Automated Capillary Electrophoresis Immunoblot for the Detection of Alpha-Synuclein in Mouse Tissue.

Background: Alpha-synuclein (aSyn) is a key player in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy. aSyn is expressed throughout the brain, and can also be detected in various peripheral tissues. In fact, initial symptoms of PD are non-motoric and include autonomic dysfunction, suggesting that the periphery might play an important role in early development of the disease. aSyn is expressed at relatively low levels in non-central tissues, which brings challenges for its detection and quantification in different tissues. Objective: Our goal was to assess the sensitivity of aSyn detection in central and peripheral mouse tissues through capillary electrophoresis (CE) immunoblot, considering the traditional SDS-PAGE immunoblot as the current standard. Methods: Tissues from central and non-central origin from wild type mice were extracted, and included midbrain, inner ear, and esophagus/stomach. aSyn detection was assessed through immunoblotting using Simple Western size-based CE and SDS-PAGE. Results: CE immunoblots show a consistent detection of aSyn in central and peripheral tissues. Through SDS-PAGE, immunoblots revealed a reliable signal corresponding to aSyn, particularly following membrane fixation. Conclusion: Our results suggest a reliable detection of aSyn in central and peripheral tissues using the CE Simple Western immunoblot system. These observations can serve as preliminary datasets when aiming to formally compare CE with SDS-PAGE, as well as for further characterization of aSyn using this technique.

Basic Science in Movement Disorders: Fueling the Engine of Translation into Clinical Practice.

Basic Science is crucial for the advancement of clinical care for Movement Disorders. Here, we provide brief updates on how basic science is important for understanding disease mechanisms, disease prevention, disease diagnosis, development of novel therapies and to establish the basis for personalized medicine. We conclude the viewpoint by a call to action to further improve interactions between clinician and basic scientists. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

α-Synuclein triggers cofilin pathology and dendritic spine impairment via a PrPC-CCR5 dependent pathway.

Cognitive dysfunction and dementia are critical symptoms of Lewy Body dementias (LBD). Specifically, alpha-synuclein (αSyn) accumulation in the hippocampus leading to synaptic dysfunction is linked to cognitive deficits in LBD. Here, we investigated the pathological impact of αSyn on hippocampal neurons. We report that either αSyn overexpression or αSyn pre-formed fibrils (PFFs) treatment triggers the formation of cofilin-actin rods, synapse disruptors, in cultured hippocampal neurons and in the hippocampus of synucleinopathy mouse models and of LBD patients. In vivo, cofilin pathology is present concomitantly with synaptic impairment and cognitive dysfunction. Rods generation prompted by αSyn involves the co-action of the cellular prion protein (PrPC) and the chemokine receptor 5 (CCR5). Importantly, we show that CCR5 inhibition, with a clinically relevant peptide antagonist, reverts dendritic spine impairment promoted by αSyn. Collectively, we detail the cellular and molecular mechanism through which αSyn disrupts hippocampal synaptic structure and we identify CCR5 as a novel therapeutic target to prevent synaptic impairment and cognitive dysfunction in LBD.

Gender-Specific Effects on the Cardiorespiratory System and Neurotoxicity of Intermittent and Permanent Low-Level Lead Exposures.

Lead exposure is a significant health concern, ranking among the top 10 most harmful substances for humans. There are no safe levels of lead exposure, and it affects multiple body systems, especially the cardiovascular and neurological systems, leading to problems such as hypertension, heart disease, cognitive deficits, and developmental delays, particularly in children. Gender differences are a crucial factor, with women's reproductive systems being especially vulnerable, resulting in fertility issues, pregnancy complications, miscarriages, and premature births. The globalization of lead exposure presents new challenges in managing this issue. Therefore, understanding the gender-specific implications is essential for developing effective treatments and public health strategies to mitigate the impact of lead-related health problems. This study examined the effects of intermittent and permanent lead exposure on both male and female animals, assessing behaviours like anxiety, locomotor activity, and long-term memory, as well as molecular changes related to astrogliosis. Additionally, physiological and autonomic evaluations were performed, focusing on baro- and chemoreceptor reflexes. The study's findings revealed that permanent lead exposure has more severe health consequences, including hypertension, anxiety, and reactive astrogliosis, affecting both genders. However, males exhibit greater cognitive, behavioural, and respiratory changes, while females are more susceptible to chemoreflex hypersensitivity. In contrast, intermittent lead exposure leads to hypertension and reactive astrogliosis in both genders. Still, females are more vulnerable to cognitive impairment, increased respiratory frequency, and chemoreflex hypersensitivity, while males show more reactive astrocytes in the hippocampus. Overall, this research emphasizes the importance of not only investigating different types of lead exposure but also considering gender differences in toxicity when addressing this public health concern.

Prion meeting 2023: implications of a growing field.

The history of human prion diseases began with the original description, by Hans Gerhard Creutzfeldt and by Alfons Maria Jakob, of patients with a severe brain disease that included speech abnormalities, confusion, and myoclonus, in a disease that was then named Creutzfeldt Jakob disease (CJD). Later, in Papua New Guinea, a disease characterized by trembling was identified, and given the name "Kuru". Neuropathological examination of the brains from CJD and Kuru patients, and of brains of sheep with scrapie disease revealed significant similarities and suggested a possible common mode of infection that, at the time, was thought to derive from an unknown virus that caused slow infections. John Stanley Griffith hypothesized that the agent causing these diseases was "probably a protein without nucleic acid" and, in 1982, Stanley Prusiner reported the identification of a proteinaceous infectious particle (coining the term prion) that was resistant to inactivation methods that were at the time standard for nucleic acids, and identified PrP as the major protein component of the infectious agent in scrapie and in Creutzfeldt-Jakob disease, classifying this also as a prion disease. Interestingly, the prion concept had been previously expanded to yeast proteins capable of replicating their conformation, seeding their own aggregation and transmitting phenotypic information. The prion concept has been more recently expanded to refer to misfolded proteins that are capable of converting a normal form of a protein into an abnormal form. The quest to understand and treat prion diseases has united a specific research community around the topic, and regular meetings (Prion Meetings) have taken place over the years to enable discussions, train junior researchers, and inspire research in the field.

Towards a biological diagnosis of PD.

Since the original description by James Parkinson, Parkinson's disease (PD) has intrigued us for over 200 years. PD is a progressive condition that is incurable so far, and affects millions of people worldwide. Over the years, our knowledge has expanded tremendously, and a range of criteria have been put forward and used to try to define PD. However, owing to the complexity of the problem, it is still not consensual how to diagnose and classify a disease that manifests with diverse features, and that responds differently to existing therapies and to those under development. We are now living a time when 'biological' information is becoming abundant, precise, and accessible enabling us to attempt to incorporate different sources of information to classify different forms of PD. These refinements are essential for basic science, as they will enable us to develop improved models for studying PD, and to implement new findings into clinical practice, as this will be the path towards effective personalized medicine.

Regional AT-8 reactive tau species correlate with intracellular Aß levels in cases of low AD neuropathologic change.

The amyloid cascade hypothesis states that Aß aggregates induce pathological changes in tau, leading to neurofibrillary tangles (NFTs) and cell death. A caveat with this hypothesis is the spatio-temporal divide between plaques and NFTs. This has been addressed by the inclusion of soluble Aß and tau species in the revised amyloid cascade hypothesis. Nevertheless, despite the potential for non-plaque Aß to contribute to tau pathology, few studies have examined relative correlative strengths between total Aß, plaque Aß and intracellular Aß with tau pathology within a single tissue cohort. Employing frozen and fixed frontal cortex grey and white matter tissue from non-AD controls (Con; n = 39) and Alzheimer's disease (AD) cases (n = 21), biochemical and immunohistochemical (IHC) measures of Aß and AT-8 phosphorylated tau were assessed. Biochemical native-state dot blots from crude tissue lysates demonstrated robust correlations between total Aß and AT-8 tau, when considered as a combined cohort (Con and AD) and when as Con and AD cases, separately. In contrast, no associations between Aß plaques and AT-8 were reported when using IHC measurements in either Con or AD cases. However, when intracellular Aß was measured via the Aß specific antibody MOAB-2, a correlative relationship with AT-8 tau was reported in non-AD controls but not in AD cases. Collectively the data suggests that accumulating intracellular Aß may influence AT-8 pathology, early in AD-related neuropathological change. Despite the lower levels of phospho-tau and Aß in controls, the robust correlative relationships observed suggest a physiological association of Aß production and tau phosphorylation, which may be modified during disease. This study is supportive of a revised amyloid cascade hypothesis and demonstrates regional associative relationships between tau pathology and intracellular Aß, but not extracellular Aß plaques.

Inhibition of 26S proteasome activity by α-synuclein is mediated by the proteasomal chaperone Rpn14/PAAF1.

Parkinson's disease (PD) is characterized by aggregation of α-synuclein (α-syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α-syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α-syn protein levels and the impact of α-syn on other components of the proteostasis network. Budding yeast Saccharomyces cerevisiae was used as eukaryotic reference organism to study the consequences of α-syn expression on protein dynamics. To address this, we investigated the impact of overexpression of α-syn and S129A variant on the abundance and stability of most yeast proteins using a genome-wide yeast library and a tandem fluorescent protein timer (tFT) reporter as a measure for protein stability. This revealed that the stability of in total 377 cellular proteins was altered by α-syn expression, and that the impact on protein stability was significantly enhanced by phosphorylation at Ser129 (pS129). The proteasome assembly chaperone Rpn14 was identified as one of the top candidates for increased protein stability by expression of pS129 α-syn. Elevated levels of Rpn14 enhanced the growth inhibition by α-syn and the accumulation of ubiquitin conjugates in the cell. We found that Rpn14 interacts physically with α-syn and stabilizes pS129 α-syn. The expression of α-syn along with elevated levels of Rpn14 or its human counterpart PAAF1 reduced the proteasome activity in yeast and in human cells, supporting that pS129 α-syn negatively affects the 26S proteasome through Rpn14. This comprehensive study into the alternations of protein homeostasis highlights the critical role of the Rpn14/PAAF1 in α-syn-mediated proteasome dysfunction.

Substitution of Met-38 to Ile in γ-synuclein found in two patients with amyotrophic lateral sclerosis induces aggregation into amyloid.

α-, ß-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson's disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member.

A biological classification of Parkinson's disease: the SynNeurGe research diagnostic criteria.

With the hope that disease-modifying treatments could target the molecular basis of Parkinson's disease, even before the onset of symptoms, we propose a biologically based classification. Our classification acknowledges the complexity and heterogeneity of the disease by use of a three-component system (SynNeurGe): presence or absence of pathological α-synuclein (S) in tissues or CSF; evidence of underlying neurodegeneration (N) defined by neuroimaging procedures; and documentation of pathogenic gene variants (G) that cause or strongly predispose to Parkinson's disease. These three components are linked to a clinical component (C), defined either by a single high-specificity clinical feature or by multiple lower-specificity clinical features. The use of a biological classification will enable advances in both basic and clinical research, and move the field closer to the precision medicine required to develop disease-modifying therapies. We emphasise the initial application of these criteria exclusively for research. We acknowledge its ethical implications, its limitations, and the need for prospective validation in future studies.

Uncovering potential diagnostic and pathophysiological roles of α-synuclein and DJ-1 in melanoma.

BACKGROUND: Melanoma, the most lethal skin cancer type, occurs more frequently in Parkinson's disease (PD), and PD is more frequent in melanoma patients, suggesting disease mechanisms overlap. α-synuclein, a protein that accumulates in PD brain, and the oncogene DJ-1, which is associated with PD autosomal recessive forms, are both elevated in melanoma cells. Whether this indicates melanoma progression or constitutes a protective response remains unclear. We hereby investigated the molecular mechanisms through which α-synuclein and DJ-1 interact, suggesting novel biomarkers and targets in melanoma. METHODS: The Cancer Genome Atlas (TCGA) expression profiles derived from UCSC Xena were used to obtain α-synuclein and DJ-1 expression and correlated with survival in skin cutaneous melanoma (SKCM). Immunohistochemistry determined the expression in metastatic melanoma lymph nodes. Protein-protein interactions (PPIs) and molecular docking assessed protein binding and affinity with chemotherapeutic drugs. Further validation was performed using in vitro cellular models and ELISA immunoassays. RESULTS: α-synuclein and DJ-1 were upregulated in primary and metastatic SKCM. Aggregated α-synuclein was selectively detected in metastatic melanoma lymph nodes. α-synuclein overexpression in SK-MEL-28 cells induced the expression of DJ-1, supporting PPI and a positive correlation in melanoma patients. Molecular docking revealed a stable protein complex, with differential binding to chemotherapy drugs such as temozolomide, dacarbazine, and doxorubicin. Parallel reduction of both proteins in temozolomide-treated SK-MEL-28 spheroids suggests drug binding may affect protein interaction and/or stability. CONCLUSION: α-synuclein, together with DJ-1, may play a role in melanoma progression and chemosensitivity, constituting novel targets for therapeutic intervention, and possible biomarkers for melanoma.

Glycation modulates superoxide dismutase 1 aggregation and toxicity in models of sporadic amyotrophic lateral sclerosis.

Different SOD1 proteoforms are implicated## in both familial and sporadic cases of Amyotrophic Lateral Sclerosis (ALS), an aging-associated disease that affects motor neurons. SOD1 is crucial to neuronal metabolism and health, regulating the oxidative stress response and the shift between oxidative-fermentative metabolism, which is important for astrocyte-neuron metabolic cooperation. Neurons have a limited capacity to metabolize methylglyoxal (MGO), a potentially toxic side product of glycolysis. MGO is highly reactive and can readily posttranslationally modify proteins, in a reaction known as glycation, impacting their normal biology. Here, we aimed to investigate the effect of glycation on the aggregation and toxicity of human SOD1WT (hSOD1WT). Cells with deficiency in MGO metabolism showed increased levels of hSOD1WT inclusions, displaying also reduced hSOD1WT activity and viability. Strikingly, we also found that the presence of hSOD1WT in stress granules increased upon MGO treatment. The treatment of recombinant hSOD1WT with MGO resulted in the formation of SDS-stable oligomers, specially trimers, and thioflavin-T positive aggregates, which can promote cell toxicity and TDP-43 pathology. Together, our results suggest that glycation may play a still underappreciated role on hSOD1WT and TDP-43 pathologies in sporadic ALS, which could open novel perspectives for therapeutic intervention.

Extracellular Vesicles for the Diagnosis of Parkinson's Disease: Systematic Review and Meta-Analysis.

Parkinson's disease (PD) biomarkers are needed by both clinicians and researchers (for diagnosis, identifying study populations, and monitoring therapeutic response). Imaging, genetic, and biochemical biomarkers have been widely studied. In recent years, extracellular vesicles (EVs) have become a promising material for biomarker development. Proteins and molecular material from any organ, including the central nervous system, can be packed into EVs and transported to the periphery into easily obtainable biological specimens like blood, urine, and saliva. We performed a systematic review and meta-analysis of articles (published before November 15, 2022) reporting biomarker assessment in EVs in PD patients and healthy controls (HCs). Biomarkers were analyzed using random effects meta-analysis and the calculated standardized mean difference (Std.MD). Several proteins and ribonucleic acids have been identified in EVs in PD patients, but only α-synuclein (aSyn) and leucine-rich repeat kinase 2 (LRRK2) were reported in sufficient studies (n = 24 and 6, respectively) to perform a meta-analysis. EV aSyn was significantly increased in neuronal L1 cell adhesion molecule (L1CAM)-positive blood EVs in PD patients compared to HCs (Std.MD = 1.84, 95% confidence interval = 0.76-2.93, P = 0.0009). Further analysis of the biological sample and EV isolation method indicated that L1CAM-IP (immunoprecipitation) directly from plasma was the best isolation method for assessing aSyn in PD patients. Upcoming neuroprotective clinical trials immediately need peripheral biomarkers for identifying individuals at risk of developing PD. Overall, the improved sensitivity of assays means they can identify biomarkers in blood that reflect changes in the brain. CNS-derived EVs in blood will likely play a major role in biomarker development in the coming years. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Proteomic analysis of the human hippocampus identifies neuronal pentraxin 1 (NPTX1) as synapto-axonal target in late-stage Parkinson's disease.

Parkinson's disease (PD) affects a significant proportion of the population over the age of 60 years, and its prevalence is increasing. While symptomatic treatment is available for motor symptoms of PD, non-motor complications such as dementia result in diminished life quality for patients and are far more difficult to treat. In this study, we analyzed PD-associated alterations in the hippocampus of PD patients, since this brain region is strongly affected by PD dementia. We focused on synapses, analyzing the proteome of post-mortal hippocampal tissue from 16 PD cases and 14 control subjects by mass spectrometry. Whole tissue lysates and synaptosomal fractions were analyzed in parallel. Differential analysis combined with bioinformatic network analyses identified neuronal pentraxin 1 (NPTX1) to be significantly dysregulated in PD and interacting with proteins of the synaptic compartment. Modulation of NPTX1 protein levels in primary hippocampal neuron cultures validated its role in synapse morphology. Our analysis suggests that NPTX1 contributes to synaptic pathology in late-stage PD and represents a putative target for novel therapeutic strategies.

Targeting Biomolecular Condensation and Protein Aggregation against Cancer.

Biomolecular condensates, membrane-less entities arising from liquid-liquid phase separation, hold dichotomous roles in health and disease. Alongside their physiological functions, these condensates can transition to a solid phase, producing amyloid-like structures implicated in degenerative diseases and cancer. This review thoroughly examines the dual nature of biomolecular condensates, spotlighting their role in cancer, particularly concerning the p53 tumor suppressor. Given that over half of the malignant tumors possess mutations in the TP53 gene, this topic carries profound implications for future cancer treatment strategies. Notably, p53 not only misfolds but also forms biomolecular condensates and aggregates analogous to other protein-based amyloids, thus significantly influencing cancer progression through loss-of-function, negative dominance, and gain-of-function pathways. The exact molecular mechanisms underpinning the gain-of-function in mutant p53 remain elusive. However, cofactors like nucleic acids and glycosaminoglycans are known to be critical players in this intersection between diseases. Importantly, we reveal that molecules capable of inhibiting mutant p53 aggregation can curtail tumor proliferation and migration. Hence, targeting phase transitions to solid-like amorphous and amyloid-like states of mutant p53 offers a promising direction for innovative cancer diagnostics and therapeutics.

Defining the Riddle in Order to Solve It: There Is More Than One "Parkinson's Disease".

BACKGROUND: More than 200 years after James Parkinsondescribed a clinical syndrome based on his astute observations, Parkinson's disease (PD) has evolved into a complex entity, akin to the heterogeneity of other complex human syndromes of the central nervous system such as dementia, motor neuron disease, multiple sclerosis, and epilepsy. Clinicians, pathologists, and basic science researchers evolved arrange of concepts andcriteria for the clinical, genetic, mechanistic, and neuropathological characterization of what, in their best judgment, constitutes PD. However, these specialists have generated and used criteria that are not necessarily aligned between their different operational definitions, which may hinder progress in solving the riddle of the distinct forms of PD and ultimately how to treat them. OBJECTIVE: This task force has identified current in consistencies between the definitions of PD and its diverse variants in different domains: clinical criteria, neuropathological classification, genetic subtyping, biomarker signatures, and mechanisms of disease. This initial effort for "defining the riddle" will lay the foundation for future attempts to better define the range of PD and its variants, as has been done and implemented for other heterogeneous neurological syndromes, such as stroke and peripheral neuropathy. We strongly advocate for a more systematic and evidence-based integration of our diverse disciplines by looking at well-defined variants of the syndrome of PD. CONCLUSION: Accuracy in defining endophenotypes of "typical PD" across these different but interrelated disciplines will enable better definition of variants and their stratification in therapeutic trials, a prerequisite for breakthroughs in the era of precision medicine. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Intermittent Lead Exposure Induces Behavioral and Cardiovascular Alterations Associated with Neuroinflammation.

The nervous system is the primary target for lead exposure and the developing brain appears to be especially susceptible, namely the hippocampus. The mechanisms of lead neurotoxicity remain unclear, but microgliosis and astrogliosis are potential candidates, leading to an inflammatory cascade and interrupting the pathways involved in hippocampal functions. Moreover, these molecular changes can be impactful as they may contribute to the pathophysiology of behavioral deficits and cardiovascular complications observed in chronic lead exposure. Nevertheless, the health effects and the underlying influence mechanism of intermittent lead exposure in the nervous and cardiovascular systems are still vague. Thus, we used a rat model of intermittent lead exposure to determine the systemic effects of lead and on microglial and astroglial activation in the hippocampal dentate gyrus throughout time. In this study, the intermittent group was exposed to lead from the fetal period until 12 weeks of age, no exposure (tap water) until 20 weeks, and a second exposure from 20 to 28 weeks of age. A control group (without lead exposure) matched in age and sex was used. At 12, 20 and 28 weeks of age, both groups were submitted to a physiological and behavioral evaluation. Behavioral tests were performed for the assessment of anxiety-like behavior and locomotor activity (open-field test), and memory (novel object recognition test). In the physiological evaluation, in an acute experiment, blood pressure, electrocardiogram, and heart and respiratory rates were recorded, and autonomic reflexes were evaluated. The expression of GFAP, Iba-1, NeuN and Synaptophysin in the hippocampal dentate gyrus was assessed. Intermittent lead exposure induced microgliosis and astrogliosis in the hippocampus of rats and changes in behavioral and cardiovascular function. We identified increases in GFAP and Iba1 markers together with presynaptic dysfunction in the hippocampus, concomitant with behavioral changes. This type of exposure produced significant long-term memory dysfunction. Regarding physiological changes, hypertension, tachypnea, baroreceptor reflex impairment and increased chemoreceptor reflex sensitivity were observed. In conclusion, the present study demonstrated the potential of lead intermittent exposure inducing reactive astrogliosis and microgliosis, along with a presynaptic loss that was accompanied by alterations of homeostatic mechanisms. This suggests that chronic neuroinflammation promoted by intermittent lead exposure since fetal period may increase the susceptibility to adverse events in individuals with pre-existing cardiovascular disease and/or in the elderly.