NFIB Haploinsufficiency Is Associated with Intellectual Disability and Macrocephaly.

The nuclear factor I (NFI) family of transcription factors play an important role in normal development of multiple organs. Three NFI family members are highly expressed in the brain, and deletions or sequence variants in two of these, NFIA and NFIX, have been associated with intellectual disability (ID) and brain malformations. NFIB, however, has not previously been implicated in human disease. Here, we present a cohort of 18 individuals with mild ID and behavioral issues who are haploinsufficient for NFIB. Ten individuals harbored overlapping microdeletions of the chromosomal 9p23-p22.2 region, ranging in size from 225 kb to 4.3 Mb. Five additional subjects had point sequence variations creating a premature termination codon, and three subjects harbored single-nucleotide variations resulting in an inactive protein as determined using an in vitro reporter assay. All individuals presented with additional variable neurodevelopmental phenotypes, including muscular hypotonia, motor and speech delay, attention deficit disorder, autism spectrum disorder, and behavioral abnormalities. While structural brain anomalies, including dysgenesis of corpus callosum, were variable, individuals most frequently presented with macrocephaly. To determine whether macrocephaly could be a functional consequence of NFIB disruption, we analyzed a cortex-specific Nfib conditional knockout mouse model, which is postnatally viable. Utilizing magnetic resonance imaging and histology, we demonstrate that Nfib conditional knockout mice have enlargement of the cerebral cortex but preservation of overall brain structure and interhemispheric connectivity. Based on our findings, we propose that haploinsufficiency of NFIB causes ID with macrocephaly.

Transcription factors NFIA and NFIB induce cellular differentiation in high-grade astrocytoma.

INTRODUCTION: Malignant astrocytomas are composed of heterogeneous cell populations. Compared to grade IV glioblastoma, low-grade astrocytomas have more differentiated cells and are associated with a better prognosis. Therefore, inducing cellular differentiation to alter the behaviour of high-grade astrocytomas may serve as a therapeutic strategy. The nuclear factor one (NFI) transcription factors are essential for normal astrocytic differentiation. Here, we investigate whether family members NFIA and NFIB act as effectors of cellular differentiation in glioblastoma. METHODS: We analysed expression of NFIA and NFIB in mRNA expression data of high-grade astrocytoma and with immunofluorescence co-staining. Furthermore, we induced NFI expression in patient-derived subcutaneous glioblastoma xenografts via in vivo electroporation. RESULTS: The expression of NFIA and NFIB is reduced in glioblastoma as compared to lower grade astrocytomas. At a cellular level, their expression is associated with differentiated and mature astrocyte-like tumour cells. In vivo analyses consistently demonstrate that expression of either NFIA or NFIB is sufficient to promote tumour cell differentiation in glioblastoma xenografts. CONCLUSION: Our findings indicate that both NFIA and NFIB may have an endogenous pro-differentiative function in astrocytomas, similar to their role in normal astrocyte differentiation. Overall, our study establishes a basis for further investigation of targeting NFI-mediated differentiation as a potential differentiation therapy.

DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation.

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.

USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X's role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.