Noncoding RNAs: Emerging regulators of behavioral complexity.

The mammalian genome encodes thousands of non-coding RNAs (ncRNAs), ranging in size from about 20 nucleotides (microRNAs or miRNAs) to kilobases (long non-coding RNAs or lncRNAs). ncRNAs contribute to a layer of gene regulation that could explain the evolution of massive phenotypic complexity even as the number of protein-coding genes remains unaltered. We propose that low conservation, poor expression, and highly restricted spatiotemporal expression patterns-conventionally considered ncRNAs may affect behavior through direct, rapid, and often sustained regulation of gene expression at the transcriptional, post-transcriptional, or translational levels. Besides these direct roles, their effect during neurodevelopment may manifest as behavioral changes later in the organism's life, especially when exposed to environmental cues like stress and seasonal changes. The lncRNAs affect behavior through diverse mechanisms like sponging of miRNAs, recruitment of chromatin modifiers, and regulation of alternative splicing. We highlight the need for synthesis between rigorously designed behavioral paradigms in model organisms and the wide diversity of behaviors documented by ethologists through field studies on organisms exquisitely adapted to their environmental niche. Comparative genomics and the latest advancements in transcriptomics provide an unprecedented scope for merging field and lab studies on model and non-model organisms to shed light on the role of ncRNAs in driving the behavioral responses of individuals and groups. We touch upon the technical challenges and contentious issues that must be resolved to fully understand the role of ncRNAs in regulating complex behavioral traits. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

A novel antisense lncRNA, ARBAG harboring an RNA destabilizing GWAS variant for C-peptide dictates the transcript isoforms of GABRA6 in cerebellum.

Human disease-associated genetic variations often map to long non-coding RNA (lncRNA) genes; however, elucidation of their functional impact is challenging. We previously identified a new genetic variant rs4454083 (A/G) residing in exon of an uncharacterized lncRNA ARBAG that strongly associates with plasma levels of C-peptide, a hormone that regulates insulin bioavailability. On the opposite strand, rs4454083 also corresponds to an intron of a cerebellum-specific GABA receptor subunit gene GABRA6 that mediates strengthening of inhibitory synapses by insulin. Here, we show that alleles of rs4454083 modulate transcript levels of the antisense gene, ARBAG, which then controls the expression of the sense gene, GABRA6. Predisposing to low C-peptide, GG (a minor allele genotype across ethnicities) stabilizes ARBAG lncRNA causing higher transcript levels in cerebellum. ARBAG lncRNA abundance leads to cleavage of GABRA6 mRNA at the complementary region, resulting in a dysfunctional GABRA6 protein that would not be recruited for synapse strengthening. Together, our findings in human cerebellar cell-line and induced Pluripotent Stem Cells (iPSCs) demonstrate biological role of a novel lncRNA in determining the ratio of mRNA isoforms of a protein-coding gene and the ability of an embedded variant in modulating lncRNA stability leading to inter-individual differences in protein expression.

A Novel Cis-Regulatory lncRNA, Kalnc2, Downregulates Kalrn Protein-Coding Transcripts in Mouse Neuronal Cells.

The KALRN gene encodes several multi-domain protein isoforms that localize to neuronal synapses, conferring the ability to grow and retract dendritic spines and shaping axonal outgrowth, dendrite morphology, and dendritic spine re-modeling. The KALRN genomic locus is implicated in several neurodevelopmental and neuropsychiatric diseases, including autism, schizophrenia, bipolar disease, and intellectual disability. We have previously shown that a novel brain-specific long non-coding RNA (lncRNA) arising from the 5' end of the kalrna gene, called durga, regulates neuronal morphology in zebrafish. Here, we characterized mammalian Kalrn loci, annotating and experimentally validating multiple novel non-coding RNAs, including linear and circular variants. Comparing the mouse and human loci, we show that certain non-coding RNAs and Kalrn protein-coding isoforms arising from the locus show similar expression dynamics during development. In humans, mice, and zebrafish, the 5' end of the Kalrn locus gives rise to a chromatin-associated lncRNA that is present in adult ovaries, besides being expressed during brain development and enriched in certain regions of the adult brain. Ectopic expression of this lncRNA led to the downregulation of all the major Kalrn mRNA isoforms. We propose that this lncRNA arising from the 5' end of the Kalrn locus is functionally the mammalian ortholog of zebrafish lncRNA durga.

Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis.

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.

Early life stressful experiences escalate aggressive behavior in adulthood via changes in transthyretin expression and function.

Escalated and inappropriate levels of aggressive behavior referred to as pathological in psychiatry can lead to violent outcomes with detrimental impact on health and society. Early life stressful experiences might increase the risk of developing pathological aggressive behavior in adulthood, though molecular mechanisms remain elusive. Here, we provide prefrontal cortex and hypothalamus specific transcriptome profiles of peripubertal stress (PPS) exposed Balb/c adult male mice exhibiting escalated aggression and adult female mice resilient to such aberrant behavioral responses. We identify transthyretin (TTR), a well known thyroid hormone transporter, as a key regulator of PPS induced escalated aggressive behavior in males. Brain-region-specific long-term changes in Ttr gene expression and thyroid hormone (TH) availability were evident in PPS induced escalated aggressive male mice, circulating TH being unaltered. Ttr promoter methylation marks were also altered being hypermethylated in hypothalamus and hypomethylated in prefrontal cortex corroborating with its expression pattern. Further, Ttr knockdown in hypothalamus resulted in escalated aggressive behavior in males without PPS and also reduced TH levels and expression of TH-responsive genes (Nrgn, Trh, and Hr). Escalated aggressive behavior along with reduced Ttr gene expression and TH levels in hypothalamus was also evident in next generation F1 male progenies. Our findings reveal that stressful experiences during puberty might trigger lasting escalated aggression by modulating TTR expression in brain. TTR can serve as a potential target in reversal of escalated aggression and related psychopathologies.

Multiple Alu Exonization in 3'UTR of a Primate-Specific Isoform of CYP20A1 Creates a Potential miRNA Sponge.

Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA-miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3'UTR. CYP20A1_Alu-LT, confirmed by 3'RACE, is an outlier in length (9 kb 3'UTR) and widely expressed. Using publically available data sets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15,928 human cortical neurons. miRanda predicts ∼4,700 miRNA recognition elements (MREs) for ∼1,000 miRNAs, primarily originated within these 3'UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. In total, 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared with random sets of differentially expressed genes (P = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA-mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.

Neev, a novel long non-coding RNA, is expressed in chaetoblasts during regeneration of Eisenia fetida.

Eisenia fetida, the common vermicomposting earthworm, shows robust regeneration of posterior segments removed by amputation. During the period of regeneration, the newly formed tissue initially contains only undifferentiated cells but subsequently differentiates into a variety of cell types including muscle, nerve and vasculature. Transcriptomics analysis, reported previously, provided a number of candidate non-coding RNAs that were induced during regeneration. We found that one such long non-coding RNA (lncRNA) is expressed in the skin, only at the base of newly formed chaetae. The spatial organization and precise arrangement of the regenerating chaetae and the cells expressing the lncRNA on the ventral side clearly support a model wherein the regenerating tissue contains a zone of growth and cell division at the tip and a zone of differentiation at the site of amputation. The temporal expression pattern of the lncRNA, named Neev, closely resembled the pattern of chitin synthase genes, implicated in chaetae formation. We found that the lncRNA has 49 sites for binding a set of four microRNAs (miRNAs) while the chitin synthase 8 mRNA has 478 sites. The over-representation of shared miRNA sites suggests that lncRNA Neev may act as a miRNA sponge to transiently de-repress chitin synthase 8 during formation of new chaetae in the regenerating segments of Eisenia fetida.

Interferon mediated neuroinflammation in polyglutamine disease is not caused by RNA toxicity.

Polyglutamine diseases are neurodegenerative diseases that occur due to the expansion of CAG repeat regions in coding sequences of genes. Previously, we have shown the formation of large protein aggregates along with activation of the interferon pathway leading to apoptosis in a cellular model of SCA17. Here, we corroborate our previous results in a tetracycline-inducible model of SCA17. Interferon gamma and lambda were upregulated in 59Q-TBP expressing cells as compared to 16Q-TBP expressing cells. Besides interferon-stimulated genes, the SCA17 model and Huntington's mice brain samples showed upregulation of RNA sensors. However, in this improved model interferon pathway activation and apoptosis preceded the formation of large polyglutamine aggregates, suggesting a role for CAG repeat RNA or soluble protein aggregates. A polyglutamine minus mutant of TBP, expressing polyCAG mRNA, was created by site directed mutagenesis of 10 potential start codons. Neither this long CAG embedded mRNA nor short polyCAG RNA could induce interferon pathway genes or cause apoptosis. polyQ-TBP induced the expression of canonical RNA sensors but the downstream transcription factor, IRF3, showed a muted response. We found that expanded CAG repeat RNA is not sufficient to account for the neuronal apoptosis. Neuronal cells sense expanded CAG repeats embedded in messenger RNAs of protein-coding genes. However, polyglutamine containing protein is responsible for the interferon-mediated neuroinflammation and cell death seen in polyglutamine disease. Thus, we delineate the inflammatory role of CAG repeats in the mRNA from the resulting polyglutamine tract in the protein. Embedded in messenger RNAs of protein-coding regions, the cell senses CAG repeat expansion and induces the expression of RNA sensors and interferon-stimulated genes.

Identification of novel circadian transcripts in the zebrafish retina.

High fecundity, transparent embryos for monitoring the rapid development of organs and the availability of a well-annotated genome has made zebrafish a model organism of choice for developmental biology and neurobiology. This vertebrate model, which is also a favourite in chronobiology studies, shows striking circadian rhythmicity in behaviour. Here, we identify novel genes in the zebrafish genome that are expressed in the zebrafish retina. We further resolve the expression pattern over time and tentatively assign specific novel transcripts to retinal bipolar cells of the inner nuclear layer. Using chemical ablation and free run experiments, we segregate the transcripts that are rhythmic when entrained by light from those that show sustained oscillations in the absence of external cues. The transcripts reported here with rigorous annotation and specific functions in circadian biology provide the groundwork for functional characterization of novel players in the zebrafish retinal clock.

Large scale changes in the transcriptome of Eisenia fetida during regeneration.

Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum Annelida, in which the genomes of the marine oligochaete Capitella telata and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.

Parentally inherited long non-coding RNA Cyrano is involved in zebrafish neurodevelopment.

Transfer of genetic material from parents to progeny via fusion of gametes is a way to ensure flow of information from one generation to the next. Apart from the genetic material, gametes provide a rich source of other factors such as RNA and proteins which can control traits of the embryo. Non-coding RNAs are not only carriers of regulatory information but can also encode memory of events of parental life. Here, we explore the possibility of parental inheritance of non-coding RNAs, especially long non-coding RNAs. Meta-analysis of RNA-seq data revealed several non-coding RNAs present in zebrafish oocyte, sperm and 2cell-stage. The embryo is transcriptionally silent at this stage, we rationalize that all the RNAs detectable at 2cell-stage are deposited either by sperm or oocyte or both and thus inherited. In the inherited pool, we noticed a conserved lncRNA, Cyrano previously known for zebrafish brain development. Knockdown of inherited Cyrano by miR-7 without changing zygotic Cyrano altered brain morphology at 24 hpf and 48 hpf. This defect could be partially rescued by injecting full length Cyrano lncRNA or a mutant resilient to knock-down by miR-7. In future, there is ample scope to check the possibility of inherited lncRNAs as carriers of memory of parental life events and building blocks that set up an initial platform for development.

microRNA dysregulation in polyglutamine toxicity of TATA-box binding protein is mediated through STAT1 in mouse neuronal cells.

BACKGROUND: Polyglutamine diseases constitute a class of neurodegenerative disorders associated with expansion of the cytosine-adenine-guanine (CAG) triplet, in protein coding genes. Expansion of a polyglutamine tract in the N-terminal of TBP is the causal mutation in SCA17. Brain sections of patients with spinocerebellar ataxia 17 (SCA17), a type of neurodegenerative disease, have been reported to contain protein aggregates of TATA-binding protein (TBP). It is also implicated in other neurodegenerative diseases like Huntington's disease, since the protein aggregates formed in such diseases also contain TBP. Dysregulation of miR-29a/b is another common feature of neurodegenerative diseases including Alzheimer's disease, Huntington's disease, and SCA17. Using a cellular model of SCA17, we identified key connections in the molecular pathway from protein aggregation to miRNA dysregulation. METHODS: Gene expression profiling was performed subsequent to the expression of TBP containing polyglutamine in a cellular model of SCA17. We studied the expression of STAT1 and other interferon-gamma dependent genes in neuronal apoptosis. The molecular pathway leading to the dysregulation of miRNA in response of protein aggregation and interferon release was investigated using RNAi-mediated knockdown of STAT1. RESULTS: We show that the accumulation of polyglutamine-TBP in the cells results in interferon-gamma release which in turn signals through STAT1 leading to downregulation of miR-29a/b. We propose that the release of interferons by cells harboring toxic protein aggregates may trigger a bystander effect resulting in loss of neurons. Interferon-gamma also led to upregulation of miR-322 although this effect is not mediated through STAT1. CONCLUSIONS: Our investigation shows that neuroinflammation could be an important player in mediating the transcriptional dysregulation of miRNA and the subsequent apoptotic effect of toxic polyglutamine-TBP. The involvement of immunomodulators in polyglutamine diseases holds special therapeutic relevance in the light of recent findings that interferon-gamma can modulate behavior.

Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters. We find that, in all motifs, overshoot correlated negatively whereas peak duration varied concavely with peak time. Differences in the other system properties were found to be mainly dependent on the nature of the controller rather than the motif structure. Protein mediated motifs showed a higher degree of adaptation i.e. a tendency to return to baseline levels; in particular, feedforward motifs exhibited perfect adaptation. RNA mediated motifs had a mild regulatory effect; they also exhibited a lower peaking tendency and mean overshoot. Protein mediated feedforward motifs showed higher overshoot and lower peak time compared to the corresponding feedback motifs.

A Novel Long Non-coding RNA, durga Modulates Dendrite Density and Expression of kalirin in Zebrafish.

Kalirin, a key player in axonal development, nerve growth and synaptic re-modeling, is implicated in many pathological conditions like schizophrenia and autism-spectrum disorders. Alternative promoters and splicing lead to functionally distinct isoforms, but the post-transcriptional regulation of Kalirin has not been studied. Here, we report a novel non-coding RNA, which we name durga, arising from the first exon of kalirin a (kalrna) in the antisense orientation in zebrafish. The kalrna and durga transcripts are barely detectable during early development, but steadily increase by 24 hours post-fertilization (hpf) as the brain develops. Over-expression of durga in the zebrafish embryo led to an increase in kalrna expression. The morphology of the neurons cultured from durga injected embryos had significantly fewer and shorter dendrites. Although durga has no apparent sequence homolog in mammals, based on gene synteny, we found a non-coding RNA arising from the 5' end of the human Kalrn gene and expressed in the human neuronal cell line, SH-SY5Y. We propose that the zebrafish lncRNA durga maintains dendritic length and density through regulation of kalrna expression and this may have further implications in mammalian systems.

Anti-HIV microRNA expression in a novel Indian cohort.

HIV-1 replication inside host cells is known to be regulated by various host factors. Host miRNAs, by virtue of its normal functioning, also regulate HIV-1 RNA expression by either directly targeting virus mRNAs or indirectly by regulating host proteins that HIV-1 uses for own replication. Therefore, it is highly possible that with differential miRNA expression, rate of disease progression will vary in HIV-1 infected individuals. In this study we have compared expression of a panel of 13 reported anti-HIV miRNAs in human PBMCs from long term non progressors (LTNPs), regular progressors and rapid progressors. We found that LTNPs have substantial lower expression of miR-382-5p that positively correlates with viral loads. Combinatorial regulation is highly probable in dictating differential disease progression as average expression of miR-382-5p and miR-155-5p can substantially distinguish LTNP individuals from regular progressors.

Novel MicroRNA signatures in HPV-mediated cervical carcinogenesis in Indian women.

This study aimed to investigate the role of miRNAs in HPV-mediated cervical pre-cancer and cancer cases in Indian population. We analysed the HPV infection and its genotypes in uterine cervical pre-cancer (n = 80), cancer (n = 200) and normal cervical samples (n = 150) by consensus sequence PCR followed by type specific PCRs. Also, microRNA profiling was done in a subset of cervical pre-cancer (n = 20), cancer cases (n = 50) and normal samples (n = 30) by real-time quantitative PCR (qRT-PCR). The prevalence of HPV infection in pre-cancer was found to be 81 % (65/80) and 94 % (188/200) in cancer cases, with most predominant high-risk HPV type-16 (HR-HPV-16) in 83 % of cancer and 91 % of pre- cancer cases, respectively. Whereas in controls, the HPV infection was found to be very low (5 %). The miRNA profiling revealed that in cervical pre-cancer, 100 miRNAs were significantly (p < 0.001) differentially expressed with 70 miRNAs upregulated and 30 miRNAs downregulated. In cervical cancer cases, 383 miRNA were found to be differentially expressed (p < 0.001), of which 350 miRNAs were upregulated and 33 miRNAs were downregulated. We also observed that 182 miRNAs were differentially expressed (p < 0.001) in HPV-16/18-positive (SiHa/HeLa) cell lines compared with HPV-negative (C33A) cell line. In addition, we identified the novel microRNAs such as miR-892b, miR-500, miR-888, miR-505 and miR-711 in cervical precancerous lesions and cervical cancer cases in Indian population. Taken together, the study demonstrates a crucial role of microRNAs in cervical cancer, which may serve as potential early diagnostic markers for cervical carcinogenesis.

Tat predominantly associates with host promoter elements in HIV-1-infected T-cells - regulatory basis of transcriptional repression of c-Rel.

HIV-1 Tat is a multifunctional regulatory protein that, in addition to its primary function of transactivating viral transcription, also tends to modulate cellular gene expression, for which the molecular mechanism remains to be clarified. We have reported earlier nuclear factor kappa B (NFκB) enhancer binding activity of Tat and proposed this DNA binding activity as a possible molecular basis for Tat-mediated regulation of cellular gene expression in infected cells. In the present study, we analyzed the genome-wide occupancy of Tat protein on host cell chromatin in HIV-1-infected T-cells to investigate a potential role of Tat on cellular gene expression. The results obtained identify a spectrum of binding sites of Tat protein on the chromatin and reveal that Tat is also recruited on a number of cellular gene promoters in HIV-1-infected T-cells, indicating its possible involvement in the regulation of gene expression of such cellular genes. Tat was identified as a repressor of one such validated gene, c-Rel, because it downregulates the expression of c-Rel in both Tat expressing and HIV-1-infected T-cells. The results also show that Tat downregulates c-Rel promoter activity by interacting with specific NFκB sites on the c-Rel promoter, thus providing a molecular basis of Tat-mediated regulation of cellular gene expression. Thus, in the present study, we have not only identified recruitment sites of Tat on the chromatin in HIV-1-infected T-cells, but also report for the first time that c-Rel is downregulated in HIV-1-infected cells specifically by interaction of Tat with NFκB binding sites on the promoter.

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA.

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice.

Several microRNAs have been implicated in neurogenesis, neuronal differentiation, neurodevelopment, and memory. Development of miRNA-based therapeutics, however, needs tools for effective miRNA modulation, tissue-specific delivery, and in vivo evidence of functional effects following the knockdown of miRNA. Expression of miR-29a is reduced in patients and animal models of several neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, and spinocerebellar ataxias. The temporal expression pattern of miR-29b during development also correlates with its protective role in neuronal survival. Here, we report the cellular and behavioral effect of in vivo, brain-specific knockdown of miR-29. We delivered specific anti-miRNAs to the mouse brain using a neurotropic peptide, thus overcoming the blood-brain-barrier and restricting the effect of knockdown to the neuronal cells. Large regions of the hippocampus and cerebellum showed massive cell death, reiterating the role of miR-29 in neuronal survival. The mice showed characteristic features of ataxia, including reduced step length. However, the apoptotic targets of miR-29, such as Puma, Bim, Bak, or Bace1, failed to show expected levels of up-regulation in mice, following knockdown of miR-29. In contrast, another miR-29 target, voltage-dependent anion channel1 (VDAC1), was found to be induced several fold in the hippocampus, cerebellum, and cortex of mice following miRNA knockdown. Partial restoration of apoptosis was achieved by down-regulation of VDAC1 in miR-29 knockdown cells. Our study suggests that regulation of VDAC1 expression by miR-29 is an important determinant of neuronal cell survival in the brain. Loss of miR-29 results in dysregulation of VDAC1, neuronal cell death, and an ataxic phenotype.