Association of CSF α-Synuclein Seeding Amplification Assay Results With Clinical Features of Possible and Probable Dementia With Lewy Bodies.

BACKGROUND AND OBJECTIVES: The clinical diagnosis of dementia with Lewy bodies (DLB) depends on identifying significant cognitive decline accompanied by core features of parkinsonism, visual hallucinations, cognitive fluctuations, and REM sleep behavior disorder (RBD). Hyposmia is one of the several supportive features. α-Synuclein seeding amplification assays (αSyn-SAAs) may enhance diagnostic accuracy by detecting pathologic αSyn seeds in CSF. In this study, we examine how different clinical features associate with CSF αSyn-SAA positivity in a large group of clinically diagnosed participants with DLB. METHODS: Cross-sectional and longitudinal CSF samples from the multicentered observational cohort study of the DLB Consortium and similar studies within the Parkinson's Disease Biomarker Program, contributed by academic medical centers in the United States, underwent αSyn-SAA testing. Participants included those clinically diagnosed with DLB and 2 control cohorts. Associations between core DLB features and olfaction with αSyn-SAA positivity were evaluated using logistic regression. RESULTS: CSF samples from 191 participants diagnosed with DLB (mean age 69.9 ± 6.8, 15% female), 50 age-matched and sex-matched clinical control participants, and 49 younger analytical control participants were analyzed. Seventy-two percent (137/191) of participants with DLB had positive αSyn-SAAs vs 4% of the control groups. Among participants with DLB, those who were αSyn-SAA-positive had lower Montreal Cognitive Assessment scores (18.8 ± 5.7 vs 21.2 ± 5.2, p = 0.01), had worse parkinsonism on the Movement Disorders Society Unified Parkinson's Disease Rating Scale part III (33.8 ± 15.1 vs 25.6 ± 16.4, p = 0.001), were more likely to report RBD (114/133 [86%] vs 33/53 [62%], p < 0.0001), and had worse hyposmia on the University of Pennsylvania Smell Identification Test (UPSIT) (94/105 [90%] below 15th percentile vs 14/44 [32%], p < 0.0001). UPSIT percentile had the highest area under the curve (0.87, 95% CI 0.81-0.94) in predicting αSyn-SAA positivity and participants scoring at or below the 15th percentile of age and sex normative values had 18.3 times higher odds (95% CI 7.52-44.6) of having a positive αSyn-SAA test. Among 82 participants with longitudinal CSF samples, 81 (99%) had the same αSyn-SAA result for initial and follow-up specimens. DISCUSSION: A substantial proportion of clinically diagnosed participants with DLB had negative αSyn-SAA results. Hyposmia was the strongest clinical predictor of αSyn-SAA positivity. Hyposmia and αSyn-SAA may have utility in improving the diagnostic assessment of individuals with potential DLB. CLASSIFICATION OF EVIDENCE: This study provided Class III evidence that CSF αSyn-SAA distinguishes patients with clinically diagnosed DLB from normal controls.

Mechanistic Insight into Intestinal α-Synuclein Aggregation in Parkinson's Disease Using a Laser-Printed Electrochemical Sensor.

Aggregated deposits of the protein α-synuclein and depleting levels of dopamine in the brain correlate with Parkinson's disease development. Treatments often focus on replenishing dopamine in the brain; however, the brain might not be the only site requiring attention. Aggregates of α-synuclein appear to accumulate in the gut years prior to the onset of any motor symptoms. Enteroendocrine cells (specialized gut epithelial cells) may be the source of intestinal α-synuclein, as they natively express this protein. Enteroendocrine cells are constantly exposed to gut bacteria and their metabolites because they border the gut lumen. These cells also express the dopamine metabolic pathway and form synapses with vagal neurons, which innervate the gut and brain. Through this connection, Parkinson's disease pathology may originate in the gut and spread to the brain over time. Effective therapeutics to prevent this disease progression are lacking due to a limited understanding of the mechanisms by which α-synuclein aggregation occurs in the gut. We previously proposed a gut bacterial metabolic pathway responsible for the initiation of α-synuclein aggregation that is dependent on the oxidation of dopamine. Here, we develop a new tool, a laser-induced graphene-based electrochemical sensor chip, to track α-synuclein aggregation and dopamine level over time. Using these sensor chips, we evaluated diet-derived catechols dihydrocaffeic acid and caffeic acid as potential inhibitors of α-synuclein aggregation. Our results suggest that these molecules inhibit dopamine oxidation. We also found that these dietary catechols inhibit α-synuclein aggregation in STC-1 enteroendocrine cells. These findings are critical next steps to reveal new avenues for targeted therapeutics to treat Parkinson's disease, specifically in the context of functional foods that may be used to reshape the gut environment.

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS.

Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

Alpha-Synuclein Interaction with UBL3 Is Upregulated by Microsomal Glutathione S-Transferase 3, Leading to Increased Extracellular Transport of the Alpha-Synuclein under Oxidative Stress.

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson's disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases.

The Pathobiology of Behavioral Changes in Multiple System Atrophy: An Update.

While cognitive impairment, which was previously considered a red flag against the clinical diagnosis of multiple system atrophy (MSA), is a common symptom of this rare neurodegenerative disorder, behavioral disorders are reported in 30 to 70% of MSA patients. They include anxiety, apathy, impaired attention, compulsive and REM sleep behavior disorders (RBD), and these conditions, like depression, are early and pervasive features in MSA, which may contribute to disease progression. Despite changing concepts of behavioral changes in this synucleinopathy, the underlying pathophysiological and biochemical mechanisms are poorly understood. While specific neuropathological data are unavailable, neuroimaging studies related anxiety disorders to changes in the cortico-limbic system, apathy (and depression) to dysfunction of prefrontal-subcortical circuits, and compulsive behaviors to impairment of basal ganglia networks and involvement of orbito-frontal circuits. Anxiety has also been related to α-synuclein (αSyn) pathology in the amygdala, RBD to striatal monoaminergic deficit, and compulsive behavior in response to dopamine agonist therapy in MSA, while the basic mechanisms of the other behavioral disorders and their relations to other non-motor dysfunctions in MSA are unknown. In view of the scarcity of functional and biochemical findings in MSA with behavioral symptoms, further neuroimaging and biochemical studies are warranted in order to obtain better insight into their pathogenesis as a basis for the development of diagnostic biomarkers and future adequate treatment modalities of these debilitating comorbidities.

High frequency electrical stimulation reduces α-synuclein levels and α-synuclein-mediated autophagy dysfunction.

Accumulation of α-synuclein (α-Syn) has been implicated in proteasome and autophagy dysfunction in Parkinson's disease (PD). High frequency electrical stimulation (HFS) mimicking clinical parameters used for deep brain stimulation (DBS) in vitro or DBS in vivo in preclinical models of PD have been found to reduce levels of α-Syn and, in certain cases, provide possible neuroprotection. However, the mechanisms by which this reduction in α-Syn improves cellular dysfunction associated with α-Syn accumulation remains elusive. Using HFS parameters that recapitulate DBS in vitro, we found that HFS led to a reduction of mutant α-Syn and thereby limited proteasome and autophagy impairments due to α-Syn. Additionally, we observed that HFS modulates via the ATP6V0C subunit of V-ATPase and mitigates α-Syn mediated autophagic dysfunction. This study highlights a role for autophagy in reduction of α-Syn due to HFS which may prove to be a viable approach to decrease pathological protein accumulation in neurodegeneration.

Retinal hyperspectral imaging in mouse models of Parkinson's disease and healthy aging.

Retinal hyperspectral imaging (HSI) is a non-invasive in vivo approach that has shown promise in Alzheimer's disease. Parkinson's disease is another neurodegenerative disease where brain pathobiology such as alpha-synuclein and iron overaccumulation have been implicated in the retina. However, it remains unknown whether HSI is altered in in vivo models of Parkinson's disease, whether it differs from healthy aging, and the mechanisms which drive these changes. To address this, we conducted HSI in two mouse models of Parkinson's disease across different ages; an alpha-synuclein overaccumulation model (hA53T transgenic line M83, A53T) and an iron deposition model (Tau knock out, TauKO). In comparison to wild-type littermates the A53T and TauKO mice both demonstrated increased reflectivity at short wavelengths ~ 450 to 600 nm. In contrast, healthy aging in three background strains exhibited the opposite effect, a decreased reflectance in the short wavelength spectrum. We also demonstrate that the Parkinson's hyperspectral signature is similar to that from an Alzheimer's disease model, 5xFAD mice. Multivariate analyses of HSI were significant when plotted against age. Moreover, when alpha-synuclein, iron or retinal nerve fibre layer thickness were added as a cofactor this improved the R2 values of the correlations in certain groups. This study demonstrates an in vivo hyperspectral signature in Parkinson's disease that is consistent in two mouse models and is distinct from healthy aging. There is also a suggestion that factors including retinal deposition of alpha-synuclein and iron may play a role in driving the Parkinson's disease hyperspectral profile and retinal nerve fibre layer thickness in advanced aging. These findings suggest that HSI may be a promising translation tool in Parkinson's disease.

Gut-Brain Axis in Focus: Polyphenols, Microbiota, and Their Influence on α-Synuclein in Parkinson's Disease.

With the recognition of the importance of the gut-brain axis in Parkinson's disease (PD) etiology, there is increased interest in developing therapeutic strategies that target α-synuclein, the hallmark abhorrent protein of PD pathogenesis, which may originate in the gut. Research has demonstrated that inhibiting the aggregation, oligomerization, and fibrillation of α-synuclein are key strategies for disease modification. Polyphenols, which are rich in fruits and vegetables, are drawing attention for their potential role in this context. In this paper, we reviewed how polyphenols influence the composition and functional capabilities of the gut microbiota and how the resulting microbial metabolites of polyphenols may potentially enhance the modulation of α-synuclein aggregation. Understanding the interaction between polyphenols and gut microbiota and identifying which specific microbes may enhance the efficacy of polyphenols is crucial for developing therapeutic strategies and precision nutrition based on the microbiome.

Serum Oligomeric α-Synuclein and p-tau181 in Progressive Supranuclear Palsy and Parkinson's Disease.

Clinical differentiation of progressive supranuclear palsy (PSP) from Parkinson's disease (PD) is challenging due to overlapping phenotypes and the late onset of specific atypical signs. Therefore, easily assessable diagnostic biomarkers are highly needed. Since PD is a synucleopathy while PSP is a tauopathy, here, we investigated the clinical usefulness of serum oligomeric-α-synuclein (o-α-synuclein) and 181Thr-phosphorylated tau (p-tau181), which are considered as the most important pathological protein forms in distinguishing between these two parkinsonisms. We assessed serum o-α-synuclein and p-tau181 by ELISA and SIMOA, respectively, in 27 PSP patients, 43 PD patients, and 39 healthy controls (HC). Moreover, we evaluated the correlation between serum biomarkers and biological and clinical features of these subjects. We did not find any difference in serum concentrations of p-tau181 and o-α-synuclein nor in the o-α-synuclein/p-tau181 ratio between groups. However, we observed that serum p-tau181 positively correlated with age in HC and PD, while serum o-α-synuclein correlated positively with disease severity in PD and negatively with age in PSP. Finally, the o-α-synuclein/p-tau181 ratio showed a negative correlation with age in PD.

Single-nucleus multi-omics of Parkinson's disease reveals a glutamatergic neuronal subtype susceptible to gene dysregulation via alteration of transcriptional networks.

The genetic architecture of Parkinson's disease (PD) is complex and multiple brain cell subtypes are involved in the neuropathological progression of the disease. Here we aimed to advance our understanding of PD genetic complexity at a cell subtype precision level. Using parallel single-nucleus (sn)RNA-seq and snATAC-seq analyses we simultaneously profiled the transcriptomic and chromatin accessibility landscapes in temporal cortex tissues from 12 PD compared to 12 control subjects at a granular single cell resolution. An integrative bioinformatic pipeline was developed and applied for the analyses of these snMulti-omics datasets. The results identified a subpopulation of cortical glutamatergic excitatory neurons with remarkably altered gene expression in PD, including differentially-expressed genes within PD risk loci identified in genome-wide association studies (GWAS). This was the only neuronal subtype showing significant and robust overexpression of SNCA. Further characterization of this neuronal-subpopulation showed upregulation of specific pathways related to axon guidance, neurite outgrowth and post-synaptic structure, and downregulated pathways involved in presynaptic organization and calcium response. Additionally, we characterized the roles of three molecular mechanisms in governing PD-associated cell subtype-specific dysregulation of gene expression: (1) changes in cis-regulatory element accessibility to transcriptional machinery; (2) changes in the abundance of master transcriptional regulators, including YY1, SP3, and KLF16; (3) candidate regulatory variants in high linkage disequilibrium with PD-GWAS genomic variants impacting transcription factor binding affinities. To our knowledge, this study is the first and the most comprehensive interrogation of the multi-omics landscape of PD at a cell-subtype resolution. Our findings provide new insights into a precise glutamatergic neuronal cell subtype, causal genes, and non-coding regulatory variants underlying the neuropathological progression of PD, paving the way for the development of cell- and gene-targeted therapeutics to halt disease progression as well as genetic biomarkers for early preclinical diagnosis.

Myricetin mitigates motor disturbance and decreases neuronal ferroptosis in a rat model of Parkinson's disease.

Ferroptosis is an iron-dependent cell death form characterized by reactive oxygen species (ROS) overgeneration and lipid peroxidation. Myricetin, a flavonoid that exists in numerous plants, exhibits potent antioxidant capacity. Given that iron accumulation and ROS-provoked dopaminergic neuron death are the two main pathological hallmarks of Parkinson's disease (PD), we aimed to investigate whether myricetin decreases neuronal death through suppressing ferroptosis. The PD models were established by intraperitoneally injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into rats and by treating SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP+), respectively. Ferroptosis was identified by assessing the levels of Fe2+, ROS, malondialdehyde (MDA), and glutathione (GSH). The results demonstrated that myricetin treatment effectively mitigated MPTP-triggered motor impairment, dopamine neuronal death, and α-synuclein (α-Syn) accumulation in PD models. Myricetin also alleviated MPTP-induced ferroptosis, as evidenced by decreased levels of Fe2+, ROS, and MDA and increased levels of GSH in the substantia nigra (SN) and serum in PD models. All these changes were reversed by erastin, a ferroptosis activator. In vitro, myricetin treatment restored SH-SY5Y cell viability and alleviated MPP+-induced SH-SY5Y cell ferroptosis. Mechanistically, myricetin accelerated nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) and subsequent glutathione peroxidase 4 (Gpx4) expression in MPP+-treated SH-SY5Y cells, two critical inhibitors of ferroptosis. Collectively, these data demonstrate that myricetin may be a potential agent for decreasing dopaminergic neuron death by inhibiting ferroptosis in PD.

Cell membrane proteome analysis in HEK293T cells challenged with α-synuclein amyloids.

Amyloids interact with plasma membranes. Extracellular amyloids cross the plasma membrane barrier. Internalized extracellular amyloids are reported to trigger amyloidogenesis of endogenous proteins in recipient cells. To what extent these extracellular and intracellular amyloids perturb the plasma membrane proteome is not investigated. Using α-synuclein as a model amyloid protein, we performed membrane shaving followed by mass spectrometry experiments to identify the conformational changes in cell surface proteins after extracellular amyloid challenge. We also performed membrane proteomics after the biogenesis of intracellular α-synuclein amyloids. Our results suggest that promiscuous interactions with extracellular amyloids stochastically alter the conformation of plasma membrane proteins. This affects the biological processes through the plasma membrane and results in loss of cell viability. Cells that survive the extracellular amyloid shock can grow normally and gradually develop intracellular amyloids which do not directly impact the plasma membrane proteome and associated biological processes. Thus, our results suggest that α-synuclein amyloids can damage the plasma membrane and related processes during cell-to-cell transfer and not during their intracellular biogenesis.

GBA1-Associated Parkinson's Disease Is a Distinct Entity.

GBA1-associated Parkinson's disease (GBA1-PD) is increasingly recognized as a distinct entity within the spectrum of parkinsonian disorders. This review explores the unique pathophysiological features, clinical progression, and genetic underpinnings that differentiate GBA1-PD from idiopathic Parkinson's disease (iPD). GBA1-PD typically presents with earlier onset and more rapid progression, with a poor response to standard PD medications. It is marked by pronounced cognitive impairment and a higher burden of non-motor symptoms compared to iPD. Additionally, patients with GBA1-PD often exhibit a broader distribution of Lewy bodies within the brain, accentuating neurodegenerative processes. The pathogenesis of GBA1-PD is closely associated with mutations in the GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase). In this review, we discuss two mechanisms by which GBA1 mutations contribute to disease development: 'haploinsufficiency,' where a single functional gene copy fails to produce a sufficient amount of GCase, and 'gain of function,' where the mutated GCase acquires harmful properties that directly impact cellular mechanisms for alpha-synuclein degradation, leading to alpha-synuclein aggregation and neuronal cell damage. Continued research is advancing our understanding of how these mechanisms contribute to the development and progression of GBA1-PD, with the 'gain of function' mechanism appearing to be the most plausible. This review also explores the implications of GBA1 mutations for therapeutic strategies, highlighting the need for early diagnosis and targeted interventions. Currently, small molecular chaperones have shown the most promising clinical results compared to other agents. This synthesis of clinical, pathological, and molecular aspects underscores the assertion that GBA1-PD is a distinct clinical and pathobiological PD phenotype, necessitating specific management and research approaches to better understand and treat this debilitating condition.

Regulation of MicroRNA-4697-3p by Parkinson's disease-associated SNP rs329648 and its impact on SNCA112 mRNA.

BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a multifaceted genetic foundation. Genome-Wide Association Studies (GWAS) have played a crucial role in pinpointing genetic variants linked to PD susceptibility. Current study aims to delve into the mechanistic aspects through which the PD-associated Single Nucleotide Polymorphism (SNP) rs329648, identified in prior GWAS, influences the pathogenesis of PD. METHODS AND RESULTS: Employing the CRISPR/Cas9-mediated genome editing mechanism, we demonstrated the association of the disease-associated allele of rs329648 with increased expression of miR-4697-3p in differentiated SH-SY5Y cells. We revealed that miR-4697-3p contributes to the formation of high molecular weight complexes of α-Synuclein (α-Syn), indicative of α-Syn aggregate formation, as evidenced by Western blot analysis. Furthermore, our study unveiled that miR-4697-3p elevates SNCA112 mRNA levels. The resultant protein product, α-Syn 112, a variant of α-Syn with 112 amino acids, is recognized for augmenting α-Syn aggregation. Notably, this regulatory effect minimally impacts the levels of full-length SNCA140 mRNA, as evidenced by qRT-PCR. Additionally, we observed a correlation between the disease-associated allele and miR-4697-3p with increased cell death, substantiated by assessments including cell viability assays, alterations in cell morphology, and TUNEL assays. CONCLUSION: Our research reveals that the disease-associated allele of rs329648 is linked to higher levels of miR-4697-3p. This increase in miR-4697-3p leads to elevated SNCA112 mRNA levels, consequently promoting the formation of α-Syn aggregates. Furthermore, miR-4697-3p appears to play a role in increased cell death, potentially contributing to the pathogenesis of PD.

Ion channel dysregulation and cellular adaptations to alpha-synuclein in stressful pacemakers of the parkinsonian brainstem.

Parkinson's disease (PD) is diagnosed by its cardinal motor symptoms that are associated with the loss of dopamine neurons in the substantia nigra pars compacta (SNc). However, PD patients suffer from various non-motor symptoms years before diagnosis. These prodromal symptoms are thought to be associated with the appearance of Lewy body pathologies (LBP) in brainstem regions such as the dorsal motor nucleus of the vagus (DMV), the locus coeruleus (LC) and others. The neurons in these regions that are vulnerable to LBP are all slow autonomous pacemaker neurons that exhibit elevated oxidative stress due to their perpetual influx of Ca2+ ions. Aggregation of toxic α-Synuclein (aSyn) - the main constituent of LBP - during the long prodromal period challenges these vulnerable neurons, presumably altering their biophysics and physiology. In contrast to pathophysiology of late stage parkinsonism which is well-documented, little is known about the pathophysiology of the brainstem during prodromal PD. In this review, we discuss ion channel dysregulation associated with aSyn aggregation in brainstem pacemaker neurons and their cellular responses to them. While toxic aSyn elevates oxidative stress in SNc and LC pacemaker neurons and exacerbates their phenotype, DMV neurons mount an adaptive response that mitigates the oxidative stress. Ion channel dysregulation and cellular adaptations may be the drivers of the prodromal symptoms of PD. For example, selective targeting of toxic aSyn to DMV pacemakers, elevates the surface density of K+ channels, which slows their firing rate, resulting in reduced parasympathetic tone to the gastrointestinal tract, which resembles the prodromal PD symptoms of dysphagia and constipation. The divergent responses of SNc & LC vs. DMV pacemaker neurons may explain why the latter outlive the former despite presenting LBPs earlier. Elucidation the brainstem pathophysiology of prodromal PD could pave the way for physiological biomarkers, earlier diagnosis and novel neuroprotective therapies for PD.

Improved Imaging Surface for Quantitative Single-Molecule Microscopy.

Preventing nonspecific binding is essential for sensitive surface-based quantitative single-molecule microscopy. Here we report a much-simplified RainX-F127 (RF-127) surface with improved passivation. This surface achieves up to 100-fold less nonspecific binding from protein aggregates compared to commonly used polyethylene glycol (PEG) surfaces. The method is compatible with common single-molecule techniques including single-molecule pull-down (SiMPull), super-resolution imaging, antibody-binding screening and single exosome visualization. This method is also able to specifically detect alpha-synuclein (α-syn) and tau aggregates from a wide range of biofluids including human serum, brain extracts, cerebrospinal fluid (CSF) and saliva. The simplicity of this method further allows the functionalization of microplates for robot-assisted high-throughput single-molecule experiments. Overall, this simple but improved surface offers a versatile platform for quantitative single-molecule microscopy without the need for specialized equipment or personnel.

Cryo-EM structures of pathogenic fibrils and their impact on neurodegenerative disease research.

Neurodegenerative diseases are commonly associated with the formation of aberrant protein aggregates within the brain, and ultrastructural analyses have revealed that the proteins within these inclusions often assemble into amyloid filaments. Cryoelectron microscopy (cryo-EM) has emerged as an effective method for determining the near-atomic structure of these disease-associated filamentous proteins, and the resulting structures have revolutionized the way we think about aberrant protein aggregation and propagation during disease progression. These structures have also revealed that individual fibril conformations may dictate different disease conditions, and this newfound knowledge has improved disease modeling in the lab and advanced the ongoing pursuit of clinical tools capable of distinguishing and targeting different pathogenic entities within living patients. In this review, we summarize some of the recently developed cryo-EM structures of ex vivo α-synuclein, tau, ß-amyloid (Aß), TAR DNA-binding protein 43 (TDP-43), and transmembrane protein 106B (TMEM106B) fibrils and discuss how these structures are being leveraged toward mechanistic research and therapeutic development.

Aggregation-resistant alpha-synuclein tetramers are reduced in the blood of Parkinson's patients.

Synucleinopathies such as Parkinson's disease (PD) are defined by the accumulation and aggregation of the α-synuclein protein in neurons, glia and other tissues. We have previously shown that destabilization of α-synuclein tetramers is associated with familial PD due to SNCA mutations and demonstrated brain-region specific alterations of α-synuclein multimers in sporadic PD patients following the classical Braak spreading theory. In this study, we assessed relative levels of disordered and higher-ordered multimeric forms of cytosolic α-synuclein in blood from familial PD with G51D mutations and sporadic PD patients. We used an adapted in vitro-cross-linking protocol for human EDTA-whole blood. The relative levels of higher-ordered α-synuclein tetramers were diminished in blood from familial PD and sporadic PD patients compared to controls. Interestingly, the relative amount of α-synuclein tetramers was already decreased in asymptomatic G51D carriers, supporting the hypothesis that α-synuclein multimer destabilization precedes the development of clinical PD. Our data, therefore suggest that measuring α-synuclein tetramers in blood may have potential as a facile biomarker assay for early detection and quantitative tracking of PD progression.

A pathophysiological biomarker combination separates Lewy body from non-Lewy body neurogenic orthostatic hypotension ​.

PURPOSE: Neurogenic orthostatic hypotension (nOH) results from deficient reflexive delivery of norepinephrine to cardiovascular receptors in response to decreased cardiac venous return. Lewy body (LB) forms of nOH are characterized by low 18F-dopamine-derived radioactivity (a measure of cardiac noradrenergic deficiency), olfactory dysfunction by the University of Pennsylvania Smell Identification Test (UPSIT), and increased deposition of alpha-synuclein (α-syn) in dermal sympathetic noradrenergic nerves by the α-syn-tyrosine hydroxylase (TH) colocalization index. This observational, cross-sectional study explored whether combinations of these biomarkers specifically identify LB forms of nOH. METHODS: Clinical laboratory data were reviewed from patients referred for evaluation at the National Institutes of Health for chronic autonomic failure between 2011 and 2023. The cutoff value for low myocardial 18F-dopamine-derived radioactivity was 6000 nCi-kg/cc-mCi, for olfactory dysfunction an UPSIT score ≤ 28, and for an increased α-syn-TH colocalization index ≥ 1.57. RESULTS: A total of 44 patients (31 LB, 13 non-LB nOH) had data for all three biomarkers. Compared to the non-LB group, the LB nOH group had low myocardial 18F-dopamine-derived radioactivity, low UPSIT scores, and high α-syn-TH colocalization indexes (p < 0.0001 each). Combining the three biomarkers completely separated the groups. Cluster analysis identified two distinct groups (p < 0.0001) independently of the clinical diagnosis, with one cluster corresponding exactly to LB nOH. CONCLUSION: LB forms of nOH feature cardiac noradrenergic deficiency, olfactory dysfunction, and increased α-syn-TH colocalization in skin biopsies. Combining the data for these variables efficiently separates LB from non-LB nOH. Independently of the clinical diagnosis, this biomarker triad identifies a pathophysiologically distinct cluster of nOH patients.

The hexosamine biosynthetic pathway rescues lysosomal dysfunction in Parkinson's disease patient iPSC derived midbrain neurons.

Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson's disease, however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5'-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson's patient midbrain cultures accumulate glucose and uridine-5'-triphosphate, while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme, GFPT2, through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate, while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological α-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity, and its failure in Parkinson's disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement.