Genetic disruption of WASHC4 drives endo-lysosomal dysfunction and cognitive-movement impairments in mice and humans.

Mutation of the Wiskott-Aldrich syndrome protein and SCAR homology (WASH) complex subunit, SWIP, is implicated in human intellectual disability, but the cellular etiology of this association is unknown. We identify the neuronal WASH complex proteome, revealing a network of endosomal proteins. To uncover how dysfunction of endosomal SWIP leads to disease, we generate a mouse model of the human WASHC4c.3056C>G mutation. Quantitative spatial proteomics analysis of SWIPP1019R mouse brain reveals that this mutation destabilizes the WASH complex and uncovers significant perturbations in both endosomal and lysosomal pathways. Cellular and histological analyses confirm that SWIPP1019R results in endo-lysosomal disruption and uncover indicators of neurodegeneration. We find that SWIPP1019R not only impacts cognition, but also causes significant progressive motor deficits in mice. A retrospective analysis of SWIPP1019R patients reveals similar movement deficits in humans. Combined, these findings support the model that WASH complex destabilization, resulting from SWIPP1019R, drives cognitive and motor impairments via endo-lysosomal dysfunction in the brain.

Cells in the brain need to regulate and transport the proteins and nutrients stored inside them. They do this by sorting and packaging the contents they want to move in compartments called endosomes, which then send these packages to other parts of the cell. If the components involved in endosome trafficking mutate, this can lead to 'traffic jams' where proteins pile up inside the cell and stop it from working normally. In 2011, researchers found that children who had a mutation in the gene for WASHC4 ­ a protein involved in endosome trafficking ­ had trouble learning. However, it remained unclear how this mutation affects the role of WASCH4 and impacts the behavior of brain cells. To answer this question, Courtland, Bradshaw et al. genetically engineered mice to carry an equivalent mutation to the one identified in humans. Experiments showed that the brain cells of the mutant mice had fewer WASHC4 proteins, and lower levels of other proteins involved in endosome trafficking. The mutant mice also had abnormally large endosomes in their brain cells and elevated levels of proteins that break down the cell's contents, resulting in a build-up of cellular debris. Together, these findings suggest that the mutation causes abnormal trafficking in brain cells. Next, Courtland, Bradshaw et al. compared the behavior of adult and young mice with and without the mutation. Mice carrying the mutation were found to have learning difficulties and showed abnormal movements which became more exaggerated as they aged, similar to people with Parkinson's disease. With this result, Courtland, Bradshaw et al. reviewed the medical records of the patients with the mutation and discovered that these children also had problems with their movement. These findings help explain what is happening inside brain cells when the gene for WASHC4 is mutated, and how disrupting endosome trafficking can lead to behavioral changes. Ultimately, understanding how learning and movement difficulties arise, on a molecular level, could lead to new therapeutic strategies to prevent, manage or treat them in the future.

Chemico-genetic discovery of astrocytic control of inhibition in vivo.

Perisynaptic astrocytic processes are an integral part of central nervous system synapses1,2; however, the molecular mechanisms that govern astrocyte-synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte-neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We find that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor effects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function.

Dysregulation of the Synaptic Cytoskeleton in the PFC Drives Neural Circuit Pathology, Leading to Social Dysfunction.

Psychiatric disorders are highly heritable pathologies of altered neural circuit functioning. How genetic mutations lead to specific neural circuit abnormalities underlying behavioral disruptions, however, remains unclear. Using circuit-selective transgenic tools and a mouse model of maladaptive social behavior (ArpC3 mutant), we identify a neural circuit mechanism driving dysfunctional social behavior. We demonstrate that circuit-selective knockout (ctKO) of the ArpC3 gene within prefrontal cortical neurons that project to the basolateral amygdala elevates the excitability of the circuit neurons, leading to disruption of socially evoked neural activity and resulting in abnormal social behavior. Optogenetic activation of this circuit in wild-type mice recapitulates the social dysfunction observed in ArpC3 mutant mice. Finally, the maladaptive sociability of ctKO mice is rescued by optogenetically silencing neurons within this circuit. These results highlight a mechanism of how a gene-to-neural circuit interaction drives altered social behavior, a common phenotype of several psychiatric disorders.

Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering.

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.