Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (approximately 10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.     

Bergmann Glia are Patterned into Topographic Molecular Zones in the Developing and Adult Mouse Cerebellum

Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. Zones are best revealed by gene expression, circuit anatomy, and cellular degeneration patterns. Thus far, the study of zones has been focused heavily on how neurons are organized. Because of this, detailed neuronal patterning maps have been established for Purkinje cells, granule cells, Golgi cells, unipolar brush cells, and also for the terminal field organization of climbing fiber and mossy fiber afferents. In comparison, however, it remains poorly understood if glial cells are also organized into zones. We have identified an Npy-Gfp BAC transgenic mouse line (Tau-Sapphire Green fluorescent protein (Gfp) is under the control of the neuropeptide Y (Npy) gene regulatory elements) that can be used to label Bergmann glial cells with Golgi-like resolution. In these adult transgenic mice, we found that Npy-Gfp expression was localized to Bergmann glia mainly in lobules VI/VII and IX/X. Using double immunofluorescence, we show that in these lobules, Npy-Gfp expression in the Bergmann glia overlaps with the pattern of the small heat shock protein HSP25, a Purkinje cell marker for zones located in lobules VI/VII and IX/X. Developmental analysis starting from the day of birth showed that HSP25 and Npy-Gfp expression follow a similar program of spatial and temporal patterning. However, loss of Npy signaling did not alter the patterning of Purkinje cell zones. We conclude that Bergmann glial cells are zonally organized and their patterns are restricted by boundaries that also confine cerebellar neurons into a topographic circuit map.     

A direct regulatory link between microRNA-137 and <Emphasis Type="Italic">SHANK2</Emphasis>: implications for neuropsychiatric disorders

Background

Mutations in the SHANK genes, which encode postsynaptic scaffolding proteins, have been linked to a spectrum of neurodevelopmental disorders. The SHANK genes and the schizophrenia-associated microRNA-137 show convergence on several levels, as they are both expressed at the synapse, influence neuronal development, and have a strong link to neurodevelopmental and neuropsychiatric disorders like intellectual disability, autism, and schizophrenia. This compiled evidence raised the question if the SHANKs might be targets of miR-137.

Methods

In silico analysis revealed a putative binding site for microRNA-137 (miR-137) in the SHANK2 3′UTR, while this was not the case for SHANK1 and SHANK3. Luciferase reporter assays were performed by overexpressing wild type and mutated SHANK2-3′UTR and miR-137 in human neuroblastoma cells and mouse primary hippocampal neurons. miR-137 was also overexpressed or inhibited in hippocampal neurons, and Shank2 expression was analyzed by quantitative real-time PCR and Western blot. Additionally, expression levels of experimentally validated miR-137 target genes were analyzed in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia and control individuals using the RNA-Seq data from the CommonMind Consortium.

Results

miR-137 directly targets the 3′UTR of SHANK2 in a site-specific manner. Overexpression of miR-137 in mouse primary hippocampal neurons significantly lowered endogenous Shank2 protein levels without detectable influence on mRNA levels. Conversely, miR-137 inhibition increased Shank2 protein expression, indicating that miR-137 regulates SHANK2 expression by repressing protein translation rather than inducing mRNA degradation.To find out if the miR-137 signaling network is altered in schizophrenia, we compared miR-137 precursor and miR-137 target gene expression in the DLPFC of schizophrenia and control individuals using the CommonMind Consortium RNA sequencing data. Differential expression of 23% (16/69) of known miR-137 target genes was detected in the DLPFC of schizophrenia individuals compared with controls. We propose that in further targets (e.g., SHANK2, as described in this paper) which are not regulated on RNA level, effects may only be detectable on protein level.

Conclusion

Our study provides evidence that a direct regulatory link exists between miR-137 and SHANK2 and supports the finding that miR-137 signaling might be altered in schizophrenia.

     

Up-regulation of <Emphasis Type="Italic">SNCA</Emphasis> gene expression: implications to synucleinopathies

Synucleinopathies are a group of neurodegenerative diseases that share a common pathological lesion of intracellular protein inclusions largely composed by aggregates of alpha-synuclein protein. Accumulating evidence, including genome wide association studies, has implicated alpha-synuclein (SNCA) gene in the etiology of synucleinopathies. However, the precise variants within SNCA gene that contribute to the sporadic forms of Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and other synucleinopathies and their molecular mechanisms of action remain elusive. It has been suggested that SNCA expression levels are critical for the development of these diseases. Here, we review several model systems that have been developed to advance the understanding of the role of SNCA expression levels in the etiology of synucleinopathies. We also describe different molecular mechanisms that regulate SNCA gene expression and discuss possible strategies for SNCA down-regulation as means for therapeutic approaches. Finally, we highlight some examples that underscore the relationships between the genetic association findings and the regulatory mechanisms of SNCA expression, which suggest that genetic variability in SNCA locus is directly responsible, at least in part, to the changes in gene expression and explain the reported associations of SNCA with synucleinopathies. Future studies utilizing induced pluripotent stem cells (iPSCs)—derived neuronal lines and genome editing by CRISPR/Cas9, will allow us to validate, characterize, and manipulate the effects of particular cis-genetic variants on SNCA expression. Moreover, this model system will enable us to compare different neuronal and glial lineages involved in synucleinopathies representing an attractive strategy to elucidate—common and specific—SNCA-genetic variants, regulatory mechanisms, and vulnerable expression levels underlying synucleinopathy spectrum disorders. This forthcoming knowledge will support the development of precision medicine for synucleinopathies.     

First large genomic inversion in familial cerebral cavernous malformation identified by whole genome sequencing

Familial cerebral cavernous malformations (CCMs) predispose to seizures and hemorrhagic stroke. Molecular genetic analyses of CCM1, CCM2, and CCM3 result in a mutation detection rate of up to 98%. However, only whole genome sequencing (WGS) in combination with the Manta algorithm for analyses of structural variants revealed a heterozygous 24 kB inversion including exon 1 of CCM2 in a 12-year-old boy with familial CCMs. Its breakpoints were fine-mapped, and quantitative analysis on RNA confirmed reduced CCM2 expression. Our data expand the spectrum of CCM mutations and indicate that the existence of a fourth CCM disease gene is rather unlikely.     

Increased cell-free mitochondrial DNA is a marker of ongoing inflammation and better neurocognitive function in virologically suppressed HIV-infected individuals

Cell-free mitochondrial DNA (mtDNA) is a highly immunogenic molecule that is associated with several inflammatory conditions and with neurocognitive impairment during untreated HIV infection. Here, we investigate how cell-free mtDNA in cerebrospinal fluid (CSF) is associated with inflammation, neuronal damage, and neurocognitive functioning in the context of long-term suppressive antiretroviral therapy (ART). We quantified the levels of cell-free mtDNA in the CSF from 41 HIV-infected individuals with completely suppressed HIV RNA levels in blood plasma (<50 copies/mL) by droplet digital PCR. We measured soluble CD14, soluble CD163, interferon γ-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), neopterin, and neurofilament light chain (NFL) by immunoassays in CSF supernatant or blood plasma. Higher levels of mtDNA in CSF were associated with higher levels of MCP-1 (r = 0.56, p < 0.01) in CSF and TNF-α (r = 0.43, p < 0.01) and IL-8 (r = 0.44, p < 0.01) in blood plasma. Subjects with a previous diagnosis of AIDS showed significantly higher levels of mtDNA (p < 0.01) than subjects without AIDS. The associations between mtDNA and MCP-1 in CSF and TNF-α in blood remained significant after adjusting for previous diagnosis of AIDS (p < 0.01). Additionally, higher levels of mtDNA were associated with a lower CD4 nadir (r = ?0.41, p < 0.01) and lower current CD4% (r = ?0.34, p = 0.03). Paradoxically, higher levels of mtDNA in CSF were significantly associated with better neurocognitive performance (r = 0.43, p = 0.02) and with less neuronal damage (i.e. lower NFL). Higher cell-free mtDNA is associated with inflammation during treated HIV infection, but the impact on neurocognitive functioning and neuronal damage remains unclear and may differ in the setting of suppressive ART.     

The molecular genetic basis of the neuronal ceroid lipofuscinoses

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the presence of autofluorescent lipopigment in neurons and other cell types. The childhood onset types display autosomal recessive inheritance. Naturally occurring animal NCLs have been described in many species including mouse, sheep and dog. In the last decade major advances have occurred in the molecular genetic analysis of the NCLs. Six disease gene loci have been mapped, and five disease genes have been isolated. Two of these encode lysosomal enzymes: CLN1 encodes palmitoyl-protein thioesterase (PPT), and CLN2 encodes tripeptidyl peptidase 1 (TPP1). The remaining three, CLN3, CLN5 and CLN8 encode putative membrane proteins of unknown function. The murine orthologue of CLN8 causes motor neuron degeneration (mnd), a mouse model of NCL. These advances have revolutionized diagnosis and classification, but a unified theory of pathogenesis and effective treatment remain elusive.     

Preliminary Study on the Role of <Emphasis Type="Italic">TMEM39A</Emphasis> Gene in Multiple Sclerosis

Genome-wide association studies (GWAS) have identified hundreds of new potential genetic risk loci associated with numerous complex diseases such as multiple sclerosis (MS). Genes which have been discovered by GWAS are now the focus of numerous ongoing studies. The goal of this study was to confirm and understand the potential role of one of such genes—transmembrane protein 39A gene (TMEM39A)—in multiple sclerosis.We showed the difference in TMEM39A messenger RNA (mRNA) expression between MS patients and controls (T ² _2;74 = 5.429; p = 0.0063). In our study, the lower mRNA expression of TMEM39A gene in patients did not correlate with a higher methylation level of the TMEM39A promoter. Moreover, a decreased level of TMEM39A mRNA was associated neither with rs1132200 nor with rs17281647. Additionally, we did not find an association between these two TMEM39A polymorphisms and the risk and progression of multiple sclerosis.Our investigation is the first which indicates that TMEM39A mRNA expression may be associated with the development and/or course of multiple sclerosis.     

<Emphasis Type="Italic">Dicer1</Emphasis> Ablation Impairs Responsiveness of Cerebellar Granule Neuron Precursors to Sonic Hedgehog and Disrupts Expression of Distinct Cell Cycle Regulator Genes

Granule neuron precursors (GNPs) proliferate under the influence of Sonic hedgehog (Shh) that is secreted by Purkinje neurons during early postnatal cerebellar development. To investigate microRNA (miRNA) function in this developmental process, we conditionally deleted the Dicer1 gene under the activity of human glial fibrillary acidic protein (hGFAP) promoter. We report that Dicer1-ablated GNPs display decreased proliferation and survival at early postnatal stages and that the proliferation defect of mutant GNPs cannot be rescued by treatment of an Shh agonist in vitro as assayed by 5-bromo-2′-deoxyuridine (BrdU) pulse labeling and Shh target gene expression detection. Further analysis reveals that the expression of distinct cell cycle regulator genes including cell cycle inhibitor, CDKN1a (p21), selectively increases in Dicer1-ablated GNPs. Subsequently, we demonstrate that miR-17-5p exhibits high expression level in the developing cerebellum and that transfection of a synthetic miR-17-5p mimic downregulates p21 protein expression in GNPs and promotes proliferation of GNPs in culture. Therefore, Dicer1 ablation impairs Shh-induced GNP proliferation by disrupting the expression of distinct cell cycle regulator genes that are targets of miR-17～92 cluster members. This study establishes a molecular link between miRNAs and cell cycle progression in the proliferating GNPs during normal cerebellar development and may facilitate miRNA application in treating medulloblastoma.     

Association Between Microglia,Inflammatory Factors,and Complement with Loss of Hippocampal Mossy Fiber Synapses Induced by Trimethyltin

Complement-associated factors are implicated in pathogen presentation, neurodegeneration, and microglia resolution of tissue injury. To characterize complement activation with microglial clearance of degenerating mossy fiber boutons, hippocampal dentate granule neurons were ablated in CD-1 mice with trimethyltin (TMT; 2.2 mg/kg, i.p.). Neuronal apoptosis was accompanied by amoeboid microglia and elevations in tumor necrosis factor [Tnfa], interleukin 1β [Il1b], and Il6 mRNA and C1q protein. Inos mRNA levels were unaltered. Silver degeneration and synaptophysin staining indicated loss of synaptic innervation to CA3 pyramidal neurons. Reactive microglia with thickened bushy morphology showed co-localization of synaptophysin+ fragments. The initial response at 2 days post-TMT included transient elevations in Tnfa, Il1b, Il6, and Inos mRNA levels. A concurrent increase at 2 days was observed in arginase-1 [Arg1], Il10, transforming growth factor β1 [Tgfb1], and chitinase 3 like-3 [Ym1] mRNA levels. At 2 days, C1q protein was evident in the CA3 with elevated C1qa, C1qb, C3, Cr3a, and Cr3b mRNA levels. mRNA levels remained elevated at 5 days, returning to control by 14 days, corresponding to silver degeneration. mRNA levels for pentraxin3 (Ptx3) were elevated on day 2 and Ptx1 was not altered. Our data suggest an association between microglia reactivity, the induction of anti-inflammatory genes concurrent with pro-inflammatory genes and the expression of complement-associated factors with the degeneration of synapses following apoptotic neuronal loss.     

Expression Analysis of Vitamin D Signaling Pathway Genes in Epileptic Patients

Vitamin D deficiency has been detected in epileptic patients. Vitamin D participates in neuroprotection, brain cell proliferation, and differentiation. Consequently, vitamin D supplementation has been suggested as an alternative treatment in epileptic patients. We aimed at assessment of vitamin D signaling pathway in epileptic patients. In the present study, we evaluated vitamin D serum concentration as well as expression of vitamin D receptor (VDR) gene and genes encoding for vitamin D activating enzyme 1-alpha-hydroxylase (CYP27B1) and deactivating enzyme 24-hyroxylase (CYP24A1) in epileptic patients compared with healthy individuals. We found significant lower levels of vitamin D in epileptic patients compared with healthy subjects. Expression analyses showed significant downregulation of VDR expression in peripheral blood of epileptic patients compared with healthy subjects (relative expression (REx)?=?0.16, P?<?0.001). However, there was no significant difference in CYP24A1 expression between epileptic patients and normal subjects. CYP27B1 expression analysis showed significant upregulation in male patients aged between 30 and 40 (REx?=?5.43, P?=?0.013). After using two-way ANCOVA for adjusting the effects of sex and age, there was a statistically significant difference in the VDR expression values between patient and control groups (P?<?0.001). Spearman’s correlation analysis showed no significant correlation between genes expression levels and patients’ age or vitamin D serum concentrations. However, we found significant correlations between VDR expression levels and CYP24A1/ CYP27B1 expression levels in epileptic patients (r?=?0.435 and P?<?0.001; r?=?0.26 and P?=?0.02 respectively). There was also a significant correlation between the expression levels of CYP24A1 and CYP27B1 (r?=?0.349 and P?=?0.001). Our study shows a possible role for VDR in the pathogenesis of epilepsy.     

Defining Trends in Global Gene Expression in Arabian Horses with Cerebellar Abiotrophy

Equine cerebellar abiotrophy (CA) is a hereditary neurodegenerative disease that affects the Purkinje neurons of the cerebellum and causes ataxia in Arabian foals. Signs of CA are typically first recognized either at birth to any time up to 6 months of age. CA is inherited as an autosomal recessive trait and is associated with a single nucleotide polymorphism (SNP) on equine chromosome 2 (13074277G>A), located in the fourth exon of TOE1 and in proximity to MUTYH on the antisense strand. We hypothesize that unraveling the functional consequences of the CA SNP using RNA-seq will elucidate the molecular pathways underlying the CA phenotype. RNA-seq (100 bp PE strand-specific) was performed in cerebellar tissue from four CA-affected and five age-matched unaffected horses. Three pipelines for differential gene expression (DE) analysis were used (Tophat2/Cuffdiff2, Kallisto/EdgeR, and Kallisto/Sleuth) with 151 significant DE genes identified by all three pipelines in CA-affected horses. TOE1 (Log₂(foldchange) = 0.92, p = 0.66) and MUTYH (Log₂(foldchange) = 1.13, p = 0.66) were not differentially expressed. Among the major pathways that were differentially expressed, genes associated with calcium homeostasis and specifically expressed in Purkinje neurons, CALB1 (Log₂(foldchange) = ?1.7, p < 0.01) and CA8 (Log₂(foldchange) = ?0.97, p < 0.01), were significantly down-regulated, confirming loss of Purkinje neurons. There was also a significant up-regulation of markers for microglial phagocytosis, TYROBP (Log₂(foldchange) = 1.99, p < 0.01) and TREM2 (Log₂(foldchange) = 2.02, p < 0.01). These findings reaffirm a loss of Purkinje neurons in CA-affected horses along with a potential secondary loss of granular neurons and activation of microglial cells.