Hermona Soreq

Alteration of the microRNA network during the progression of Alzheimer's disease

An overview of miRNAs altered in Alzheimers disease (AD) was established by profiling the hippocampus of a cohort of 41 late‐onset AD (LOAD) patients and 23 controls, showing deregulation of 35 miRNAs. Profiling of miRNAs in the prefrontal cortex of a second independent cohort of 49 patients grouped by Braak stages revealed 41 deregulated miRNAs. We focused on miR‐132‐3p which is strongly altered in both brain areas. Downregulation of this miRNA occurs already at Braak stages III and IV, before loss of neuron‐specific miRNAs. Next‐generation sequencing confirmed a strong decrease of miR‐132‐3p and of three family‐related miRNAs encoded by the same miRNA cluster on chromosome 17. Deregulation of miR‐132‐3p in AD brain appears to occur mainly in neurons displaying Tau hyper‐phosphorylation. We provide evidence that miR‐132‐3p may contribute to disease progression through aberrant regulation of mRNA targets in the Tau network. The transcription factor (TF) FOXO1a appears to be a key target of miR‐132‐3p in this pathway.

Hippocampal microRNA-132 mediates stress-inducible cognitive deficits through its acetylcholinesterase target

Diverse stress stimuli induce long-lasting cognitive deficits, but the underlying molecular mechanisms are still incompletely understood. Here, we report three different stress models demonstrating that stress-inducible increases in microRNA-132 (miR-132) and consequent decreases in its acetylcholinesterase (AChE) target are causally involved. In a mild model of predator scent-induced anxiety, we demonstrate long-lasting hippocampal elevation of miR-132, accompanied by and associated with reduced AChE activity. Using lentiviral-mediated suppression of “synaptic” AChE-S mRNA, we quantified footshock stress-inducible changes in miR-132 and AChE and its corresponding cognitive damages. Stressed mice showed long-lasting impairments in the Morris water maze. In contrast, pre-stress injected AChE-suppressing lentivirus, but not a control virus, reduced hippocampal levels of both miR-132 and AChE and maintained similar cognitive performance to that of naïve, non-stressed mice. To dissociate between miR-132 and synaptic AChE-S as potential causes for stress-inducible cognitive deficits, we further used engineered TgR mice with enforced over-expression of the soluble “readthrough” AChE-R variant without the 3′-untranslated region binding site for miR-132. TgR mice displayed excess AChE-R in hippocampal neurons, enhanced c-fos labeling and correspondingly intensified reaction to the cholinergic agonist pilocarpine. They further showed excessive hippocampal expression of miR-132, accompanied by reduced host AChE-S mRNA and the GTPase activator p250GAP target of miR-132. At the behavioral level, TgR mice showed abnormal nocturnal locomotion patterns and serial maze mal-performance in spite of their reduced AChE-S levels. Our findings attribute stress-inducible cognitive impairments to cholinergic-mediated induction of miR-132 and consequently suppressed ACHE-S, opening venues for intercepting these miR-132-mediated damages.

Cholinergic-associated loss of hnRNP-A/B in Alzheimer's disease impairs cortical splicing and cognitive function in mice

Genetic studies link inherited errors in RNA metabolism to familial neurodegenerative disease. Here, we report such errors and the underlying mechanism in sporadic Alzheimers disease (AD). AD entorhinal cortices presented globally impaired exon exclusions and selective loss of the hnRNP A/B splicing factors. Supporting functional relevance, hnRNP A/B knockdown induced alternative splicing impairments and dendrite loss in primary neurons, and memory and electrocorticographic impairments in mice. Transgenic mice with disease‐associated mutations in APP or Tau displayed no alterations in hnRNP A/B suggesting that its loss in AD is independent of Aβ and Tau toxicity. However, cholinergic excitation increased hnRNP A/B levels while in vivo neurotoxin‐mediated destruction of cholinergic neurons caused cortical AD‐like decrease in hnRNP A/B and recapitulated the alternative splicing pattern of AD patients. Our findings present cholinergic‐mediated hnRNP A/B loss and impaired RNA metabolism as important mechanisms involved in AD.

Long Non-Coding RNA and Alternative Splicing Modulations in Parkinson's Leukocytes Identified by RNA Sequencing

The continuously prolonged human lifespan is accompanied by increase in neurodegenerative diseases incidence, calling for the development of inexpensive blood-based diagnostics. Analyzing blood cell transcripts by RNA-Seq is a robust means to identify novel biomarkers that rapidly becomes a commonplace. However, there is lack of tools to discover novel exons, junctions and splicing events and to precisely and sensitively assess differential splicing through RNA-Seq data analysis and across RNA-Seq platforms. Here, we present a new and comprehensive computational workflow for whole-transcriptome RNA-Seq analysis, using an updated version of the software AltAnalyze, to identify both known and novel high-confidence alternative splicing events, and to integrate them with both protein-domains and microRNA binding annotations. We applied the novel workflow on RNA-Seq data from Parkinsons disease (PD) patients leukocytes pre- and post- Deep Brain Stimulation (DBS) treatment and compared to healthy controls. Disease-mediated changes included decreased usage of alternative promoters and N-termini, 5′-end variations and mutually-exclusive exons. The PD regulated FUS and HNRNP A/B included prion-like domains regulated regions. We also present here a workflow to identify and analyze long non-coding RNAs (lncRNAs) via RNA-Seq data. We identified reduced lncRNA expression and selective PD-induced changes in 13 of over 6,000 detected leukocyte lncRNAs, four of which were inversely altered post-DBS. These included the U1 spliceosomal lncRNA and RP11-462G22.1, each entailing sequence complementarity to numerous microRNAs. Analysis of RNA-Seq from PD and unaffected controls brains revealed over 7,000 brain-expressed lncRNAs, of which 3,495 were co-expressed in the leukocytes including U1, which showed both leukocyte and brain increases. Furthermore, qRT-PCR validations confirmed these co-increases in PD leukocytes and two brain regions, the amygdala and substantia-nigra, compared to controls. This novel workflow allows deep multi-level inspection of RNA-Seq datasets and provides a comprehensive new resource for understanding disease transcriptome modifications in PD and other neurodegenerative diseases.

MicroRNA-132 modulates cholinergic signaling and inflammation in human inflammatory bowel disease.

Background:MicroRNA-132 (miR-132) targets acetylcholinesterase (AChE) and potentiates the cholinergic blockade of inflammatory reactions in cultured cells and experimental mice, but the implications of this interaction to human inflammatory disease remained unexplored. This study aimed to test whether miR-132 is causally involved in anti-inflammatory reactions of patients with inflammatory bowel disease (IBD) and modulates vagal tone and consequently inflammation in patients with IBD. Methods:We prospectively measured inflammation readouts and the cholinergic status (total capacity for hydrolyzing acetylcholine in ones circulation), and AChE activity in 2 independent cohorts of patients with IBD and quantified miR-132 levels in intestinal tissue biopsies removed at colonoscopy from inflamed and apparently quiescent tissues of tested volunteers. Results:MiR-132 levels are higher in inflamed compared with apparently quiescent intestinal biopsies from patients with IBD. Correspondingly, the cholinergic status and AChE activity was significantly lower in patients with IBD suffering from moderate–severe disease as compared with healthy controls or patient with IBD presenting low disease severity. Patients with IBD (n = 16) presented lower AChE activity compared with healthy controls (n = 33; 289 ± 128 AU versus 391 ± 102 AU, P = 0.001), and a negative correlation between AChE activity and C-reactive protein levels (r = −0.47, P = 0.01). Corroborating these observations in an additional cohort of participants, C-reactive protein and AChE activity were negatively correlated in patients with moderate–severe disease (n = 16; r = −0.6, P = 0.04) and positively correlated in healthy controls (n = 74, r = 0.24, P = 0.046). Conclusions:Taken together, these findings support an inflammation-dependent homeostatic role for the regulation by miR-132 of AChE in IBD, opening new venues for therapeutic interference.

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.

Competing targets of microRNA-608 affect anxiety and hypertension

MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA–target interaction can simultaneously affect multiple other miRNA–target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3′-untranslated region (3′UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.

Heterogeneous nuclear ribonucleoprotein A1 in health and neurodegenerative disease: from structural insights to post-transcriptional regulatory roles.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of conserved nuclear proteins that associate with nascent RNA polymerase II transcripts to yield hnRNP particles, playing key roles in mRNA metabolism, DNA-related functions and microRNA biogenesis. HnRNPs accompany transcripts from stages of transcriptional regulation through splicing and post-transcriptional regulation, and are believed to affect the majority of expressed genes in mammals. Most hnRNP mRNA transcripts undergo alternative splicing and post-translational modifications, to yield a remarkable diversity of proteins with numerous functional elements that work in concert in their multiple functions. Therefore, mis-regulation of hnRNPs leads to different maladies. Here, we focus on the role of one of the best-known members of this protein family, hnRNP A1 in RNA metabolism, and address recent works that note its multileveled involvement in several neurodegenerative disorders. Initially discovered as a DNA binding protein, hnRNP A1 includes two RNA recognition motifs, and post-translational modifications of these and other regions in this multifunctional protein alter both its nuclear pore shuttling properties and its RNA interactions and affect transcription, mRNA splicing and microRNA biogenesis. HnRNP A1 plays several key roles in neuronal functioning and its depletion, either due to debilitated cholinergic neurotransmission or under autoimmune reactions causes drastic changes in RNA metabolism. Consequently, hnRNP A1 decline contributes to the severity of symptoms in several neurodegenerative diseases, including Alzheimers disease (AD), spinal muscular atrophy (SMA), fronto-temporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), hereditary spastic paraparesis (HSP) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). At the translational level, these properties of hnRNP A1 led to massive research efforts aimed at developing RNA-targeted therapeutic tools such as splicing-modulating oligonucleotides with promising pharmaceutical potential. HnRNP A1 thus presents an intriguing example for the complexity and importance of heteronuclear ribonucleoproteins in health and disease. This article is part of a Special Issue entitled RNA and splicing regulation in neurodegeneration.

Cholinergic involvement and manipulation approaches in multiple system disorders.

Within the autonomic system, acetylcholine signaling contributes simultaneously and interactively to cognitive, behavioral, muscle and immune functions. Therefore, manipulating cholinergic parameters such as the activities of the acetylcholine hydrolyzing enzymes in body fluids or the corresponding transcript levels in blood leukocytes can change the global status of the autonomic system in treated individuals. Specifically, cholinesterase activities are subject to rapid and effective changes. The enzyme activity baseline increases with age and body mass index and depends on gender and ethnic origin. Also, the corresponding DNA (for detecting mutations) and RNA (for measuring specific mRNA transcripts) of cholinergic genes present individual variability. In leukocytes, acetylcholine inhibits the production of pro-inflammatory cytokines, suggesting relevance of cholinergic parameters to both the basal levels and to disease-induced inflammation. Inversely, acetylcholine levels increase under various stress stimuli, inducing changes in autonomic system molecules (e.g., pro-inflammatory cytokines) which can penetrate the brain; therefore, manipulating these levels can also effect brain reactions, mainly of anxiety, depression and pain. Additionally, neurodegenerative diseases often involve exacerbated inflammation, depression and anxiety, providing a focus interest group for cholinergic manipulations. In Alzheimers disease, the systemic cholinergic impairments reflect premature death of cholinergic neurons. The decline of cholinesterases in the serum of Parkinsons disease and post- stroke patients, discovery of the relevant microRNAs and the growing range of use of anticholinesterase medications all call for critical re-inspection of established and novel approaches for manipulating cholinergic parameters.

Predicted overlapping microRNA regulators of acetylcholine packaging and degradation in neuroinflammation-related disorders

MicroRNAs (miRNAs) can notably control many targets each and regulate entire cellular pathways, but whether miRNAs can regulate complete neurotransmission processes is largely unknown. Here, we report that miRNAs with complementary sequence motifs to the key genes involved in acetylcholine (ACh) synthesis and/or packaging show massive overlap with those regulating ACh degradation. To address this topic, we first searched for miRNAs that could target the 3′-untranslated regions of the choline acetyltransferase (ChAT) gene that controls ACh synthesis; the vesicular ACh transporter (VAChT), encoded from an intron in the ChAT gene and the ACh hydrolyzing genes acetyl- and/or butyrylcholinesterase (AChE, BChE). Intriguingly, we found that many of the miRNAs targeting these genes are primate-specific, and that changes in their levels associate with inflammation, anxiety, brain damage, cardiac, neurodegenerative, or pain-related syndromes. To validate the in vivo relevance of this dual interaction, we selected the evolutionarily conserved miR-186, which targets both the stress-inducible soluble “readthrough” variant AChE-R and the major peripheral cholinesterase BChE. We exposed mice to predator scent stress and searched for potential associations between consequent changes in their miR-186, AChE-R, and BChE levels. Both intestinal miR-186 as well as BChE and AChE-R activities were conspicuously elevated 1 week post-exposure, highlighting the previously unknown involvement of miR-186 and BChE in psychological stress responses. Overlapping miRNA regulation emerges from our findings as a recently evolved surveillance mechanism over cholinergic neurotransmission in health and disease; and the corresponding miRNA details and disease relevance may serve as a useful resource for studying the molecular mechanisms underlying this surveillance.