Lilach Soreq

Small RNA sequencing-microarray analyses in Parkinson leukocytes reveal deep brain stimulation-induced splicing changes that classify brain region transcriptomes

MicroRNAs (miRNAs) are key post transcriptional regulators of their multiple target genes. However, the detailed profile of miRNA expression in Parkinsons disease, the second most common neurodegenerative disease worldwide and the first motor disorder has not been charted yet. Here, we report comprehensive miRNA profiling by next-generation small-RNA sequencing, combined with targets inspection by splice-junction and exon arrays interrogating leukocyte RNA in Parkinsons disease patients before and after deep brain stimulation (DBS) treatment and of matched healthy control volunteers (HC). RNA-Seq analysis identified 254 miRNAs and 79 passenger strand forms as expressed in blood leukocytes, 16 of which were modified in patients pre-treatment as compared to HC. 11 miRNAs were modified following brain stimulation 5 of which were changed inversely to the disease induced changes. Stimulation cessation further induced changes in 11 miRNAs. Transcript isoform abundance analysis yielded 332 changed isoforms in patients compared to HC, which classified brain transcriptomes of 47 PD and control independent microarrays. Functional enrichment analysis highlighted mitochondrion organization. DBS induced 155 splice changes, enriched in ubiquitin homeostasis. Cellular composition analysis revealed immune cell activity pre and post treatment. Overall, 217 disease and 74 treatment alternative isoforms were predictably targeted by modified miRNAs within both 3′ and 5′ untranslated ends and coding sequence sites. The stimulation-induced network sustained 4 miRNAs and 7 transcripts of the disease network. We believe that the presented dynamic networks provide a novel avenue for identifying disease and treatment-related therapeutic targets. Furthermore, the identification of these networks is a major step forward in the road for understanding the molecular basis for neurological and neurodegenerative diseases and assessment of the impact of brain stimulation on human diseases.

Major Shifts in Glial Regional Identity Are a Transcriptional Hallmark of Human Brain Aging

Summary Gene expression studies suggest that aging of the human brain is determined by a complex interplay of molecular events, although both its region- and cell-type-specific consequences remain poorly understood. Here, we extensively characterized aging-altered gene expression changes across ten human brain regions from 480 individuals ranging in age from 16 to 106 years. We show that astrocyte- and oligodendrocyte-specific genes, but not neuron-specific genes, shift their regional expression patterns upon aging, particularly in the hippocampus and substantia nigra, while the expression of microglia- and endothelial-specific genes increase in all brain regions. In line with these changes, high-resolution immunohistochemistry demonstrated decreased numbers of oligodendrocytes and of neuronal subpopulations in the aging brain cortex. Finally, glial-specific genes predict age with greater precision than neuron-specific genes, thus highlighting the need for greater mechanistic understanding of neuron-glia interactions in aging and late-life diseases.

Advanced microarray analysis highlights modified neuro-immune signaling in nucleated blood cells from Parkinson's disease patients.

Laboratory tests for Parkinsons disease (PD) were recently extended to microarray analyses of nucleated blood cells. Here, we report the use of statistical and gene ontology tools to re-examine these microarray data. Distribution plots and PCA mapping enabled removal of several outliers out of the 105 analyzed PD and control samples, which improved the discriminative power for PD blood cells compared to healthy and neurological disease controls. Combined with gene ontology tests, our findings point at neuro-immune signaling-related transcripts as distinctly expressed early in PD progress and call for exploiting microarray tests also for follow-up of PD treatment efficacy.

Math1 target genes are enriched with evolutionarily conserved clustered E-box binding sites

The basic helix-loop-helix (bHLH) transcription factor Math1 and its orthologs are fundamental for proper development of various neuronal subpopulations, such as cerebellar granule cells, D1 interneurons in the spinal cord, and inner ear hair cells. Although crucial for neurogenesis, the mechanisms by which Math1 specifically recognizes its direct targets are not fully understood. To search for direct and indirect target genes and signaling pathways controlled by Math1, we analyzed the effect of Math1 knockout on the expression profile of multiple genes in the embryonic cerebellum. Eighteen differentially expressed transcripts were identified and found to belong to a few developmentally-related functional groups, such as transcriptional regulation, proliferation, organogenesis, signal transduction, and apoptosis. Importantly, genomic analysis of E-box motifs has identifieda significant enrichment and clustering of MATH1-binding E-boxes only in a subset of differentially expressed genes (Nr2f6, Hras1, and Hes5) in both mouse and man. Moreover, Math1 was shown by chromatin immuno-precipitation (ChIP) to bind, and by a luciferase reporter assay to activate transcription, of an upstream genomic fragment of Nr2f6. Taken together, we propose that when putative direct targets of Math1 are being selected for detailed studies on DNA microarray hybridization, the enrichment and clustering of binding E-boxes in multiple species may be helpful criteria. Our findings may be useful to the study of other bHLH transcription factors, many of which control the development of the nervous system.

Exon Arrays Reveal Alternative Splicing Aberrations in Parkinson's Disease Leukocytes

Background: Parkinson’s disease (PD) is the second most frequent neurodegenerative disease worldwide. Clinical diagnosis can only be made when the vast majority of the dopaminergic cell population has died. However, the cause(s) for sporadic PD is/are yet unclear. Transcript changes have recently been described in PD patients’ whole blood cells, but corresponding splicing patterns remained unknown. Objective: To search for alternative splicing aberrations in PD patients’ blood leukocytes. Methods: We applied exon microarrays to profile PD patients’ blood leukocyte mRNA. Exon level splicing analysis served as a basis for downstream classification and functional analyses. Results: Patients and carefully matched controls were classified by the splicing exon profiles of their leukocyte transcripts. Specifically, many exons were downregulated in PD patients compared to controls. Functional analysis highlighted aberrant splicing of PD-related transcripts and impaired NF-ĸB cascade and immune response. Conclusion: PD patient’s blood leukocytes exhibit alternative splicing of numerous transcripts. The aberrant alternative splicing in PD patients’ blood cells has potential implications for early diagnosis and future therapeutics.

Meta-analysis of genetic and environmental Parkinson's disease models reveals a common role of mitochondrial protection pathways.

Both genetic and environmental factors trigger risks of and protection from Parkinsons disease, the second most common neurodegenerative syndrome, but possible inter-relationships between these risk and protection processes were not yet explored. By examining gene expression changes in the brains of mice under multiple treatments that increase or attenuate PD symptoms we detected underlying disease and protection-associated genes and pathways. In search for potential links between these different genes and pathways, we conducted meta-analysis on 131 brain region transcriptomes from mice over-expressing native or mutated α-synuclein (SNCA) with or without the protective HSP70 chaperone, or exposed to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), with or without the protective acetylcholinesterase (AChE-R) variant. All these models showed shared risk-inducible and protection-suppressible transcript modifications. Self-organized map (SOM) classification revealed risk- and protection-associated alterations in nuclear and mitochondrial metal ion-regulated transcripts, respectively; Gene Ontology based analysis validated these pathways. To complement this approach, and identify potential outcome damages, we further searched for shared functional enrichments in the lists of genes detected in young SNCA mutant or in old SNCA mutants and MPTP-exposed mice. This post-hoc functional analysis identified early-onset changes in Parkinsonian, immune and alternative splicing pathways which shifted into late-onset or exposure-associated NFkB-mediated neuro-inflammation. Our study suggests metal ions-mediated cross-talk between nuclear and mitochondrial pathways by both environmental and genetic risk and protective factors involved in Parkinsons disease, which eventually culminates in neuro-inflammation. Together, these findings offer new insights and novel targets for therapeutic interference with the gene-environment interactions underlying sporadic PD.

Identifying alternative hyper-splicing signatures in MG-thymoma by exon arrays.

Background The vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes. Methodology/Principal Findings We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO) statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events. Conclusions/Significance Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatability gene HLA-DRB1 and interleukin (IL)19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF2 and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH), Reverse Transcription–Polymerase Chain Reaction (RT-PCR) and mass spectrometry (MS) followed by peptide sequencing. Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in MG-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches.

Cholinergic Surveillance over Hippocampal RNA Metabolism and Alzheimer's-Like Pathology

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimers disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aβ1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimers-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.

Deep brain stimulation modulates nonsense-mediated RNA decay in Parkinson’s patients leukocytes

BackgroundNonsense-Mediated decay (NMD) selectively degrades mRNA transcripts that carry premature stop codons. NMD is often triggered by alternative splicing (AS) modifications introducing such codons. NMD plays an important regulatory role in brain neurons, but the in vivo dynamics of AS and NMD changes in neurological diseases and under treatment were scarcely explored.ResultsHere, we report exon arrays analysis of leukocyte mRNA AS events prior to and following Deep Brain Stimulation (DBS) neurosurgery, which efficiently improves the motor symptoms of Parkinson’s disease (PD), the leading movement disorder, and is increasingly applied to treat other diseases. We also analyzed publicly available exon array dataset of whole blood cells from mixed early and advanced PD patients. Our in-house exon array dataset of leukocyte transcripts was derived from advanced PD patients’ pre- and post-DBS stimulation and matched healthy control volunteers. The mixed cohort exhibited 146 AS changes in 136 transcripts compared to controls, including 9 NMD protein-level assessed events. In comparison, PD patients from our advanced cohort differed from healthy controls by 319 AS events in 280 transcripts, assessed as inducing 27 protein-level NMD events. DBS stimulation induced 254 AS events in 229 genes as compared to the pre-DBS state including 44 NMD inductions. A short, one hour electrical stimulus cessation caused 234 AS changes in 125 genes compared to ON-stimulus state, 22 of these were assessed for NMD. Functional analysis highlighted disease-induced DNA damage and inflammatory control and its reversal under ON and OFF stimulus as well as alternative splicing in all the tested states.ConclusionsThe study findings indicate a potential role for NMD both in PD and following electrical brain stimulation. Furthermore, our current observations entail future implications for developing therapies for PD, and for interfering with the impaired molecular mechanisms that underlie PD and other neurodegenerative and neurological disorders, as well as DBS-treatable conditions in general.

Deep brain stimulation induces rapidly reversible transcript changes in Parkinson's leucocytes

Subthalamic deep brain stimulation (DBS) reversibly modulates Parkinsons disease (PD) motor symptoms, providing an unusual opportunity to compare leucocyte transcripts in the same individuals before and after neurosurgery and 1 hr after stimulus cessation (ON‐ and OFF‐stimulus). Here, we report DBS‐induced reversibility and OFF‐stimulus restoration in 12 of 16 molecular functions and 3 of 4 biological processes shown in exon microarrays to be differentially expressed between PD patients and controls, post‐DBS from pre‐DBS and OFF from ON states. Intriguingly, 6 of 18 inflammation and immune‐related functions exhibited reversibility, and the extent of stimulus‐induced changes correlated with the neurological DBS efficacy, suggesting mechanistic implications. A minimal list of 29 transcripts that changed in all three comparisons between states discriminated pre‐surgery and OFF states from post‐surgery and controls. Six of these transcripts were found to be able to distinguish between PD patients and both healthy controls and patients with other neurological diseases in a previously published whole blood 3’ array data study of early PD patients. Our findings support the future use of this approach for identifying targets for therapeutic intervention and assessing the efficacy of current and new treatments in this and other neurological diseases.