Heat shock promotes inclusion body formation of mutant huntingtin (mHtt) and alleviates mHtt-induced transcription factor dysfunction.

PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in individuals with and models of Huntington's disease (HD). The role of IB versus diffusible mHtt in neurotoxicity remains unclear. Using a ponasterone (PA)-inducible cell model of HD, here we evaluated the effects of heat shock on the appearance and functional outcome of Htt103QExon1-EGFP expression. Quantitative image analysis indicated that 80-90% of this mHtt protein initially appears as "diffuse" signals in the cytosol, with IBs forming at high mHtt expression. A 2-h heat shock during the PA induction reduced the diffuse signal, but greatly increased mHtt IB formation in both cytosol and nucleus. Dose- and time-dependent mHtt expression suggested that nucleated polymerization drives IB formation. RNA-mediated knockdown of heat shock protein 70 (HSP70) and heat shock cognate 70 protein (HSC70) provided evidence for their involvement in promoting diffuse mHtt to form IBs. Reporter gene assays assessing the impacts of diffuse versus IB mHtt showed concordance of diffuse mHtt expression with the repression of heat shock factor 1, cAMP-responsive element-binding protein (CREB), and NF-κB activity. CREB repression was reversed by heat shock coinciding with mHtt IB formation. In an embryonic striatal neuron-derived HD model, the chemical chaperone sorbitol similarly promoted the structuring of diffuse mHtt into IBs and supported cell survival under stress. Our results provide evidence that mHtt IB formation is a chaperone-supported cellular coping mechanism that depletes diffusible mHtt conformers, alleviates transcription factor dysfunction, and promotes neuron survival.

Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging.

Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (Pr). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets Pr remain to be defined. To address this, we developed QuaSOR, a super-resolution method for determining Pr from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. We mapped the Pr onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). We find that Pr varies greatly between synapses made by a single axon, quantify the contribution of key AZ proteins to Pr diversity and find that one of these, Complexin, suppresses spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission.

Homologous organization of cerebellar pathways to sensory, motor, and associative forebrain.

Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here, we quantify pathways between the cerebellum and forebrain by using transsynaptic tracing viruses and a whole-brain analysis pipeline. With retrograde tracing, we find that most descending paths originate from the somatomotor cortex. Anterograde tracing of ascending paths encompasses most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei. In the neocortex, sensorimotor regions contain the most labeled neurons, but we find higher densities in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlate with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of the cerebellar cortex to diverse forebrain targets. We conclude that shared areas of the cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function.

Normal cognitive and social development require posterior cerebellar activity.

Cognitive and social capacities require postnatal experience, yet the pathways by which experience guides development are unknown. Here we show that the normal development of motor and nonmotor capacities requires cerebellar activity. Using chemogenetic perturbation of molecular layer interneurons to attenuate cerebellar output in mice, we found that activity of posterior regions in juvenile life modulates adult expression of eyeblink conditioning (paravermal lobule VI, crus I), reversal learning (lobule VI), persistive behavior and novelty-seeking (lobule VII), and social preference (crus I/II). Perturbation in adult life altered only a subset of phenotypes. Both adult and juvenile disruption left gait metrics largely unaffected. Contributions to phenotypes increased with the amount of lobule inactivated. Using an anterograde transsynaptic tracer, we found that posterior cerebellum made strong connections with prelimbic, orbitofrontal, and anterior cingulate cortex. These findings provide anatomical substrates for the clinical observation that cerebellar injury increases the risk of autism.