Increased burden of rare risk variants across gene expression networks predisposes to sporadic Parkinson's disease.

Alpha-synuclein (αSyn) is an intrinsically disordered protein that accumulates in the brains of patients with Parkinson's disease and forms intraneuronal inclusions called Lewy Bodies. While the mechanism underlying the dysregulation of αSyn in Parkinson's disease is unclear, it is thought that prionoid cell-to-cell propagation of αSyn has an important role. Through a high throughput screen, we recently identified 38 genes whose knock down modulates αSyn propagation. Follow up experiments were undertaken for two of those genes, TAX1BP1 and ADAMTS19, to study the mechanism with which they regulate αSyn homeostasis. We used a recently developed M17D neuroblastoma cell line expressing triple mutant (E35K+E46K+E61K) "3K" αSyn under doxycycline induction. 3K αSyn spontaneously forms inclusions that show ultrastructural similarities to Lewy Bodies. Experiments using that cell line showed that TAX1BP1 and ADAMTS19 regulate how αSyn interacts with lipids and phase separates into inclusions, respectively, adding to the growing body of evidence implicating those processes in Parkinson's disease. Through RNA sequencing, we identified several genes that are differentially expressed after knock-down of TAX1BP1 or ADAMTS19. Burden analysis revealed that those differentially expressed genes (DEGs) carry an increased frequency of rare risk variants in Parkinson's disease patients versus healthy controls, an effect that was independently replicated across two separate cohorts (GP2 and AMP-PD). Weighted gene co-expression network analysis (WGCNA) showed that the DEGs cluster within modules in regions of the brain that develop high degrees of αSyn pathology (basal ganglia, cortex). We propose a novel model for the genetic architecture of sporadic Parkinson's disease: increased burden of risk variants across genetic networks dysregulates pathways underlying αSyn homeostasis, thereby leading to pathology and neurodegeneration.

Effects of antimicrobial exposure on the risk of Parkinson's disease.

BACKGROUND: We aimed to assess how antimicrobial exposure affects Parkinson's disease (PD) risk. METHODS: A nested case-control study was performed to examine the association between antimicrobial exposure and newly diagnosed PD using the Clinical Practice Research Datalink (CPRD). Each PD case was matched by age, sex, and year of diagnosis (index date) to up to 15 controls. Number of prescribed antimicrobial courses was assessed 1-5, 6-10, and 11-15 years prior to the index date. Logistic regression with generalized estimating equations (GEE) was used to estimate odds ratios (ORs) and false discovery rate-adjusted p-values between antimicrobial exposure and risk of PD. RESULTS: We compared 12,557 PD cases with 80,804 matched controls. We found an inverse dose-response relationship between number of penicillin courses and PD risk across multiple time periods (5+ courses, 1-5 years prior: OR 0.85, 95 % CI 0.76-0.95, p = 0.043; 6-10 years prior: OR 0.84, 95 % CI: 0.73-0.95, p = 0.059; 11-15 years prior: OR 0.87, 95 % CI 0.74-1.02, p = 0.291). The number of macrolide courses was inversely but not significantly associated with PD risk 1-5 years prior to the index date (OR 0.89-0.91, 95 % CI: 0.79-0.99, adjusted p = 0.140-0.167). Exposure to ≥2 courses of antifungals 1-5 years prior was associated with an increased risk of PD (OR 1.16, 95 % CI: 1.06-1.27, p = 0.020). CONCLUSIONS: In a large UK-representative population, the risk of PD was modestly lower among adults who had previously received multiple courses of penicillins in the last 15 years and modestly higher among those exposed to antifungal medicines in recent years.

Post-translational Modifications of α-Synuclein, their Therapeutic Potential and Crosstalk in Health and Neurodegenerative Diseases.

α-Synuclein (α-Syn) aggregation in Lewy bodies and Lewy neurites has emerged as a key pathogenetic feature in Parkinson's disease (PD), Dementia with Lewy Bodies and Multiple System Atrophy. Various factors, including post-translational modifications (PTMs), can influence the propensity of α-Syn to misfold and aggregate. PTMs are biochemical modifications of a protein that occur during or after translation and are typically mediated by enzymes. PTMs modulate several characteristics of proteins including their structure, activity, localization, and stability. α-Syn undergoes various post-translational modifications, including phosphorylation, ubiquitination, SUMOylation, acetylation, glycation, O-GlcNAcylation, nitration, oxidation, polyamination, arginylation, and truncation. Different PTMs of a protein can physically interact with one another or work together to influence a particular physiological or pathological feature in a process known as PTMs crosstalk. The development of detection techniques for the co-occurrence of PTMs in recent years has uncovered previously unappreciated mechanisms of their crosstalk. This has led to the emergence of evidence supporting an association between α-Syn PTMs crosstalk and synucleinopathies. In this review, we provide a comprehensive evaluation of α-Syn PTMs, their impact on misfolding and pathogenicity, the pharmacological means of targeting them, and their potential as biomarkers of disease. We also highlight the importance of the crosstalk between these PTMs in α-Syn function and aggregation. Insight into these PTMS and the complexities of their crosstalk can improve our understanding of the pathogenesis of synucleinopathies and identify novel targets of therapeutic potential. Significance Statement α-Synuclein as a key pathogenic protein in Parkinson's disease and other synucleinopathies, making it a leading therapeutic target for disease modification. Multiple post-translational modifications occur at various sites in α-Synuclein and alter its biophysical and pathological properties, some interacting with one another to add to the complexity of the pathogenicity of this protein. This review details these modifications, their implications in disease and potential therapeutic opportunities.

Pathogenetic Contributions and Therapeutic Implications of Transglutaminase 2 in Neurodegenerative Diseases.

Neurodegenerative diseases encompass a heterogeneous group of disorders that afflict millions of people worldwide. Characteristic protein aggregates are histopathological hallmark features of these disorders, including Amyloid ß (Aß)-containing plaques and tau-containing neurofibrillary tangles in Alzheimer's disease, α-Synuclein (α-Syn)-containing Lewy bodies and Lewy neurites in Parkinson's disease and dementia with Lewy bodies, and mutant huntingtin (mHTT) in nuclear inclusions in Huntington's disease. These various aggregates are found in specific brain regions that are impacted by neurodegeneration and associated with clinical manifestations. Transglutaminase (TG2) (also known as tissue transglutaminase) is the most ubiquitously expressed member of the transglutaminase family with protein crosslinking activity. To date, Aß, tau, α-Syn, and mHTT have been determined to be substrates of TG2, leading to their aggregation and implicating the involvement of TG2 in several pathophysiological events in neurodegenerative disorders. In this review, we summarize the biochemistry and physiologic functions of TG2 and describe recent advances in the pathogenetic role of TG2 in these diseases. We also review TG2 inhibitors tested in clinical trials and discuss recent TG2-targeting approaches, which offer new perspectives for the design of future highly potent and selective drugs with improved brain delivery as a disease-modifying treatment for neurodegenerative disorders.

NIH Toolbox performance of persons with Parkinson's disease according to GBA1 and STN-DBS status.

OBJECTIVE: Mutations in the glucocerebrosidase (GBA1) gene and subthalamic nucleus deep brain stimulation (STN-DBS) are independently associated with cognitive dysfunction in persons with Parkinson's disease (PwP). We hypothesized that PwP with both GBA1 mutations and STN-DBS are at greater risk of cognitive dysfunction than PwP with only GBA1 mutations or STN-DBS, or neither. In this study, we determined the pattern of cognitive dysfunction in PwP based on GBA1 mutation status and STN-DBS treatment. METHODS: PwP who are GBA1 mutation carriers with or without DBS (GBA1+DBS+, GBA1+DBS-), and noncarriers with or without DBS (GBA1-DBS+, GBA1-DBS-) were included. Using the NIH Toolbox, cross-sectional differences in response inhibition, processing speed, and episodic memory were compared using analysis of variance with adjustment for relevant covariates. RESULTS: Data were available for 9 GBA1+DBS+, 14 GBA1+DBS-, 17 GBA1-DBS+, and 26 GBA1-DBS- PwP. In this cross-sectional study, after adjusting for covariates, we found that performance on the Flanker test (measure of response inhibition) was lower in GBA1+DBS+ PwP compared with GBA1-DBS+ PwP (P = 0.030). INTERPRETATION: PwP who carry GBA1 mutations and have STN-DBS have greater impaired response inhibition compared with PwP with STN-DBS but without GBA1 mutations. Longitudinal data, including preoperative scores, are required to definitively determine whether GBA1 mutation carriers respond differently to STN-DBS, particularly in the domain of response inhibition.

Decreasing the intrinsically disordered protein α-synuclein levels by targeting its structured mRNA with a ribonuclease-targeting chimera.

α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

Experimental Neurotherapeutics: Surfing the Tidal Wave of New Opportunities.

The subspecialty of experimental neurotherapeutics trains neurologists in discovering and developing new treatments for neurologic diseases. Based on development of exciting new treatments for genetic and inflammatory diseases, we predict that there will be many other breakthroughs. The job market has expanded rapidly in academia, the pharmaceutical industry, government, and not-for-profit sectors; many new opportunities can be anticipated. The burgeoning opportunities in the field mandate that training address the challenges of overcoming obstacles in therapeutic discovery, implementation science, and development of affordable and equitably available treatments. ANN NEUROL 2023;93:427-430.

Tumor suppressor PALB2 maintains redox and mitochondrial homeostasis in the brain and cooperates with ATG7/autophagy to suppress neurodegeneration.

The PALB2 tumor suppressor plays key roles in DNA repair and has been implicated in redox homeostasis. Autophagy maintains mitochondrial quality, mitigates oxidative stress and suppresses neurodegeneration. Here we show that Palb2 deletion in the mouse brain leads to mild motor deficits and that co-deletion of Palb2 with the essential autophagy gene Atg7 accelerates and exacerbates neurodegeneration induced by ATG7 loss. Palb2 deletion leads to elevated DNA damage, oxidative stress and mitochondrial markers, especially in Purkinje cells, and co-deletion of Palb2 and Atg7 results in accelerated Purkinje cell loss. Further analyses suggest that the accelerated Purkinje cell loss and severe neurodegeneration in the double deletion mice are due to excessive oxidative stress and mitochondrial dysfunction, rather than DNA damage, and partially dependent on p53 activity. Our studies uncover a role of PALB2 in mitochondrial homeostasis and a cooperation between PALB2 and ATG7/autophagy in maintaining redox and mitochondrial homeostasis essential for neuronal survival.

Parkinson Disease and Subthalamic Nucleus Deep Brain Stimulation: Cognitive Effects in GBA Mutation Carriers.

OBJECTIVE: This study was undertaken to compare the rate of change in cognition between glucocerebrosidase (GBA) mutation carriers and noncarriers with and without subthalamic nucleus deep brain stimulation (STN-DBS) in Parkinson disease. METHODS: Clinical and genetic data from 12 datasets were examined. Global cognition was assessed using the Mattis Dementia Rating Scale (MDRS). Subjects were examined for mutations in GBA and categorized as GBA carriers with or without DBS (GBA+DBS+, GBA+DBS-), and noncarriers with or without DBS (GBA-DBS+, GBA-DBS-). GBA mutation carriers were subcategorized according to mutation severity (risk variant, mild, severe). Linear mixed modeling was used to compare rate of change in MDRS scores over time among the groups according to GBA and DBS status and then according to GBA severity and DBS status. RESULTS: Data were available for 366 subjects (58 GBA+DBS+, 82 GBA+DBS-, 98 GBA-DBS+, and 128 GBA-DBS- subjects), who were longitudinally followed (range = 36-60 months after surgery). Using the MDRS, GBA+DBS+ subjects declined on average 2.02 points/yr more than GBA-DBS- subjects (95% confidence interval [CI] = -2.35 to -1.69), 1.71 points/yr more than GBA+DBS- subjects (95% CI = -2.14 to -1.28), and 1.49 points/yr more than GBA-DBS+ subjects (95% CI = -1.80 to -1.18). INTERPRETATION: Although not randomized, this composite analysis suggests that the combined effects of GBA mutations and STN-DBS negatively impact cognition. We advise that DBS candidates be screened for GBA mutations as part of the presurgical decision-making process. We advise that GBA mutation carriers be counseled regarding potential risks associated with STN-DBS so that alternative options may be considered. ANN NEUROL 2022;91:424-435.

Therapeutics in the Pipeline Targeting α-Synuclein for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and the fastest growing neurologic disease in the world, yet no disease-modifying therapy is available for this disabling condition. Multiple lines of evidence implicate the protein α-synuclein (α-Syn) in the pathogenesis of PD, and as such, there is intense interest in targeting α-Syn for potential disease modification. α-Syn is also a key pathogenic protein in other synucleionpathies, most commonly dementia with Lewy bodies. Thus, therapeutics targeting this protein will have utility in these disorders as well. Here we discuss the various approaches that are being investigated to prevent and mitigate α-Syn toxicity in PD, including clearing its pathologic aggregates from the brain using immunization strategies, inhibiting its misfolding and aggregation, reducing its expression level, enhancing cellular clearance mechanisms, preventing its cell-to-cell transmission within the brain and perhaps from the periphery, and targeting other proteins associated with or implicated in PD that contribute to α-Syn toxicity. We also discuss the therapeutics in the pipeline that harness these strategies. Finally, we discuss the challenges and opportunities for the field in the discovery and development of therapeutics for disease modification in PD. SIGNIFICANCE STATEMENT: PD is the second most common neurodegenerative disorder, for which disease-modifying therapies remain a major unmet need. A large body of evidence points to α-synuclein as a key pathogenic protein in this disease as well as in dementia with Lewy bodies, making it of leading therapeutic interest. This review discusses the various approaches being investigated and progress made to date toward discovering and developing therapeutics that would slow and stop progression of these disabling diseases.

Silica-coated magnetic-nanoparticle-induced cytotoxicity is reduced in microglia by glutathione and citrate identified using integrated omics.

BACKGROUND: Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue. METHODS: Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network. RESULTS: Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes (from 41,214 identified genes), and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus. CONCLUSIONS: Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity.

MicroRNA-7 Protects Against Neurodegeneration Induced by α-Synuclein Preformed Fibrils in the Mouse Brain.

α-Synuclein is a key protein in the pathogenesis of Parkinson's disease as it accumulates in fibrillar form in affected brain regions. Misfolded α-synuclein seeds recruit monomeric α-synuclein to form aggregates, which can spread to anatomically connected brain regions, a phenomenon that correlates with clinical disease progression. Thus, downregulating α-synuclein levels could reduce seeding and inhibit aggregate formation and propagation. We previously reported that microRNA-7 (miR-7) protects neuronal cells by downregulating α-synuclein expression through its effect on the 3'-untranslated region of SNCA mRNA; however, whether miR-7 blocks α-synuclein seeding and propagation in vivo remains unknown. Here, we induced miR-7 overexpression in the mouse striatum unilaterally by infusing adeno-associated virus 1 (AAV-miR-7) followed by inoculation with recombinant α-synuclein preformed fibrils (PFF) a month later. Compared with control mice injected with non-targeting AAV-miR-NT followed by PFF, AAV-miR-7 pre-injected mice exhibited lower levels of monomeric and high-molecular-weight α-synuclein species in the striatum, and reduced amount of phosphorylated α-synuclein in the striatum and in nigral dopamine neurons. Accordingly, AAV-miR-7-injected mice had less pronounced degeneration of the nigrostriatal pathway and better behavioral performance. The neuroinflammatory reaction to α-synuclein PFF inoculation was also significantly attenuated. These data suggest that miR-7 inhibits the formation and propagation of pathological α-synuclein and protects against neurodegeneration induced by PFF. Collectively, these findings support the potential of miR-7 as a disease modifying biologic agent for Parkinson's disease and related α-synucleinopathies.

Striatal ΔFosB gene suppression inhibits the development of abnormal involuntary movements induced by L-Dopa in rats.

L-Dopa-induced dyskinesia (LID) is associated with the upregulation of striatal ∆FosB in animal models and patients with Parkinson's disease (PD). A mechanistic role of ∆FosB is suspected because its transgenic overexpression leads to the early appearance of LID in rodents and primates. This study in rodents is aimed at exploring the therapeutic potential of striatal ∆FosB gene suppression to control LID in patients with PD. To determine the effect of reducing striatal ∆FosB expression, we used RNAi gene knockdown in a rat model of PD and assessed abnormal involuntary movements (AIMs) in response to L-Dopa. Rats with dopamine depletion received striatal injections of rAAV-∆FosB shRNA or a control virus before exposure to chronic L-Dopa treatment. The development of AIMs during the entire L-Dopa treatment period was markedly inhibited by ∆FosB gene knockdown and its associated molecular changes. The antiparkinsonian action of L-Dopa was unchanged by ∆FosB gene knockdown. These results suggest a major role for ∆FosB in the development of LID and support exploring strategies to reduce striatal ∆FosB levels in patients with PD.

Metabolome Changes in Cerebral Ischemia.

Cerebral ischemia is caused by perturbations in blood flow to the brain that trigger sequential and complex metabolic and cellular pathologies. This leads to brain tissue damage, including neuronal cell death and cerebral infarction, manifesting clinically as ischemic stroke, which is the cause of considerable morbidity and mortality worldwide. To analyze the underlying biological mechanisms and identify potential biomarkers of ischemic stroke, various in vitro and in vivo experimental models have been established investigating different molecular aspects, such as genes, microRNAs, and proteins. Yet, the metabolic and cellular pathologies of ischemic brain injury remain not fully elucidated, and the relationships among various pathological mechanisms are difficult to establish due to the heterogeneity and complexity of the disease. Metabolome-based techniques can provide clues about the cellular pathologic status of a condition as metabolic disturbances can represent an endpoint in biological phenomena. A number of investigations have analyzed metabolic changes in samples from cerebral ischemia patients and from various in vivo and in vitro models. We previously analyzed levels of amino acids and organic acids, as well as polyamine distribution in an in vivo rat model, and identified relationships between metabolic changes and cellular functions through bioinformatics tools. This review focuses on the metabolic and cellular changes in cerebral ischemia that offer a deeper understanding of the pathology underlying ischemic strokes and contribute to the development of new diagnostic and therapeutic approaches.

Transglutaminase 2 Depletion Attenuates α-Synuclein Mediated Toxicity in Mice.

α-Synuclein (α-Syn) is a key pathogenic protein in α-synucleinopathies including Parkinson disease (PD) and Dementia with Lewy Bodies. The aggregation of α-Syn is believed to be deleterious and a critical step leading to neuronal dysfunction and death. One of the factors that may contribute to the initial steps of this aggregation is crosslinking through transglutaminase 2 (TG2). We previously demonstrated that overexpression of TG2 exacerbates α-Syn toxicity in mice and yeast by increasing the higher-order species of α-Syn. Herein, we investigated whether deletion of the TG2 encoding gene could mitigate the toxicity of α-Syn in vivo. Compared with α-Syn transgenic (SynTg) mice, TG2 null /α-Syn transgenic mice (TG2KO/SynTg) exhibited a reduced amount of phosphorylated α-Syn aggregates and fewer proteinase K-resistant α-Syn aggregates in sections of brain tissue. Neuritic processes that are depleted in SynTg mice compared to wild-type mice were preserved in double TG2KO/SynTg mice. Additionally, the neuroinflammatory reaction to α-Syn was attenuated in TG2KO/SynTg animals. These neuropathological markers of diminished α-Syn toxicity in the absence of TG2 were associated with better motor performance on the rotarod and balance beam. These results suggest that deleting TG2 reduces the toxicity of α-Syn in vivo and improves the behavioral performance of SynTg mice. Accordingly, these findings collectively support pharmacological inhibition of TG2 as a potential disease modifying therapeutic strategy for α-synucleinopathies.