Dynamic changes in murine forebrain miR-211 expression associate with cholinergic imbalances and epileptiform activity.

Epilepsy is a common neurological disease, manifested in unprovoked recurrent seizures. Epileptogenesis may develop due to genetic or pharmacological origins or following injury, but it remains unclear how the unaffected brain escapes this susceptibility to seizures. Here, we report that dynamic changes in forebrain microRNA (miR)-211 in the mouse brain shift the threshold for spontaneous and pharmacologically induced seizures alongside changes in the cholinergic pathway genes, implicating this miR in the avoidance of seizures. We identified miR-211 as a putative attenuator of cholinergic-mediated seizures by intersecting forebrain miR profiles that were Argonaute precipitated, synaptic vesicle target enriched, or differentially expressed under pilocarpine-induced seizures, and validated TGFBR2 and the nicotinic antiinflammatory acetylcholine receptor nAChRa7 as murine and human miR-211 targets, respectively. To explore the link between miR-211 and epilepsy, we engineered dTg-211 mice with doxycycline-suppressible forebrain overexpression of miR-211. These mice reacted to doxycycline exposure by spontaneous electrocorticography-documented nonconvulsive seizures, accompanied by forebrain accumulation of the convulsive seizures mediating miR-134. RNA sequencing demonstrated in doxycycline-treated dTg-211 cortices overrepresentation of synaptic activity, Ca2+ transmembrane transport, TGFBR2 signaling, and cholinergic synapse pathways. Additionally, a cholinergic dysregulated mouse model overexpressing a miR refractory acetylcholinesterase-R splice variant showed a parallel propensity for convulsions, miR-211 decreases, and miR-134 elevation. Our findings demonstrate that in mice, dynamic miR-211 decreases induce hypersynchronization and nonconvulsive and convulsive seizures, accompanied by expression changes in cholinergic and TGFBR2 pathways as well as in miR-134. Realizing the importance of miR-211 dynamics opens new venues for translational diagnosis of and interference with epilepsy.

TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis.

See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.

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 Alzheimer's 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 Aß1-42. 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 Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.

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 Alzheimer's 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'.

What can Drosophila teach us about iron-accumulation neurodegenerative disorders?

The present review focuses on the development and use of Drosophila for modeling neurodegenerative disorders that involve iron accumulation, in particular Friedrich's Ataxia (FRDA) and Neurodegeneration with Iron accumulation (NBIA). Several Drosophila models of such disorders have been introduced successfully in the last few years. Here we review these models and note on the feasibility and advantages of using fly models for understanding the molecular and cellular bases of these devastating diseases.

Molecular Regulatory Pathways Link Sepsis With Metabolic Syndrome: Non-coding RNA Elements Underlying the Sepsis/Metabolic Cross-Talk.

Sepsis and metabolic syndrome (MetS) are both inflammation-related entities with high impact for human health and the consequences of concussions. Both represent imbalanced parasympathetic/cholinergic response to insulting triggers and variably uncontrolled inflammation that indicates shared upstream regulators, including short microRNAs (miRs) and long non-coding RNAs (lncRNAs). These may cross talk across multiple systems, leading to complex molecular and clinical outcomes. Notably, biomedical and RNA-sequencing based analyses both highlight new links between the acquired and inherited pathogenic, cardiac and inflammatory traits of sepsis/MetS. Those include the HOTAIR and MIAT lncRNAs and their targets, such as miR-122, -150, -155, -182, -197, -375, -608 and HLA-DRA. Implicating non-coding RNA regulators in sepsis and MetS may delineate novel high-value biomarkers and targets for intervention.

Antisense miR-132 blockade via the AChE-R splice variant mitigates cortical inflammation.

MicroRNA (miR)-132 brain-to-body messages suppress inflammation by targeting acetylcholinesterase (AChE), but the target specificity of 3'-AChE splice variants and the signaling pathways involved remain unknown. Using surface plasmon resonance (SPR), we identified preferential miR-132 targeting of soluble AChE-R over synaptic-bound AChE-S, potentiating miR-132-mediated brain and body cholinergic suppression of pro-inflammatory cytokines. Inversely, bacterial lipopolysaccharide (LPS) reduced multiple miR-132 targets, suppressed AChE-S more than AChE-R and elevated inflammatory hallmarks. Furthermore, blockade of peripheral miR-132 by chemically protected AM132 antisense oligonucleotide elevated muscle AChE-R 10-fold over AChE-S, and cortical miRNA-sequencing demonstrated inverse brain changes by AM132 and LPS in immune-related miRs and neurotransmission and cholinergic signaling pathways. In neuromuscular junctions, AM132 co-elevated the nicotinic acetylcholine receptor and AChE, re-balancing neurotransmission and reaching mild muscle incoordination. Our findings demonstrate preferential miR-132-induced modulation of AChE-R which ignites bidirectional brain and body anti-inflammatory regulation, underscoring splice-variant miR-132 specificity as a new complexity level in inflammatory surveillance.

AChE and RACK1 promote the anti-inflammatory properties of fluoxetine.

Selective serotonin reuptake inhibitors (SSRIs) show anti-inflammatory effects, suggesting a possible interaction with both Toll-like-receptor 4 (TLR4) responses and cholinergic signaling through as yet unclear molecular mechanism(s). Our results of structural modeling support the concept that the antidepressant fluoxetine physically interacts with the TLR4-myeloid differentiation factor-2 complex at the same site as bacterial lipopolysaccharide (LPS). We also demonstrate reduced LPS-induced pro-inflammatory interleukin-6 and tumor necrosis factor alpha in human peripheral blood mononuclear cells preincubated with fluoxetine. Furthermore, we show that fluoxetine intercepts the LPS-induced decreases in intracellular acetylcholinesterase (AChE-S) and that AChE-S interacts with the nuclear factor kappa B (NFκB)-activating intracellular receptor for activated C kinase 1 (RACK1). This interaction may prevent NFκB activation by residual RACK1 and its interacting protein kinase PKCßII. Our findings attribute the anti-inflammatory properties of SSRI to surface membrane interference with leukocyte TLR4 activation accompanied by intracellular limitation of pathogen-inducible changes in AChE-S, RACK1, and PKCßII.

A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies) that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.