Heterogeneous Nuclear Ribonucleoproteins: Implications in Neurological Diseases

Heterogenous nuclear ribonucleoproteins (hnRNPs) are a complex and functionally diverse family of RNA binding proteins with multifarious roles. They are involved, directly or indirectly, in alternative splicing, transcriptional and translational regulation, stress granule formation, cell cycle regulation, and axonal transport. It is unsurprising, given their heavy involvement in maintaining functional integrity of the cell, that their dysfunction has neurological implications. However, compared to their more established roles in cancer, the evidence of hnRNP implication in neurological diseases is still in its infancy. This review aims to consolidate the evidences for hnRNP involvement in neurological diseases, with a focus on spinal muscular atrophy (SMA), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), multiple sclerosis (MS), congenital myasthenic syndrome (CMS), and fragile X-associated tremor/ataxia syndrome (FXTAS). Understanding more about hnRNP involvement in neurological diseases can further elucidate the pathomechanisms involved in these diseases and perhaps guide future therapeutic advances

White matter DNA methylation profiling reveals deregulation of HIP1, LMAN2, MOBP, and other loci in multiple system atrophy

Multiple system atrophy (MSA) is a fatal late-onset neurodegenerative disease. Although presenting with distinct pathological hallmarks, which in MSA consist of glial cytoplasmic inclusions (GCIs) containing fbrillar α-synuclein in oligodendrocytes, both MSA and Parkinson’s disease are α-synucleinopathies. Pathologically, MSA can be categorized into striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA) or mixed subtypes. Despite extensive research, the regional vulnerability of the brain to MSA pathology remains poorly understood. Genetic, epigenetic and environmental factors have been proposed to explain which brain regions are afected by MSA, and to what extent. Here, we explored for the frst time epigenetic changes in post-mortem brain tissue from MSA cases. We conducted a case–control study, and profled DNA methylation in white mater from three brain regions characterized by severe-to-mild GCIs burden in the MSA mixed subtype (cerebellum, frontal lobe and occipital lobe). Our genome-wide approach using Illumina MethylationEPIC arrays and a powerful cross-region analysis identifed 157 CpG sites and 79 genomic regions where DNA methylation was signifcantly altered in the MSA mixed-subtype cases. HIP1, LMAN2 and MOBP were amongst the most diferentially methylated loci. We replicated these fndings in an independent cohort and further demonstrated that DNA methylation profles were perturbed in MSA mixed subtype, and also to variable degrees in the other pathological subtypes (OPCA and SND). Finally, our comethylation network analysis revealed several molecular signatures (modules) signifcantly associated with MSA (disease status and pathological subtypes), and with neurodegeneration in the cerebellum. Importantly, the co-methylation module having the strongest association with MSA included a CpG in SNCA, the gene encoding α-synuclein. Altogether, our results provide the frst evidence for DNA methylation changes contributing to the molecular processes altered in MSA, some of which are shared with other neurodegenerative diseases, and highlight potential novel routes for diagnosis and therapeutic interventions

Heterogeneous nuclear ribonucleoproteins R and Q accumulate in pathological inclusions in FTLD-FUS

Frontotemporal lobar degeneration (FTLD) is pathologically subdivided based on the presence of particular pathological proteins that are identified in inclusion bodies observed post-mortem. The FTLD-FUS subgroup is defined by the presence of the fused in sarcoma protein (FUS) in pathological inclusions. FUS is a heterogeneous nuclear ribonucleoprotein (hnRNP) protein and a member of the FET (FUS, EWS, TAF15) protein family. It shuttles between the nucleus and cytoplasm, and has been implicated in many cellular functions including translation, splicing, and RNA transport. EWS, TAF15 and the nuclear import receptor transportin have been shown to co-accumulate with FUS in neuronal inclusions specifically in FTLD-FUS, with transportin-positive inclusions most frequently observed. Here, we report the identification of hnRNP R and hnRNP Q in neuronal cytoplasmic and intranuclear inclusions in the frontal cortex and hippocampus of FTLD-FUS patients, as frequently as transportin. hnRNP R and hnRNP Q were not found in the characteristic pathological inclusions observed in FTLD-TDP (subtypes A-C). Additionally, we studied the expression of hnRNP R in the frontal and temporal cortices from patients with FTLD and found significantly increased expression of the heterogeneous nuclear ribonucleoprotein R in several FTLD disease groups. Our identification of the frequent presence of hnRNP R and hnRNP Q in FTLD-FUS inclusions suggests a potential role for these hnRNPs in FTLD-FUS pathogenesis and supports the role of dysfunctional RNA metabolism in FTLD

Cerebral mitochondrial electron transport chain dysfunction in multiple system atrophy and Parkinson's disease.

Multiple system atrophy (MSA) is a neurodegenerative disease characterised by glial cytoplasmic inclusions (GCIs), containing α-synuclein. Mutated COQ2, encoding an enzyme essential for co-enzyme Q10 (CoQ10) biosynthesis, has been associated with MSA. CoQ10 is an electron carrier in the mitochondrial electron transport chain (ETC) and antioxidant. It has been shown to be deficient in MSA brain tissue, thus implicating mitochondrial dysfunction in MSA. To investigate mitochondrial dysfunction in MSA further we examined ETC activity in MSA and control brain tissue, compared with Parkinson's disease (PD) where mitochondrial dysfunction is known to be important. Using cerebellar and occipital white matter ETC complex I, II/III and IV activities were measured spectrophotometrically, selected individual components of the ETC were assessed by immunoblotting and cellular complex IV activity was analysed by enzyme histochemistry. We show decreased complex II/III activity with increased complex I and IV activity in MSA cerebellar white matter. This corresponds with the deficit in CoQ10 previously described in MSA and reflects the high regional pathological burden of GCIs. This study highlights mitochondrial dysfunction in MSA pathogenesis, suggests an influence on selective regional vulnerability to disease and points to shared disease mechanisms in α-synucleinopathies

MOBP and HIP1 in multiple system atrophy: new α‐synuclein partners in glial cytoplasmic inclusions implicated in the disease pathogenesis

Aims: MSA is a fatal neurodegenerative disease. Similar to Parkinson’s disease (PD), MSA is an α‐synucleinopathy, and its pathological hallmark consists of glial cytoplasmic inclusions (GCIs) containing α‐synuclein in oligodendrocytes. We previously identified consistent changes in MOBP and HIP1 DNA methylation status in MSA. We hypothesized that if differential DNA methylation at these loci is mechanistically relevant for MSA, it should have downstream consequences on gene regulation. / Methods: We investigated the relationship between MOBP and HIP1 DNA methylation and mRNA levels in cerebellar white matter from MSA and healthy controls. Additionally, we analysed protein expression using western blotting, immunohistochemistry and proximity ligation assays. / Results: We found decreased MOBP mRNA levels significantly correlated with increased DNA methylation in MSA. For HIP1, we found a distinct relationship between DNA methylation and gene expression levels in MSA compared to healthy controls, suggesting this locus may be subjected to epigenetic remodelling in MSA. Although soluble protein levels for MOBP and HIP1 in cerebellar white matter were not significantly different between MSA cases and controls, we found striking differences between MSA and other neurodegenerative diseases, including PD and Huntington’s disease. We also found that MOBP and HIP1 are mislocalized into the GCIs in MSA, where they appear to interact with α‐synuclein. / Conclusions: This study supports a role for DNA methylation in downregulation of MOBP mRNA in MSA. Most importantly, the identification of MOBP and HIP1 as new constituents of GCIs emphasizes the relevance of these two loci to the pathogenesis of MSA

Immunohistochemical and Molecular Investigations Show Alteration in the Inflammatory Profile of Multiple System Atrophy Brain

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterized by aggregation of α-synuclein in oligodendrocytes to form glial cytoplasmic inclusions. According to the distribution of neurodegeneration, MSA is subtyped as striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA), or as combination of these 2 (mixed MSA). In the current study, we aimed to investigate regional microglial populations and gene expression in the 3 different MSA subtypes. Microscopy with microglial marker Iba-1 combined with either proinflammatory marker CD68 or anti-inflammatory marker Arginase-1 was analyzed in control, SND, and OPCA cases (n = 5) using paraffin embedded sections. Western immunoblotting and cytokine array were used to determine protein expression in MSA and control brain regions. Gene expression was investigated using the NanoString nCounter Human Inflammation panel v2 mRNA Expression Assay. Analysis of neuropathological subtypes of MSA demonstrated a significant increase in microglia in the substantia nigra of OPCA cases. There was no difference in the microglial activation state in any region. Cytokine expression in MSA was comparable with controls. Decreased expression of CX3CL1 precursor protein and significantly greater CX3CR1 protein was found in MSA. NanoString analysis revealed the >2-fold greater expression of ARG1, MASP1, NOX4, PTGDR2, and C6 in MSA

Structural and functional conservation of non-lumenized lymphatic endothelial cells in the mammalian leptomeninges

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-β, the toxic driver of Alzheimer's disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges

Identification of multiple system atrophy mimicking Parkinson's disease or progressive supranuclear palsy

WWe studied a subset of patients with autopsy-confirmed multiple system atrophy who presented a clinical picture that closely resembled either Parkinson’s disease or progressive supranuclear palsy. These mimics are not captured by the current diagnostic criteria for multiple system atrophy. Among 218 autopsy-proven multiple system atrophy cases reviewed, 177 (81.2%) were clinically diagnosed and pathologically confirmed as multiple system atrophy (i.e. typical cases), while the remaining 41 (18.8%) had received an alternative clinical diagnosis, including Parkinson’s disease (i.e. Parkinson’s disease mimics; n = 16) and progressive supranuclear palsy (i.e. progressive supranuclear palsy mimics; n = 17). We also reviewed the clinical records of another 105 patients with pathologically confirmed Parkinson’s disease or progressive supranuclear palsy, who had received a correct final clinical diagnosis (i.e. Parkinson’s disease, n = 35; progressive supranuclear palsy-Richardson syndrome, n = 35; and progressive supranuclear palsy-parkinsonism, n = 35). We investigated 12 red flag features that would support a diagnosis of multiple system atrophy according to the current diagnostic criteria. Compared with typical multiple system atrophy, Parkinson’s disease mimics more frequently had a good levodopa response and visual hallucinations. Vertical gaze palsy and apraxia of eyelid opening were more commonly observed in progressive supranuclear palsy mimics. Multiple logistic regression analysis revealed an increased likelihood of having multiple system atrophy [Parkinson’s disease mimic versus typical Parkinson’s disease, odds ratio (OR): 8.1; progressive supranuclear palsy mimic versus typical progressive supranuclear palsy, OR: 2.3] if a patient developed any one of seven selected red flag features in the first 10 years of disease. Severe autonomic dysfunction (orthostatic hypotension and/or urinary incontinence with the need for a urinary catheter) was more frequent in clinically atypical multiple system atrophy than other parkinsonian disorders (Parkinson’s disease mimic versus typical Parkinson’s disease, OR: 4.1; progressive supranuclear palsy mimic versus typical progressive supranuclear palsy, OR: 8.8). The atypical multiple system atrophy cases more frequently had autonomic dysfunction within 3 years of symptom onset than the pathologically confirmed patients with Parkinson’s disease or progressive supranuclear palsy (Parkinson’s disease mimic versus typical Parkinson’s disease, OR: 4.7; progressive supranuclear palsy mimic versus typical progressive supranuclear palsy, OR: 2.7). Using all included clinical features and 21 early clinical features within 3 years of symptom onset, we developed decision tree algorithms with combinations of clinical pointers to differentiate clinically atypical cases of multiple system atrophy from Parkinson’s disease or progressive supranuclear palsy

Genome Wide Transcriptome Analysis of Dendritic Cells Identifies Genes with Altered Expression in Psoriasis

Activation of dendritic cells by different pathogens induces the secretion of proinflammatory mediators resulting in local inflammation. Importantly, innate immunity must be properly controlled, as its continuous activation leads to the development of chronic inflammatory diseases such as psoriasis. Lipopolysaccharide (LPS) or peptidoglycan (PGN) induced tolerance, a phenomenon of transient unresponsiveness of cells to repeated or prolonged stimulation, proved valuable model for the study of chronic inflammation. Thus, the aim of this study was the identification of the transcriptional diversity of primary human immature dendritic cells (iDCs) upon PGN induced tolerance. Using SAGESeq approach, a tag-based transcriptome sequencing method, we investigated gene expression changes of primary human iDCs upon stimulation or restimulation with Staphylococcus aureus derived PGN, a widely used TLR2 ligand. Based on the expression pattern of the altered genes, we identified non-tolerizeable and tolerizeable genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (Kegg) analysis showed marked enrichment of immune-, cell cycle- and apoptosis related genes. In parallel to the marked induction of proinflammatory mediators, negative feedback regulators of innate immunity, such as TNFAIP3, TNFAIP8, Tyro3 and Mer are markedly downregulated in tolerant cells. We also demonstrate, that the expression pattern of TNFAIP3 and TNFAIP8 is altered in both lesional, and non-lesional skin of psoriatic patients. Finally, we show that pretreatment of immature dendritic cells with anti-TNF-α inhibits the expression of IL-6 and CCL1 in tolerant iDCs and partially releases the suppression of TNFAIP8. Our findings suggest that after PGN stimulation/restimulation the host cell utilizes different mechanisms in order to maintain critical balance between inflammation and tolerance. Importantly, the transcriptome sequencing of stimulated/restimulated iDCs identified numerous genes with altered expression to date not associated with role in chronic inflammation, underlying the relevance of our in vitro model for further characterization of IFNprimed iDCs