Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson's disease pathogenesis.

Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.

Epidemiology of Parkinson's Disease in Rural Gujarat, India.

INTRODUCTION: In India, there have been only few published studies of Parkinson's disease (PD) showing a wide range of prevalence. We conducted this study to determine the prevalence of PD in the rural population of Gujarat, in the western region of India. METHODS: This cross-sectional descriptive study was conducted in the villages of Anand, a district of Gujarat, India, between September 2019 and February 2020. This study used a multistep approach including a screening questionnaire and video recording followed by clinical examination by a neurologist, laboratory evaluation, and brain imaging to evaluate patients with PD. RESULTS: A total population of 18,896 was screened. The overall crude prevalence of PD was 42.3 per 100,000, and the prevalence over the age of 60 was 308.9 per 100,000 which showed the trend of increasing disease prevalence with age. Their mean duration of illness was 39.3 ± 27.3 months, and more than half of patients with PD had multiple associated nonmotor symptoms and nearly one-third had comorbid anxiety or depression. Environmental factors are important in the pathogenesis of PD, but there was no clear association between patients with PD and certain variables including consumption of well water, exposure to pesticides or other toxins, smoking cigarettes, and drinking alcohol or coffee in our study. CONCLUSIONS: The present study showed the current epidemiological data of PD from Gujarat, in western India. Further studies across different regions in India need to be encouraged for better understanding of PD prevalence in the Indian population.

Dorsal vagal nucleus involvement relates to QTc-prolongation after acute medullary infarction.

BACKGROUND: Infarction of the medulla has been associated with prolongation of the QTc, severe arrhythmia, and sudden cardiac death, yet the precise anatomical substrate remains uncertain. AIMS: We sought to determine the possible anatomical structures relating to QTc-prolongation in patients with acute medullary infarction. METHODS: We included 12 subjects with acute ischemic medullary infarction on brain MRI, who presented within 4.5 h from the last known well time, with a 90-day follow-up. For an unbiased lesion analysis, medullary infarcts were manually outlined on diffusion weighted MRI and co-registered with an anatomical atlas. RESULTS: Nine out of 12 had QTc-prolongation. Qualitative and semi-quantitative comparisons were made between infarct location and QTc-prolongation. Among patients with QTc-prolongation, the greatest degree of congruence of the infarct location was over the dorsal vagal nucleus (DVN, 8 out of 9). There was a significant correlation between the number of sections showing infarction of the DVN and presence of QTc-prolongation (r = .582, p = .047). Among patients without QTc-prolongation, the maximum lesion overlap included the medial aspect of the gigantocelluar reticular nucleus of the reticular formation. CONCLUSION: We found that the DVN is a key anatomical substrate related to QTc-prolongation. Further studies with more patients and high-resolution, volumetric MRI are needed to confirm our findings.

Characterization of a Parkinson's disease rat model using an upgraded paraquat exposure paradigm.

Animal models of human diseases are crucial experimental tools to investigate the mechanisms involved in disease pathogenesis and to develop new therapies. In spite of the numerous animal models currently available that reproduce several neuropathological features of Parkinson disease (PD), it is challenging to have one that consistently recapitulates human PD conditions in both motor behaviors and biochemical pathological outcomes. Given that, we have implemented a new paradigm to expose rats to a chronic low dose of paraquat (PQ), using osmotic minipumps and characterized the developed pathologic features over time. The PQ exposure paradigm used lead to a rodent model of PD depicting progressive nigrostriatal dopaminergic neurodegeneration, characterized by a 41% significant loss of dopaminergic neuron in the substantia nigra pars compacta (SNpc), a significant decrease of 18% and 40% of dopamine levels in striatum at week 5 and 8, respectively, and a significant 1.5-fold decrease in motor performance. We observed a significant increase of microglia activation state, sustained levels of α-synucleinopathy and increased oxidative stress markers in the SNpc. In summary, this is an explorative study that allowed to characterize an improved PQ-based rat model that recapitulates cardinal features of PD and may represent an attractive tool to investigate several mechanisms underlying the various aspects of PD pathogenesis as well as for the validation of the efficacy of new therapeutic approaches that targets different mechanisms involved in PD neurodegeneration.

Endogenous Alpha-Synuclein Protein Analysis from Human Brain Tissues Using Single-Molecule Pull-Down Assay.

Alpha-synuclein (α-SYN) is a central molecule in Parkinson's disease pathogenesis. Despite several studies, the molecular nature of endogenous α-SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of biospecimens. Here, we introduce α-SYN single-molecule pull-down (α-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual α-SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. This powerful single-molecule assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation.

BACKGROUND: Histamine is an amine widely known as a peripheral inflammatory mediator and as a neurotransmitter in the central nervous system. Recently, it has been suggested that histamine acts as an innate modulator of microglial activity. Herein, we aimed to disclose the role of histamine in microglial phagocytic activity and reactive oxygen species (ROS) production and to explore the consequences of histamine-induced neuroinflammation in dopaminergic (DA) neuronal survival. METHODS: The effect of histamine on phagocytosis was assessed both in vitro by using a murine N9 microglial cell line and primary microglial cell cultures and in vivo. Cells were exposed to IgG-opsonized latex beads or phosphatidylserine (PS) liposomes to evaluate Fcγ or PS receptor-mediated microglial phagocytosis, respectively. ROS production and protein levels of NADPH oxidases and Rac1 were assessed as a measure of oxidative stress. DA neuronal survival was evaluated in vivo by counting the number of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) of mice. RESULTS: We found that histamine triggers microglial phagocytosis via histamine receptor 1 (H1R) activation and ROS production via H1R and H4R activation. By using apocynin, a broad NADPH oxidase (Nox) inhibitor, and Nox1 knockout mice, we found that the Nox1 signaling pathway is involved in both phagocytosis and ROS production induced by histamine in vitro. Interestingly, both apocynin and annexin V (used as inhibitor of PS-induced phagocytosis) fully abolished the DA neurotoxicity induced by the injection of histamine in the SN of adult mice in vivo. Blockade of H1R protected against histamine-induced Nox1 expression and death of DA neurons in vivo. CONCLUSIONS: Overall, our results highlight the relevance of histamine in the modulation of microglial activity that ultimately may interfere with neuronal survival in the context of Parkinson's disease (PD) and, eventually, other neurodegenerative diseases which are accompanied by microglia-induced neuroinflammation. Importantly, our results also open promising new perspectives for the therapeutic use of H1R antagonists to treat or ameliorate neurodegenerative processes.

NADPH oxidase 1 mediates α-synucleinopathy in Parkinson's disease.

Accumulation of misfolded α-synuclein is the pathological hallmark of Parkinson's disease (PD). Nevertheless, little is known about the mechanism contributing to α-synuclein aggregation and its further toxicity to dopaminergic neurons. Since oxidative stress can increase the expression and aggregation levels of α-synuclein, NADPH oxidases (Noxs), which are responsible for reactive oxygen species generation, could be major players in α-synucleinopathy. Previously, we demonstrated that Nox1 is expressed in dopaminergic neurons of the PD animal models as well as postmortem brain tissue of PD patients, and is responsible for oxidative stress and subsequent neuronal degeneration. Here, using paraquat (PQ)-based in vitro and in vivo PD models, we show that Nox1 has a crucial role in modulating the behavior of α-synuclein expression and aggregation in dopaminergic neurons. We observed in differentiated human dopaminergic cells that Nox1 and α-synuclein expressions are increased under PQ exposure. Nox1 knockdown significantly reduced both α-synuclein expression and aggregation, supporting the role of Nox1 in this process. Furthermore, in rats exposed to PQ, the selective knockdown of Nox1 in the substantia nigra, using adeno-associated virus encoding Nox1-specific shRNA, largely attenuated the PQ-mediated increase of α-synuclein and ubiquitin expression levels as well as α-synuclein aggregates (proteinase K resistant) and A11 oligomers. Significant reductions in oxidative stress level and dopaminergic neuronal loss were also observed. Our data reveal a new mechanism by which α-synuclein becomes a neuropathologic protein through Nox1-mediated oxidative stress. This finding may be used to generate new therapeutic interventions that slower the rate of α-synuclein aggregation and the progression of PD pathogenesis.

PKCδ mediates paraquat-induced Nox1 expression in dopaminergic neurons.

Our previous works have shown that the (NADPH) oxidase (Nox) enzyme, in particular Nox1, plays an important role in oxidative stress and subsequent dopaminergic cell death elicited by paraquat (PQ). In non-neuronal and glial cells, protein kinase C δ (PKCδ) shows the ability to regulate the activity of the Nox system. Herein we aimed to investigate if also in dopaminergic neurons exposed to PQ, PKCδ can regulate Nox1 expression. The chemical inhibitor, rottlerin, and short interference RNA (siRNA) were used to inhibit or selectively knockdown PKCδ, respectively. The studies were performed using the immortalized rat mesencephalic dopaminergic cell line (N27 cells) exposed to PQ, after pre-incubation with rottlerin or transfected with PKCδ-siRNA. We observed that inhibition or knockdown of PKCδ significantly reduced PQ induced Nox1 transcript and protein levels, ROS generation and subsequent dopaminergic cell death. The results suggest that PKCδ plays a role in the regulation of Nox1-mediated oxidative stress elicited by PQ and could have a role in the pathogenesis of Parkinson's disease.

Gene Therapy in Amyotrophic Lateral Sclerosis.

Since the discovery of Cu/Zn superoxide dismutase (SOD1) gene mutation, in 1993, as the first genetic abnormality in amyotrophic lateral sclerosis (ALS), over 50 genes have been identified as either cause or modifier in ALS and ALS/frontotemporal dementia (FTD) spectrum disease. Mutations in C9orf72, SOD1, TAR DNA binding protein 43 (TARDBP), and fused in sarcoma (FUS) genes are the four most common ones. During the last three decades, tremendous effort has been made worldwide to reveal biological pathways underlying the pathogenesis of these gene mutations in ALS/FTD. Accordingly, targeting etiologic genes (i.e., gene therapies) to suppress their toxic effects have been investigated widely. It includes four major strategies: (i) removal or inhibition of abnormal transcribed RNA using microRNA or antisense oligonucleotides (ASOs), (ii) degradation of abnormal mRNA using RNA interference (RNAi), (iii) decrease or inhibition of mutant proteins (e.g., using antibodies against misfolded proteins), and (iv) DNA genome editing with methods such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas). The promising results of these studies have led to the application of some of these strategies into ALS clinical trials, especially for C9orf72 and SOD1. In this paper, we will overview advances in gene therapy in ALS/FTD, focusing on C9orf72, SOD1, TARDBP, and FUS genes.

Regulation of Αlpha-Synuclein Gene (SNCA) by Epigenetic Modifier TET1 in Parkinson Disease.

PURPOSE: Deregulation of SNCA encoding α-synuclein (α-SYN) has been associated with both the familial and sporadic forms of Parkinson disease (PD). Epigenetic regulation plays a crucial role in PD. The intron1 of SNCA harbors a large unmethylated CpG island. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), a CpG island binding protein, can repress gene expression by occupying hypomethylated CpG-rich promoters, and therefore SNCA could be a target for TET1. We investigated whether TET1 binds to SNCA-intron1 and regulates gene expression. METHODS: The dopaminergic neuronal cell line, ReNcell VM, was used. Reverse transcription-polymerase chain reaction (RT-PCR), real time-quantitative PCR, Western blot, dot-blot, and Chromatin immunoprecipitation were conducted. The substantia nigra tissues of postmortem PD samples were used to confirm the level of TET1 expression. RESULTS: In the human dopaminergic cell line, ReNcell VM, overexpression of the DNA-binding domain of TET1 (TET1-CXXC) led to significant repression of α-SYN. On the contrary, knocking down of TET1 led to significantly higher expression of α-SYN. However, overexpression of the DNA-hydroxymethylating catalytic domain of TET1 failed to change the expression of α-SYN. Altogether, we showed that TET1 is a repressor for SNCA, and a CXXC domain of TET1 is the primary mediator for this repressive action independent of its hydroxymethylation activity. TET1 levels in PD patients are significantly lower than that in the controls. CONCLUSION: We identified that TET1 acts as a repressor for SNCA by binding the intron1 regions of the gene. As a high level of α-SYN is strongly implicated in the pathogenesis of PD, discovering a repressor for the gene encoding α-SYN is highly important for developing novel therapeutic strategies for the disease.

Overview of stem cells therapy in amyotrophic lateral sclerosis.

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of upper and lower motor neurons with high burden on society. Despite tremendous efforts over the last several decades, there is still no definite cure for ALS. Up to now, only two disease-modifying agents, riluzole and edaravone, are approved by U.S. Food and Drug Administration (FDA) for ALS treatment, which only modestly improves survival and disease progression. Major challenging issues to find an effective therapy are heterogeneity in the pathogenesis and genetic variability of ALS. As such, stem cell therapy has been recently a focus of both preclinical and clinical investigations of ALS. This is because stem cells have multifaceted features that can potentially target multiple pathogenic mechanisms in ALS even though its underlying mechanisms are not completely elucidated. Methods & Results: Here, we will have an overview of stem cell therapy in ALS, including their therapeutic mechanisms, the results of recent clinical trials as well as ongoing clinical trials. In addition, we will further discuss complications and limitations of stem cell therapy in ALS. Conclusion: The determination of whether stem cells offer a viable treatment strategy for ALS rests on well-designed and appropriately powered future clinical trials. Randomized, double-blinded, and sham-controlled studies would be valuable.

Targeted attenuation of elevated histone marks at SNCA alleviates α-synuclein in Parkinson's disease.

Epigenetic deregulation of α-synuclein plays a key role in Parkinson's disease (PD). Analysis of the SNCA promoter using the ENCODE database revealed the presence of important histone post-translational modifications (PTMs) including transcription-promoting marks, H3K4me3 and H3K27ac, and repressive mark, H3K27me3. We investigated these histone marks in post-mortem brains of controls and PD patients and observed that only H3K4me3 was significantly elevated at the SNCA promoter of the substantia nigra (SN) of PD patients both in punch biopsy and in NeuN-positive neuronal nuclei samples. To understand the importance of H3K4me3 in regulation of α-synuclein, we developed CRISPR/dCas9-based locus-specific H3K4me3 demethylating system where the catalytic domain of JARID1A was recruited to the SNCA promoter. This CRISPR/dCas9 SunTag-JARID1A significantly reduced H3K4me3 at SNCA promoter and concomitantly decreased α-synuclein both in the neuronal cell line SH-SY5Y and idiopathic PD-iPSC derived dopaminergic neurons. In sum, this study indicates that α-synuclein expression in PD is controlled by SNCA's histone PTMs and modulation of the histone landscape of SNCA can reduce α-synuclein expression.

A novel extended form of alpha-synuclein 3'UTR in the human brain.

Alpha-synuclein (α-SYN) is one of the key contributors in Parkinson's disease (PD) pathogenesis. Despite the fact that increased α-SYN levels are considered one of the key contributors in developing PD, the molecular mechanisms underlying the regulation of α-SYN still needs to be elucidated. Since the 3' untranslated regions (3'UTRs) of messenger RNAs (mRNAs) have important roles in translation, localization, and stability of mRNAs through RNA binding proteins (RBPs) and microRNAs (miRNAs), it is important to identify the exact length of 3'UTRs of transcripts in order to understand the precise regulation of gene expression. Currently annotated human α-SYN mRNA has a relatively long 3'UTR (2529 nucleotides [nt]) with several isoforms. RNA-sequencing and epigenomics data have suggested, however, the possible existence of even longer transcripts which extend beyond the annotated α-SYN 3'UTR sequence. Here, we have discovered the novel extended form of α-SYN 3'UTR (3775 nt) in the substantia nigra of human postmortem brain samples, induced pluripotent stem cell (iPSC)-derived dopaminergic neurons, and other human neuronal cell lines. Interestingly, the longer variant reduced α-SYN translation. The extended α-SYN 3'UTR was significantly lower in iPSC-derived dopaminergic neurons from sporadic PD patients than controls. On the other hand, α-SYN protein levels were much higher in PD cases, showing the strong negative correlation with the extended 3'UTR. These suggest that dysregulation of the extended α-SYN 3'UTR might contribute to the pathogenesis of PD.

Mitochondrial ROS-mediated post-transcriptional regulation of α-synuclein through miR-7 and miR-153.

Dysregulation of human alpha-synuclein (α-SYN) is one of the major contributors in the pathogenesis of Parkinson's disease. 1-methyl-4-phenylpyridinium (MPP+) is well known neurotoxin which increases α-SYN expression and causes dopaminergic neuronal death. Increasing evidence suggests microRNAs (miRNAs), especially miRNA-7 and miR-153, have important role in the regulation of α-SYN translation and they can prevent MPP+-mediated neuronal death. Here, we examined whether MPP+-mediated upregulation of α-SYN expression is directly related to miRNA-7 and miR-153. First, we established HEK293/TR cells stably expressing both miR-7 and miR-153. Human α-SYN 3'-UTR containing target sites for both miRNAs was cloned next to a luciferase reporter construct. To control the total levels of reporter mRNA, a tetracycline-inducible system was used. Compared to wild-type HEK293/TR cells, cells overexpressing both miRNAs demonstrated about 75% reduction in luciferase activity. MPP+ treatment, however, significantly increased luciferase activity of human α-SYN 3'-UTR. Either quenching mitochondrial reactive oxygen species (ROS) or translational inhibition significantly reduced MPP+-mediated luciferase activity, suggesting mitochondrial ROS is responsible for MPP+-induced α-SYN translation. Together, our results suggest that MPP+-mediated increased α-SYN levels are contributed by mitochondrial ROS-mediated de novo protein synthesis which is regulated by miRNA-7 and miR-153.

Polymorphism of Nitric Oxide Synthase 1 Affects the Clinical Phenotypes of Ischemic Stroke in Korean Population.

OBJECTIVE: To investigate whether four single nucleotide polymorphisms (SNPs) rs2293054 [Ile734Ile], rs1047735 [His902His], rs2293044 [Val1353Val], rs2682826 (3'UTR) of nitric oxide synthase 1 (NOS1) are associated with the development and clinical phenotypes of ischemic stroke. METHODS: We enrolled 120 ischemic stroke patients and 314 control subjects. Ischemic stroke patients were divided into subgroups according to the scores of the National Institutes of Health Stroke Survey (NIHSS, <6 and ≥6) and Modified Barthel Index (MBI, <60 and ≥60). SNPStats, SNPAnalyzer, and HelixTree programs were used to calculate odds ratios (ORs), 95% confidence intervals (CIs), and p-values. Multiple logistic regression models were performed to analyze genetic data. RESULTS: No SNPs of the NOS1 gene were found to be associated with ischemic stroke. However, in an analysis of clinical phenotypes, we found that rs2293054 was associated with the NIHSS scores of ischemic stroke patients in codominant (p=0.019), dominant (p=0.007), overdominant (p=0.033), and log-additive (p=0.0048) models. Also, rs2682826 revealed a significant association in the recessive model (p=0.034). In allele frequency analysis, we also found that the T alleles of rs2293054 were associated with lower NIHSS scores (p=0.007). Respectively, rs2293054 had a significant association in the MBI scores of ischemic stroke in codominant (p=0.038), dominant (p=0.031), overdominant (p=0.045), and log-additive (p=0.04) models. CONCLUSION: These results suggest that NOS1 may be related to the clinical phenotypes of ischemic stroke in Korean population.

Transcriptional mutagenesis by 8-oxodG in α-synuclein aggregation and the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease associated with selective loss of dopaminergic neurons. The characteristic hallmark of the disease is intracytoplasmic proteinacious inclusion bodies called Lewy bodies, primarily consisting of a presynaptic protein α-synuclein. Oxidative stress-mediated damage to macromolecules have been shown to occur frequently in PD. Oxidative damage to DNA in the form of oxidized guanine (8-oxodG) accumulates in both the mitochondrial and nuclear DNA of dopaminergic neurons of the substantia nigra in PD. 8-oxodG-mediated transcriptional mutagenesis has been shown to have the potential to alter phenotype of cells through production of mutant pool of proteins. This review comprehensively summarizes the role of oxidative stress-mediated damage incurred during neurodegeneration, and highlights the scope of transcriptional mutagenesis event in leading to α-synuclein aggregation as seen in PD.

Rodent models of Parkinson's disease: beyond the motor symptomatology.

Parkinson's disease (PD) is classically characterized by motor symptoms; however, non-motor symptoms (NMS) are increasingly recognized as relevant in disease-state, given the associated alterations in mood (depression and anxiety) and cognition. Here, particularly in regards to NMS, we aimed to compare the motor, emotional and cognitive behavior of three animal models of PD that trigger dopaminergic (DAergic) degeneration on both brain hemispheres: (i) the 6-hydroxydopamine (6-OHDA, 8 or 6 µg) lesion model; (ii) the paraquat (PQ) induced model, and (iii) a genetic model based on α-synuclein overexpression (α-syn). 6-OHDA and α-syn vector were injected bilaterally in the substantia nigra pars compacta (SNpc) of adult male Wistar rats; as for PQ delivery, micro-osmotic pumps were implanted in the interscapular region. Motor deficits were observed in all models, with histological analysis of tyrosine hydroxylase positive cells in the SNpc revealing a significant loss of DAergic neurons in all animal models. In addition, the α-syn animal model also presented a reduction in exploratory activity, and the 6-OHDA and PQ animals displayed a significant increase in both depressive- and anxiety-like behavior. Interestingly, cognitive impairment (working memory) was only observed in the 6-OHDA model. Overall, these PD models are suitable for mimicking the motor symptoms associated to PD, with each encompassing other relevant NMS components of the disorder that may prove beneficial for further studies in PD.