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Summary: The complex functions of neuronal synapses depend on their tightly interconnected protein network, and their dysregulation is implicated in the pathogenesis of autism spectrum disorders and schizophrenia. However, it remains unclear how synaptic molecular networks are altered biochemically in these disorders. Here, we apply multiplexed imaging to probe the effects of RNAi knockdown of 16 autism- and schizophrenia-associated genes on the simultaneous joint distribution of 10 synaptic proteins, observing several protein composition phenotypes associated with these risk genes. We apply Bayesian network analysis to infer hierarchical dependencies among eight excitatory synaptic proteins, yielding predictive relationships that can only be accessed with single-synapse, multiprotein measurements performed simultaneously in situ. Finally, we find that central features of the network are affected similarly across several distinct gene knockdowns. These results offer insight into the convergent molecular etiology of these widespread disorders and provide a general framework to probe subcellular molecular networks.

Summary: Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis.

Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here, authors show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity and can be targeted to rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion