Sustained afferent signals evoke adaptive cellular responses that may ultimately underlie synaptic plasticity. One such adaptive response could be initiated by micro-RNA (miRNA) transcription. miRNAs comprise a large family of regulatory molecules that are derived from DNA, but not translated into proteins. They modulate the expression of protein-coding mRNAs by repressing their translation or enhancing their degradation. Since a single miRNA modulates expression of 5-30 target mRNA's, miRNA transcription could influence a wide array of cellular responses. MicroRNAs regulate aspects of developmental plasticity and apoptosis. They are also implicated in microbial defense and oncogenesis. Here we will identify miRNAs in the mouse flocculus whose transcription is modulated by sustained optokinetically-evoked climbing fiber activity. We will identify the genes whose mRNA expression is repressed by the identified miRNAs. We will test the hypothesis that transcription of a subset of miRNAs in the cerebellar flocculus is modulated by optokinetically-evoked climbing fiber activity. These miRNAs regulate the expression of several protein-coding mRNAs in the cerebellum by repressing their translation or enhancing their degradation. The proposed research has three objectives: First, we will expose mice to long-term (24h) unidirectional HOKS. After 24h of HOKS we will remove the flocculi of stimulated mice, extract the RNA and run samples from the two flocculi side by side on a miRNA microarray. We will identify cell types in which changes of miRNA are induced with hybridization histochemistry. We will analyze the time course of induced changes in miRNAs using 'real time'RT-PCR for whole floccular RNA samples. Once the cellular origin of the induced miRNA change is determined, we will use laser-capture microscopy to obtain cell specific samples of RNA. Second, we will link miRNAs to cellular function by microinjecting specific miRNA inhibitors into the cerebellum. The microinjected miRNA inhibitors should increase the expression of the mRNAs that are targets of the specific miRNAs for which the inhibitors are designed. mRNA enhancement will be compared with predictions based on complementary miRNA nucleotide motifs of the microinjected inhibitor. Third, we will characterize a protein, 14-3-3-8, on the phosphorylation of other proteins that are differentially regulated in the flocculus in response to HOKS. We have already shown that during HOKS the transcription of 14-3-3-8 mRNA decreases in the flocculus ipsilateral to the eye stimulated in the PxA direction. Our preliminary data show that 14-3-3-8 is negatively regulated by a differentially expressed miRNA, miR335. The transcription of miR335 increases during HOKS in the flocculus ipsilateral to the eye stimulated in the PxA direction. Consequently, miR335 could account for the observed HOKS-induced repression of 14-3-3-8 expression. We will clarify the functions of 14-3-3-8 by identifying other proteins with which it interacts in the cerebellum. We will use these techniques to test the repressive effects of other miRNAs on cellular function in the cerebellum. The ultimate goal of the proposed research is tol identify families of proteins regulated by single miRNAs. It will also identify promising specific molecular targets for diagnostic and therapeutic intervention in patients with various categories of spinocerebellar ataxias.

Public Health Relevance

The proposed research will clarify how a subset of RNA contributes to motor learning by the cerebellum. It will show how proteins necessary for cellular function are changed in accordance with experience. It may identify specific molecular targets for diagnostic and therapeutic intervention in patients with various categories of spinocerebellar ataxias.

National Institute of Health (NIH)
National Institute on Deafness and Other Communication Disorders (NIDCD)
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Sensorimotor Integration Study Section (SMI)
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Platt, Christopher
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Oregon Health and Science University
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Barmack, N H; Yakhnitsa, V (2015) Climbing fibers mediate vestibular modulation of both ""complex"" and ""simple spikes"" in Purkinje cells. Cerebellum 14:597-612
Barmack, N H; Yakhnitsa, V (2013) Modulated discharge of Purkinje and stellate cells persists after unilateral loss of vestibular primary afferent mossy fibers in mice. J Neurophysiol 110:2257-74
Qian, Z; Micorescu, M; Yakhnitsa, V et al. (2012) Climbing fiber activity reduces 14-3-3-? regulated GABA(A) receptor phosphorylation in cerebellar Purkinje cells. Neuroscience 201:34-45
Qian, Zuyuan; Yakhnitsa, Vadim; Barmack, Neal H (2011) Climbing fiber-evoked Purkinje cell discharge reduces expression of GABA(A) receptor-associated protein and decreases its interaction with GABA(A) receptors. J Neurochem 117:197-208
Barmack, Neal H; Yakhnitsa, Vadim (2011) Microlesions of the inferior olive reduce vestibular modulation of Purkinje cell complex and simple spikes in mouse cerebellum. J Neurosci 31:9824-35
Pettorossi, V E; Ferraresi, A; Botti, F M et al. (2011) Head position modulates optokinetic nystagmus. Exp Brain Res 213:141-52
Barmack, Neal H; Yakhnitsa, Vadim (2011) Topsy turvy: functions of climbing and mossy fibers in the vestibulo-cerebellum. Neuroscientist 17:221-36
Barmack, N H; Qian, Z; Yakhnitsa, V (2010) Climbing fibers induce microRNA transcription in cerebellar Purkinje cells. Neuroscience 171:655-65
Barmack, Neal H; Yakhnitsa, Vadim (2008) Functions of interneurons in mouse cerebellum. J Neurosci 28:1140-52
Barmack, N H; Yakhnitsa, V (2008) Distribution of granule cells projecting to focal Purkinje cells in mouse uvula-nodulus. Neuroscience 156:216-21

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