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Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause neurodevelopmental disorders (NDDs). Here, we report on 5 individuals with mutations in SMARCD1, presenting with developmental delay, intellectual disability, hypotonia, feeding difficulties, and small hands and feet. The mutations were proven to be de novo in 4 of the 5 individuals, by trio exome sequencing. Mutations in other SWI/SNF components cause Coffin-Siris syndrome and Nicolaides-Baraitser syndrome, or other syndromic and non-syndromic NDDs. Although the individuals presented here have dysmorphisms and some clinical overlap with these syndromes, they lack the typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, we investigated the Drosophila ortholog, Bap60, in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom body specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, we identify a NDD caused by SMARCD1 mutations and establish a role for the SMARCD1 ortholog Bap60 in regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development that is essential in establishing neuronal circuits that are required for learning and memory.

Proteinase-Activated Receptors (PARs) are a four-member family of G-protein coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by the serine proteinases such as thrombin, trypsin and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types including the platelets, vascular smooth muscle cells and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane with spherical membrane protrusions that resemble plasma membrane blebbing. Since non-apoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/Cas9 mediated gene editing approaches we establish that PAR4-dependent membrane blebbing occurs independently of the Gαq/11 and Gαi signaling pathways and is dependent on signaling via the β-arrestin-1/-2 pathway and RhoA signaling. In order to gain a more comprehensive understanding of β-arrestin mediated signaling downstream of PAR4 and to guide future studies, we undertook RNAseq analysis of PAR4 activated genes in control cells and in cells lacking β-arrestin-1/-2. A list of differentially expressed genes was generated followed by Gene Ontology (GO) and enrichment analysis revealing PAR4 regulation of genes involved in processes including blood coagulation and circulation, cell-cell adhesion, sensory perception and neuron-neuron synaptic transmission, terms that relates back to known function of PAR4 and that are consistent with our finding of membrane blebbing triggered by PAR4 activation. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA- and β-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4. Finally through CRISPR/Cas9-mediated targeting and RNA-seq analysis we catalogue here PAR4-dependent transcription that is dependent on β-arrestin.

Proteinase-Activated Receptors (PARs) are a four-member family of G-protein coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types including platelets, vascular smooth muscle cells and neuronal cells. In studying PAR4 signalling and trafficking, we observed dynamic changes in the cell membrane with spherical membrane protrusions that resemble plasma membrane blebbing. Since non-apoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release we sought to elucidate the signalling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/Cas9-mediated gene-editing approaches we establish that PAR4-dependent membrane blebbing occurs independently of the Gαq/11and Gαisignalling pathways and is dependent on signalling via the β-arrestin-1/-2 and RhoA signalling pathways. In order to gain a more comprehensive understanding of β-arrestin-mediated signalling downstream of PAR4 and to guide future studies, we undertook RNA-seq analysis of PAR4 activated genes in control cells and in cells lacking β-arrestin-1/-2. A list of differentially expressed genes was generated followed by Gene Ontology (GO) and enrichment analysis, revealing PAR4 regulation of genes involved in processes including blood coagulation and circulation, cell-cell adhesion, sensory perception and neuron-neuron synaptic transmission-terms that relate back to known functions of PAR4 and are consistent with our finding of membrane blebbing triggered by PAR4 activation. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function.Significance StatementWe find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA- and β-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4. Finally through CRISPR/Cas9-mediated targeting and RNA-seq analysis we catalogue here PAR4-dependent transcription that is dependent on β-arrestin.

Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause syndromic intellectual disability (ID). Here, we report on 5 individuals with mutations in the SMARCD1 gene, presenting with ID, developmental delay, hypotonia, feeding difficulties, and small extremities. The mutations were proven to be de novo in 4 of the 5 individuals. Mutations in other SWI/SNF components cause Coffin-Siris, Nicolaides-Baraitser, or other syndromic ID disorders. Although the individuals presented here have some clinical overlap with these disorders, they lack the typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, we investigated the Drosophila ortholog, Bap60, in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom body specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, we identify SMARCD1 mutations as a novel cause of ID, and establish a role for the SMARCD1 ortholog Bap60 in regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development that is essential in establishing neuronal circuits that are required for learning and memory.

Defects in neuronal migration cause brain malformations, which are associated with intellectual disability (ID) and epilepsy. Using exome sequencing, we identified compound heterozygous variants (p.Arg71His and p. Leu729ThrfsTer6) in TMTC3, encoding transmembrane and tetratricopeptide repeat containing 3, in four siblings with nocturnal seizures and ID. Three of the four siblings have periventricular nodular heterotopia (PVNH), a common brain malformation caused by failure of neurons to migrate from the ventricular zone to the cortex. Expression analysis using patient-derived cells confirmed reduced TMTC3 transcript levels and loss of the TMTC3 protein compared to parental and control cells. As TMTC3 function is currently unexplored in the brain, we gathered support for a neurobiological role for TMTC3 by generating flies with post-mitotic neuron-specific knockdown of the highly conserved Drosophila melanogaster TMTC3 ortholog, CG4050/tmtc3. Neuron-specific knockdown of tmtc3 in flies resulted in increased susceptibility to induced seizures. Importantly, this phenotype was rescued by neuron-specific expression of human TMTC3, suggesting a role for TMTC3 in seizure biology. In addition, we observed co-localization of TMTC3 in the rat brain with vesicular GABA transporter (VGAT), a presynaptic marker for inhibitory synapses. TMTC3 is localized at VGAT positive pre-synaptic terminals and boutons in the rat hypothalamus and piriform cortex, suggesting a role for TMTC3 in the regulation of GABAergic inhibitory synapses. TMTC3 did not co-localize with Vglut2, a presynaptic marker for excitatory neurons. Our data identified TMTC3 as a synaptic protein that is involved in PVNH with ID and epilepsy, in addition to its previously described association with cobblestone lissencephaly.