty-four patients with mainly peripheral neuropathies were used as controls. Patients underwent skin biopsy for immunofluorescence and RT-QuIC; CSF was examined in patients who underwent lumbar puncture for diagnostic purposes. Immunofluorescence and RT-QuIC analysis were made blinded to the clinical diagnosis. RESULTS: Immunofluorescence showed reproducible results between 2 pairs of neighboring skin samples. Both immunofluorescence and RT-QuIC showed high sensitivity and specificity in discriminating synucleinopathies from non-synucleinopathies and controls but immunofluorescence presented higher diagnostic accuracy. Immunofluorescence presented a good level of agreement with RT-QuIC in both skin and CSF in synucleinopathies. CONCLUSIONS: Both immunofluorescence and RT-QuIC showed high diagnostic accuracy, although immunofluorescence displayed the better value as well as optimal reproducibility; they presented a good level of agreement in synucleinopathies, supporting the use of less invasive tests such as skin immunofluorescence or RT-QuIC instead of CSF RT-QuIC as a diagnostic tool for synucleinopathies. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that immunofluorescence or RT-QuIC accurately distinguish synucleinopathies from non-synucleinopathies.

Dermal and cardiac autonomic fiber involvement in Parkinson's disease and multiple system atrophy.

Pathological aggregates of alpha-synuclein in peripheral dermal nerve fibers can be detected in patients with idiopathic Parkinson's disease and multiple system atrophy. This study combines skin biopsy staining for p-alpha-synuclein depositions and radionuclide imaging of the heart with [123I]-metaiodobenzylguanidine to explore peripheral denervation in both diseases. To this purpose, 42 patients with a clinical diagnosis of Parkinson's disease or multiple system atrophy were enrolled. All patients underwent a standardized clinical work-up including neurological evaluation, neurography, and blood samples. Skin biopsies were obtained from the distal and proximal leg, back, and neck for immunofluorescence double labeling with anti-p-alpha-synuclein and anti-PGP9.5. All patients underwent myocardial [123I]-metaiodobenzylguanidine scintigraphy. Dermal p-alpha-synuclein was observed in 47.6% of Parkinson's disease patients and was mainly found in autonomic structures. 81.0% of multiple system atrophy patients had deposits with most of cases in somatosensory fibers. The [123I]-metaiodobenzylguanidine heart-to-mediastinum ratio was lower in Parkinson's disease than in multiple system atrophy patients (1.94 ± 0.63 vs. 2.91 ± 0.96; p < 0.0001). Irrespective of the diagnosis, uptake was lower in patients with than without p-alpha-synuclein in autonomic structures (1.42 ± 0.51 vs. 2.74 ± 0.83; p < 0.0001). Rare cases of Parkinson's disease with p-alpha-synuclein in somatosensory fibers and multiple system atrophy patients with deposits in autonomic structures or both fiber types presented with clinically overlapping features. In conclusion, this study suggests that alpha-synuclein contributes to peripheral neurodegeneration and mediates the impairment of cardiac sympathetic neurons in patients with synucleinopathies. Furthermore, it indicates that Parkinson's disease and multiple system atrophy share pathophysiologic mechanisms of peripheral nervous system dysfunction with a clinical overlap.

The Alpha-Synuclein RT-QuIC Products Generated by the Olfactory Mucosa of Patients with Parkinson's Disease and Multiple System Atrophy Induce Inflammatory Responses in SH-SY5Y Cells.

Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by two distinct strains of disease-associated α-synuclein (αSynD). Recently, we have shown that olfactory mucosa (OM) samples of patients with PD and MSA can seed the aggregation of recombinant α-synuclein by means of Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC). Remarkably, the biochemical and morphological properties of the final α-synuclein aggregates significantly differed between PD and MSA seeded samples. Here, these aggregates were given to neuron-like differentiated SH-SY5Y cells and distinct inflammatory responses were observed. To deepen whether the morphological features of α-synuclein aggregates were responsible for this variable SH-SY5Y inflammatory response, we generated three biochemically and morphologically distinct α-synuclein aggregates starting from recombinant α-synuclein that were used to seed αSyn_RT-QuIC reaction; the final reaction products were used to stimulate SH-SY5Y cells. Our study showed that, in contrast to OM samples of PD and MSA patients, the artificial aggregates did not transfer their distinctive features to the αSyn_RT-QuIC products and the latter induced analogous inflammatory responses in cells. Thus, the natural composition of the αSynD strains but also other specific factors in OM tissue can substantially modulate the biochemical, morphological and inflammatory features of the αSyn_RT-QuIC products.

Viral-based rodent and nonhuman primate models of multiple system atrophy: Fidelity to the human disease.

Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histological analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA.

Comparison of 123I-MIBG scintigraphy and phosphorylated α-synuclein skin deposits in synucleinopathies.

INTRODUCTION: Cardiac [123I]metaiodobenzylguanidine scintigraphy (123I-MIBG) is considered a useful test in differentiating multiple system atrophy (MSA) and Lewy body disorders (LBD), including idiopathic Parkinson's disease (IPD), dementia with Lewy bodies (DLB) and pure autonomic failure (PAF). The detection of skin nerve phosphorylated α-synuclein (p-α-syn) deposits could be an alternative marker in vivo. We sought to compare 123I-MIBG scintigraphy and skin biopsy findings in α-synucleinopathies. METHODS: We studied 54 patients (7 DLB, 21 IPD, 13 PAF, 13 MSA) who underwent 123I-MIBG scintigraphy and skin biopsy to evaluate cardiac innervation and skin p-α-syn deposition, respectively. RESULTS: Cardiac denervation was observed in 90.5% IPD, 100% DLB and PAF and in none of the MSA patients (P < 0.0001) whereas p-α-syn deposits were detected in all DLB and PAF, in 95.2% of IPD and 69.2% of MSA patients (P = 0.02). However, the analysis of skin structures disclosed a different distribution of the deposits in somatic subepidermal plexus and autonomic fibers among groups, showing that p-α-syn deposits rarely affected the autonomic fibers in MSA as opposed to LBD. Studying the p-α-syn deposition in autonomic nerves, concordance among I123-MIBG scintigraphy and skin biopsy results was observed in 100% of DLB and PAF, 95.2% IPD and 92.3% MSA patients. I123-MIBG scintigraphy and autonomic p-α-syn deposits analysis both showed a sensitivity of 97.5% and a specificity of 100% and 92.3%, respectively, in distinguishing LBD and MSA. CONCLUSION: Skin biopsy and 123-MIBG scintigraphy can be considered alternative tests for the differential diagnosis of IPD, PAF and DLB versus MSA.

Quantitative susceptibility mapping depicts severe myelin deficit and iron deposition in a transgenic model of multiple system atrophy.

Despite internationally established diagnostic criteria, multiple system atrophy (MSA) is frequently misdiagnosed, particularly at disease onset. While neuropathological changes such as demyelination and iron deposition are typically detected in MSA, these structural hallmarks were so far only demonstrated post-mortem. Here, we examine whether myelin deficit observed in a transgenic murine model of MSA can be visualized and quantified in vivo using specific magnetic resonance imaging (MRI) approaches. Reduced myelin content was measured histologically in prototypical white matter as well as mixed grey-white matter regions i.e. corpus callosum, anterior commissure, and striatum of transgenic mice overexpressing human α-synuclein under the control of the myelin basic protein promotor (MBP29-hα-syn mice). Correspondingly, in vivo quantitative susceptibility mapping (QSM) showed a strongly reduced susceptibility contrast in white matter regions and T2-weighted MR imaging revealed a significantly reduced grey-white matter contrast in MBP29-hα-syn mice. In addition, morphological analysis suggested a pronounced, white matter-specific deposition of iron in MBP29-hα-syn mice. Importantly, in vivo MRI results were matched by comprehensive structural characterization of myelin, iron, and axonal directionality. Taken together, our results provide strong evidence that QSM is a very sensitive tool measuring changes in myelin density in conjunction with iron deposition in MBP29-hα-syn mice. This multimodal neuroimaging approach may pave the way towards a novel non-invasive technique to detect crucial neuropathological changes specifically associated with MSA.

FTY720-Mitoxy reduces synucleinopathy and neuroinflammation, restores behavior and mitochondria function, and increases GDNF expression in Multiple System Atrophy mouse models.

Multiple system atrophy (MSA) is a fatal disorder with no effective treatment. MSA pathology is characterized by α-synuclein (aSyn) accumulation in oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). aSyn accumulation in oligodendrocytes forms the pathognomonic glial cytoplasmic inclusions (GCIs) of MSA. MSA aSyn pathology is also associated with motor and autonomic dysfunction, including an impaired ability to sweat. MSA patients have abnormal CNS expression of glial-cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Our prior studies using the parent compound FTY720, a food and drug administration (FDA) approved immunosuppressive for multiple sclerosis, reveal that FTY720 protects parkinsonian mice by increasing BDNF. Our FTY720-derivative, FTY720-Mitoxy, is known to increase expression of oligodendrocyte BDNF, GDNF, and nerve growth factor (NGF) but does not reduce levels of circulating lymphocytes as it is not phosphorylated so cannot modulate sphingosine 1 phosphate receptors (S1PRs). To preclinically assess FTY720-Mitoxy for MSA, we used mice expressing human aSyn in oligodendrocytes under a 2,' 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter. CNP-aSyn transgenic (Tg) mice develop motor dysfunction between 7 and 9 mo, and progressive GCI pathology. Using liquid chromatography-mass spectrometry (LC-MS/MS) and enzymatic assays, we confirmed that FTY720-Mitoxy was stable and active. Vehicle or FTY720-Mitoxy (1.1 mg/kg/day) was delivered to wild type (WT) or Tg littermates from 8.5-11.5 mo by osmotic pump. We behaviorally assessed their movement by rotarod and sweat production by starch­iodine test. Postmortem tissues were evaluated by qPCR for BDNF, GDNF, NGF and GDNF-receptor RET mRNA and for aSyn, BDNF, GDNF, and Iba1 protein by immunoblot. MicroRNAs (miRNAs) were also assessed by qPCR. FTY720-Mitoxy normalized movement, sweat function and soleus muscle mass in 11.5 mo Tg MSA mice. FTY720-Mitoxy also increased levels of brain GDNF and reduced brain miR-96-5p, a miRNA that acts to decrease GDNF expression. Moreover, FTY720-Mitoxy blocked aSyn pathology measured by sequential protein extraction and immunoblot, and microglial activation assessed by immunohistochemistry and immunoblot. In the 3-nitropropionic acid (3NP) toxin model of MSA, FTY720-Mitoxy protected movement and mitochondria in WT and CNP-aSyn Tg littermates. Our data confirm potent in vivo protection by FTY720-Mitoxy, supporting its further evaluation as a potential therapy for MSA and related synucleinopathies.

Stabilization of α-synuclein oligomers using formaldehyde.

The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type α-synuclein (α-syn) aggregates within degenerating brain cells. α-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating α-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized α-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific α-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked α-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked α-syn oligomers could serve as important calibrators to facilitate comparative and standardized α-syn biomarker studies going forward.

Quantifying iron deposition in the cerebellar subtype of multiple system atrophy and spinocerebellar ataxia type 6 by quantitative susceptibility mapping.

We used quantitative susceptibility mapping (QSM) to assess the brain iron deposition in 28 patients with the cerebellar subtype of multiple system atrophy (MSA-C), nine patients with spinocerebellar ataxia type 6 (SCA6), and 23 healthy controls. Two reviewers independently measured the mean QSM values in brain structures including the putamen, globus pallidus, caudate nucleus, red nucleus, substantia nigra, and cerebellar dentate nucleus. A receiver operating characteristics (ROC) analysis was performed to assess the diagnostic usefulness of the QSM measurements. The QSM values in the substantia nigra were significantly higher in the MSA-C group compared to the HC group (p = .007). The QSM values in the cerebellar dentate nucleus were significantly higher in MSA-C than those in the SCA6 and HC groups (p < .001), and significantly lower in the SCA6 patients compared to the HCs (p = .027). The QSM values in the cerebellar dentate nucleus were correlated with disease duration in MSA-C, but inversely correlated with disease duration in SCA6. In the ROC analysis, the QSM values in the cerebellar dentate nucleus showed excellent accuracy for differentiating MSA-C from SCA6 (area under curve [AUC], 0.925), and good accuracy for differentiating MSA-C from healthy controls (AUC 0.834). QSM can identify increased susceptibility of the substantia nigra and cerebellar dentate nucleus in MSA-C patients. These results suggest that an increase in iron accumulation in the cerebellar dentate nucleus may be secondary to the neurodegeneration associated with MSA-C.

Transcription factor EB overexpression prevents neurodegeneration in experimental synucleinopathies.

The synucleinopathies Parkinson's disease (PD) and Multiple system atrophy (MSA) - characterized by α-synuclein intracytoplasmic inclusions into, respectively, neurons and oligodendrocytes - are associated with impairment of the autophagy-lysosomal pathways (ALP). Increased expression of the master regulator of ALP, transcription factor EB (TFEB), is hypothesized to promote the clearance of WT α-synuclein and survival of dopaminergic neurons. Here, we explore the efficacy of targeted TFEB overexpression either in neurons or oligodendrocytes to reduce the pathological burden of α-synuclein in a PD rat model and a MSA mouse model. While TFEB neuronal expression was sufficient to prevent neurodegeneration in the PD model, we show that only TFEB oligodendroglial overexpression leads to neuroprotective effects in the MSA model. These beneficial effects were associated with a decreased accumulation of α-synuclein into oligodendrocytes through recovery of the ALP machinery. Our study demonstrates that the cell type where α-synuclein aggregates dictates the target of TFEB overexpression in order to be protective, paving the way for adapted therapies.

Abnormal α-synuclein deposits in skin nerves: intra- and inter-laboratory reproducibility.

BACKGROUND AND PURPOSE: Visualization of phosphorylated α-synuclein at serine 129 (p-syn) in skin nerves is a promising test for the in vivo diagnosis of synucleinopathies. Here the aim was to establish the intra- and inter-laboratory reproducibility of measurement of intraneural p-syn immunoreactivity in two laboratories with major expertise (Würzburg and Bologna). METHODS: In total, 43 patients affected by Parkinson's disease (PD 21 patients), dementia with Lewy bodies (DLB 1), rapid eye movement sleep behaviour disorder (RBD 11), multiple system atrophy (MSA-P 4) and small fibre neuropathy (SFN 6) were enrolled. Skin biopsy was performed at the C7 paravertebral spine region and distal skin sites (thigh or leg). The analysis was standardized in both laboratories and carried out blinded on a single skin section double stained with antibodies to p-syn and the pan-axonal marker protein gene product 9.5. Fifty skin sections were randomly selected for the analysis: 25 from C7 and 25 from distal sites. Differently classified sections were re-evaluated to understand the reasons for the discrepancy. RESULTS: The intra-laboratory analysis showed an excellent reproducibility both in Würzburg (concordance of classification 100% of sections; K = 1; P < 0.001) and Bologna (96% of sections; K = 0.92; P < 0.001). Inter-laboratory analysis showed reproducibility in 45 sections (90%; K = 0.8; P < 0.001) and a different classification in five sections, which was mainly due to fragmented skin samples or weak fluorescent signals. CONCLUSIONS: Analysis of p-syn showed excellent inter- and intra-laboratory reproducibility supporting the reliability of this technique. The few ascertained discordances were important to further improve the standardization of this technique.

Investigating the neuroprotective effect of AAV-mediated ß-synuclein overexpression in a transgenic model of synucleinopathy.

Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases characterized by inclusions mainly composed of α-synuclein (α-syn) aggregates. The objective of this study was to investigate if ß-synuclein (ß-syn) overexpression could have beneficial effects by inhibiting the aggregation of α-syn. The M83 transgenic mouse is a model of synucleinopathy, which develops severe motor symptoms associated with aggregation of α-syn. M83 neonate or adult mice were injected with adeno-associated virus vectors carrying the human ß-syn gene (AAVß-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. The M83 disease was - or not - accelerated using extracts of M83 brains injected with brain extract from mouse (M83) or human (MSA) origins. AAV vectors expression was confirmed using Western blot and ELISA technics. AAV mediated ß-syn overexpression did not delay the disease onset or reduce the α-syn phosphorylated at serine 129 levels detected by ELISA, regardless of the AAV injection route and the inoculation of brain extracts. Instead, a proteinase-K resistant ß-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing ß-syn after inoculation of AAVß-syn. This study indicated for the first time that viral vector-mediated ß-syn overexpression could form aggregates in a model of synucleinopathy.

NLRP3 Inflammasome-Related Proteins Are Upregulated in the Putamen of Patients With Multiple System Atrophy.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia, and autonomic dysfunction. Microglial infiltration is an important mediator in MSA. The nucleotide-binding domain, leucine-rich repeats-containing family, pyrin domain-containing-3 (NLRP3) inflammasome complex, comprising NLRP3, apoptotic speck protein containing a caspase recruitment domain (ASC), and cysteine aspartic acid protease 1 (Caspase 1), regulates microglial inflammation in several neurodegenerative diseases. However, its role in MSA remains unknown. This study aimed to investigate the role of the NLRP3 inflammasome in MSA. Immunohistochemical staining of postmortem brains from 11 cases of MSA, 5 of Parkinson disease, and 6 age-matched controls were assessed. The relationships among α-synuclein deposition, microglial infiltration, and NLRP3 inflammasome-related proteins (NLRP3, ASC, and Caspase 1) were quantitatively analyzed. Double-labeling immunofluorescence staining confirmed colocalization of NLRP3 inflammasome-related proteins and Cluster of Differentiation 68. We demonstrated that the density of microglia expressing NLRP3 inflammasome-related proteins was increased in the putamina of MSA cases and was significantly related to the deposition of phosphorylated α-synuclein-positive glial cytoplasmic inclusions, tyrosine hydroxylase-positive fiber loss, and gliosis of glial fibrillary acidic protein-positive astrocytes. Our study suggests that the NLRP3 inflammasome is significantly upregulated and correlates with the neurodegenerative process in MSA.