Mechanisms of motor superperformance

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MECHANISMS OF MOTOR SUPERPERFORMANCE: ABSTRACT Clinical experience and world population-level data indicate that most neurological disability stems from motor dysfunction. Yet, spontaneous superperformer mutations occur in a variety of species including man, illustrating that the intrinsic motor capacity of the organism can be augmented. We set out to identify similar mutations by rotarod screening of 33,806 laboratory mice harboring chemically induced random mutations with the goal of mechanistically explaining and, eventually, pharmacologically enabling the phenomenon of motor superperformance. In this context, we have discovered that a mutation in an unsuspected gene, Rif1 (Replication Timing Regulatory Factor 1), confers supernormal motor ability. Using clustered regularly interspaced short palindromic repeats (CRISPR) Rif1-mutant mice, we have determined that: 1) mutant superperformance is a selective phenotype, with distinct changes in motor features quantifiable by our novel analysis method, but no other effects upon rigorous behavioral testing, and 2) the mutation also leads to accelerated motor recovery from stroke. The superperformance mechanism, however, is unknown: although Rif1 participates in DNA repair and in transcriptional regulation via G4 folded DNA structural stabilization, little is known about its function in the nervous system. There is precedent that DNA repair may be associated to synaptic transmission strength, while DNA G4 regulation could enhance the transcription of genes active in the motor system. We have refined this hypothetical framework by identifying several consequences of the Rif1 mutation: a) Increased cerebellar Purkinje cell firing regularity and local field potential changes in the non- moving mouse, which can influence movement precision, with change of these neurophysiological parameters upon locomotion on a treadmill; b) Increased resistance to DNA-damaging radiation; c) Increased resistance to G4 stabilization; d) Overexpression of a fraction of the cerebellar (but not forebrain or spinal cord) synaptic transcriptome including potential Rif1 mutation mediators such as Kcnma1, Kif5c and Nab2; e) These transcripts may be relevant to the phenotype because we show that loss of function mutations in them degrade motor performance, whereas f) Cerebellar injection of adenovirus-containing Nab2 induces superperformance. This proposal unifies this body of work by postulating that the Rif1 mutation modifies DNA repair and/or G4 DNA folding resulting in upregulation of synaptic transcripts, with either one or both mechanisms augmenting the precision of cerebellar synapse activity relevant to movement control. To this effect, we will conduct neurophysiological studies, determine the transcriptome in single cells, alter Kcnma1, Kif5c and Nab2 expression, and investigate DNA repair and DNA G4 regulation to test which of these mechanisms enable the superperformance phenotype. Our goal is to initiate and steer the mechanistic investigation (by us or any others) of Rif1 in the brain for ultimate therapeutic gain if appropriate.