Knockout of the longevity gene Klotho perturbs aging and Alzheimer's disease-linked brain microRNAs and tRNA fragments.

Overexpression of the longevity gene Klotho prolongs lifespan, while its knockout shortens lifespan and impairs cognition via perturbation of myelination and synapse formation. However, comprehensive analysis of Klotho knockout effects on mammalian brain transcriptomics is lacking. Here, we report that Klotho knockout alters the levels of aging- and cognition related mRNAs, long non-coding RNAs, microRNAs and tRNA fragments. These include altered neuronal and glial regulators in murine models of aging and Alzheimer's disease and in human Alzheimer's disease post-mortem brains. We further demonstrate interaction of the knockout-elevated tRNA fragments with the spliceosome, possibly affecting RNA processing. Last, we present cell type-specific short RNA-seq datasets from FACS-sorted neurons and microglia of live human brain tissue demonstrating in-depth cell-type association of Klotho knockout-perturbed microRNAs. Together, our findings reveal multiple RNA transcripts in both neurons and glia from murine and human brain that are perturbed in Klotho deficiency and are aging- and neurodegeneration-related.

Ribosomal protein L24 mediates mammalian microRNA processing in an evolutionarily conserved manner.

To investigate the mechanism(s) underlying the expression of primate-specific microRNAs (miRs), we sought DNA regulatory elements and proteins mediating expression of the primate-specific hsa-miR-608 (miR-608), which is located in the SEMA4G gene and facilitates the cholinergic blockade of inflammation by targeting acetylcholinesterase mRNA. 'Humanized' mice carrying pre-miR-608 flanked by 250 bases of endogenous sequences inserted into the murine Sema4g gene successfully expressed miR-608. Moreover, by flanking miR-608 by shortened fragments of its human genome region we identified an active independent promoter within the 150 nucleotides 5' to pre-miR-608, which elevated mature miR-608 levels by 100-fold in transfected mouse- and human-originated cells. This highlighted a regulatory role of the 5' flank as enabling miR-608 expression. Moreover, pull-down of the 150-base 5' sequence revealed its interaction with ribosomal protein L24 (RPL24), implicating an additional mechanism controlling miR-608 levels. Furthermore, RPL24 knockdown altered the expression of multiple miRs, and RPL24 immunoprecipitation indicated that up- or down-regulation of the mature miRs depended on whether their precursors bind RPL24 directly. Finally, further tests showed that RPL24 interacts directly with DDX5, a component of the large microprocessor complex, to inhibit miR processing. Our findings reveal that RPL24, which has previously been shown to play a role in miR processing in Arabidopsis thaliana, has a similar evolutionarily conserved function in miR biogenesis in mammals. We thus characterize a novel extra-ribosomal role of RPL24 in primate miR regulation.

Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via ß-Klotho and perilipin 2.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5'tRF transfer RNA fragments and microRNA miR-194-5p. METHODS: Combined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5'tRF and miR-194-5p. RESULTS: Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5'tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5'tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5'tRF mimic elevated mRNA levels of the metabolic regulator ß-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5'tRF levels. CONCLUSION: Our findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5'tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis.

Altered levels of variant cholinesterase transcripts contribute to the imbalanced cholinergic signaling in Alzheimer's and Parkinson's disease.

Acetylcholinesterase and butyrylcholinesterase (AChE and BChE) are involved in modulating cholinergic signaling, but their roles in Alzheimer's and Parkinson's diseases (AD and PD) remain unclear. We identified a higher frequency of the functionally impaired BCHE-K variant (rs1803274) in AD and PD compared to controls and lower than in the GTEx dataset of healthy individuals (n = 651); in comparison, the prevalence of the 5'-UTR (rs1126680) and intron 2 (rs55781031) single-nucleotide polymorphisms (SNPs) of BCHE and ACHE's 3'-UTR (rs17228616) which disrupt AChE mRNA targeting by miR-608 remained unchanged. qPCR validations confirmed lower levels of the dominant splice variant encoding the "synaptic" membrane-bound ACHE-S in human post-mortem superior temporal gyrus samples from AD and in substantia nigra (but not amygdala) samples from PD patients (n = 79, n = 67) compared to controls, potentially reflecting region-specific loss of cholinergic neurons. In contradistinction, the non-dominant "readthrough" AChE-R mRNA variant encoding for soluble AChE was elevated (p < 0.05) in the AD superior temporal gyrus and the PD amygdala, but not in the neuron-deprived substantia nigra. Elevated levels of BChE (p < 0.001) were seen in AD superior temporal gyrus. Finally, all three ACHE splice variants, AChE-S, AChE-R, and N-extended AChE, were elevated in cholinergic-differentiated human neuroblastoma cells, with exposure to the oxidative stress agent paraquat strongly downregulating AChE-S and BChE, inverse to their upregulation under exposure to the antioxidant simvastatin. The multi-leveled changes in cholinesterase balance highlight the role of post-transcriptional regulation in neurodegeneration. (235).

A Parkinson's disease CircRNAs Resource reveals a link between circSLC8A1 and oxidative stress.

Circular RNAs (circRNAs) are brain-abundant RNAs of mostly unknown functions. To seek their roles in Parkinson's disease (PD), we generated an RNA sequencing resource of several brain region tissues from dozens of PD and control donors. In the healthy substantia nigra (SN), circRNAs accumulate in an age-dependent manner, but in the PD SN this correlation is lost and the total number of circRNAs reduced. In contrast, the levels of circRNAs are increased in the other studied brain regions of PD patients. We also found circSLC8A1 to increase in the SN of PD individuals. CircSLC8A1 carries 7 binding sites for miR-128 and is strongly bound to the microRNA effector protein Ago2. Indeed, RNA targets of miR-128 are also increased in PD individuals, suggesting that circSLC8A1 regulates miR-128 function and/or activity. CircSLC8A1 levels also increased in cultured cells exposed to the oxidative stress-inducing agent paraquat but were decreased in cells treated with the neuroprotective antioxidant regulator drug Simvastatin. Together, our work links circSLC8A1 to oxidative stress-related Parkinsonism and suggests further exploration of its molecular function in PD.

A lncRNA survey finds increases in neuroprotective LINC-PINT in Parkinson's disease substantia nigra.

Recent reports highlight regulatory functions of long noncoding RNAs (lncRNAs) in neurodegeneration and aging, but biomedical implications remain limited. Here, we report an rRNA-depletion-based long RNA-Sequencing Resource of 65 substantia nigra, amygdala, and medial temporal gyrus samples from Parkinson's disease (PD) and matched control brains. Using a lncRNA-focused analysis approach to identify functionally important transcripts, we discovered and prioritized many lncRNAs dysregulated in PD. Those included pronounced elevation of the P53-induced noncoding transcript LINC-PINT in the substantia nigra of PD patients, as well as in additional models of oxidative stress and PD. Intriguingly, we found that LINC-PINT is a primarily neuronal transcript which showed conspicuous increases in maturing primary culture neurons. LINC-PINT also accumulated in several brain regions of Alzheimer's and Huntington's disease patients and decreased with healthy brain aging, suggesting a general role in aging and neurodegeneration for this lncRNA. RNAi-mediated depletion of LINC-PINT exacerbated the death of cultured N2A and SH-SY5Y cells exposed to oxidative stress, highlighting a previously undiscovered neuroprotective role for this tumor-inducible lncRNA in the brains of patients with neurodegenerative disorders.

NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress.

Recent reports attribute numerous regulatory functions to the nuclear paraspeckle-forming long noncoding RNA, nuclear enriched assembly transcript 1 (NEAT1), but the implications of its involvement in Parkinson's disease (PD) remain controversial. To address this issue, we assessed NEAT1 expression levels and cell type patterns in the substantia nigra (SN) from 53 donors with and without PD, as well as in interference tissue culture tests followed by multiple in-house and web-available models of PD. PCR quantification identified elevated levels of NEAT1 expression in the PD SN compared with control brains, an elevation that was reproducible across a multitude of disease models. In situ RNA hybridization supported neuron-specific formation of NEAT1-based paraspeckles at the SN and demonstrated coincreases of NEAT1 and paraspeckles in cultured cells under paraquat (PQ)-induced oxidative stress. Furthermore, neuroprotective agents, including fenofibrate and simvastatin, induced NEAT1 up-regulation, whereas RNA interference-mediated depletion of NEAT1 exacerbated death of PQ-exposed cells in a leucine-rich repeat kinase 2-mediated manner. Our findings highlight a novel protective role for NEAT1 in PD and suggest a previously unknown mechanism for the neuroprotective traits of widely used preventive therapeutics.-Simchovitz, A., Hanan, M., Niederhoffer, N., Madrer, N., Yayon, N., Bennett, E. R., Greenberg, D. S., Kadener, S., Soreq, H. NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress.

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.

miRNA-132 induces hepatic steatosis and hyperlipidaemia by synergistic multitarget suppression.

OBJECTIVE: Both non-alcoholic fatty liver disease (NAFLD) and the multitarget complexity of microRNA (miR) suppression have recently raised much interest, but the in vivo impact and context-dependence of hepatic miR-target interactions are incompletely understood. Assessing the relative in vivo contributions of specific targets to miR-mediated phenotypes is pivotal for investigating metabolic processes. DESIGN: We quantified fatty liver parameters and the levels of miR-132 and its targets in novel transgenic mice overexpressing miR-132, in liver tissues from patients with NAFLD, and in diverse mouse models of hepatic steatosis. We tested the causal nature of miR-132 excess in these phenotypes by injecting diet-induced obese mice with antisense oligonucleotide suppressors of miR-132 or its target genes, and measured changes in metabolic parameters and transcripts. RESULTS: Transgenic mice overexpressing miR-132 showed a severe fatty liver phenotype and increased body weight, serum low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and liver triglycerides, accompanied by decreases in validated miR-132 targets and increases in lipogenesis and lipid accumulation-related transcripts. Likewise, liver samples from both patients with NAFLD and mouse models of hepatic steatosis or non-alcoholic steatohepatitis (NASH) displayed dramatic increases in miR-132 and varying decreases in miR-132 targets compared with controls. Furthermore, injecting diet-induced obese mice with anti-miR-132 oligonucleotides, but not suppressing its individual targets, reversed the hepatic miR-132 excess and hyperlipidemic phenotype. CONCLUSIONS: Our findings identify miR-132 as a key regulator of hepatic lipid homeostasis, functioning in a context-dependent fashion via suppression of multiple targets and with cumulative synergistic effects. This indicates reduction of miR-132 levels as a possible treatment of hepatic steatosis.