LIS1 determines cleavage plane positioning by regulating actomyosin-mediated cell membrane contractility.

Heterozygous loss of human PAFAH1B1 (coding for LIS1) results in the disruption of neurogenesis and neuronal migration via dysregulation of microtubule (MT) stability and dynein motor function/localization that alters mitotic spindle orientation, chromosomal segregation, and nuclear migration. Recently, human- induced pluripotent stem cell (iPSC) models revealed an important role for LIS1 in controlling the length of terminal cell divisions of outer radial glial (oRG) progenitors, suggesting cellular functions of LIS1 in regulating neural progenitor cell (NPC) daughter cell separation. Here, we examined the late mitotic stages NPCs in vivo and mouse embryonic fibroblasts (MEFs) in vitro from Pafah1b1-deficient mutants. Pafah1b1-deficient neocortical NPCs and MEFs similarly exhibited cleavage plane displacement with mislocalization of furrow-associated markers, associated with actomyosin dysfunction and cell membrane hyper-contractility. Thus, it suggests LIS1 acts as a key molecular link connecting MTs/dynein and actomyosin, ensuring that cell membrane contractility is tightly controlled to execute proper daughter cell separation.

"Females Are Not Just 'Protected' Males": Sex-Specific Vulnerabilities in Placenta and Brain after Prenatal Immune Disruption.

Current perceptions of genetic and environmental vulnerabilities in the developing fetus are biased toward male outcomes. An argument is made that males are more vulnerable to gestational complications and neurodevelopmental disorders, the implication being that an understanding of disrupted development in males is sufficient to understand causal mechanisms that are assumed to be similar but attenuated in females. Here we examine this assumption in the context of immune-driven alterations in fetal brain development and related outcomes in female and male mice. Pregnant C57BL/6 mice were treated with low-dose lipopolysaccharide at embryonic day 12.5. Placental pathology, acute fetal brain inflammation and hypoxia, long-term changes in adult cortex cytoarchitecture, altered densities and ratio of excitatory (Satb2+) to inhibitory (parvalbumin+) neuronal subtypes, postnatal growth, and behavior outcomes were compared between male and female offspring. We find that while males experience more pronounced placental pathology, fetal brain hypoxia, depleted PV and Satb2+ densities, and social and learning-related behavioral abnormalities, females exhibit unique acute inflammatory signaling in fetal brain, postnatal growth delay, opposite alterations in cortical PV densities, changes in juvenile behavior, delayed postnatal body growth, and elevated anxiety-related behavior as adults. While males are more severely impacted by prenatal immune disruption by several measures, females exposed to the same insult exhibit a unique set of vulnerabilities and developmental consequences that is not present in males. Our results clearly outline disparate sex-specific features of prenatal vulnerability to inflammatory insults and warn against the casual extrapolation of male disease mechanisms to females.

LIS1 controls mitosis and mitotic spindle organization via the LIS1-NDEL1-dynein complex.

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore-microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1-dynein complex. Overexpression of NDEL1-dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1-NDEL1-dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division.

Stereotypical alterations in cortical patterning are associated with maternal illness-induced placental dysfunction.

We have previously shown in mice that cytokine-mediated damage to the placenta can temporarily limit the flow of nutrients and oxygen to the fetus. The placental vulnerability is pronounced before embryonic day 11, when even mild immune challenge results in fetal loss. As gestation progresses, the placenta becomes increasingly resilient to maternal inflammation, but there is a narrow window in gestation when the placenta is still vulnerable to immune challenge yet resistant enough to allow for fetal survival. This gestational window correlates with early cortical neurogenesis in the fetal brain. Here, we show that maternal illness during this period selectively alters the abundance and laminar positioning of neuronal subtypes influenced by the Tbr1, Satb2, and Ctip2/Fezf2 patterning axis. The disturbances also lead to a laminar imbalance in the proportions of projection neurons and interneurons in the adult and are sufficient to cause changes in social behavior and cognition. These data illustrate how the timing of an illness-related placental vulnerability causes developmental alterations in neuroanatomical systems and behaviors that are relevant to autism spectrum disorders.

Cytoskeleton in action: lissencephaly, a neuronal migration disorder.

During neocortical development, the extensive migratory movements of neurons from their place of birth to their final location are essential for the coordinated wiring of synaptic circuits and proper neurological function. Failure or delay in neuronal migration causes severe abnormalities in cortical layering, which consequently results in human lissencephaly ('smooth brain'), a neuronal migration disorder. The brains of lissencephaly patients have less-convoluted gyri in the cerebral cortex with impaired cortical lamination of neurons. Since microtubule (MT) and actin-associated proteins play important functions in regulating the dynamics of MT and actin cytoskeletons during neuronal migration, genetic mutations or deletions of crucial genes involved in cytoskeletal processes lead to lissencephaly in human and neuronal migration defects in mouse. During neuronal migration, MT organization and transport are controlled by platelet-activating factor acetylhydrolase isoform 1b regulatory subunit 1 (PAFAH1B1, formerly known as LIS1, Lissencephaly-1), doublecortin (DCX), YWHAE, and tubulin. Actin stress fibers are modulated by PAFAH1B1 (LIS1), DCX, RELN, and VLDLR (very low-density lipoprotein receptor)/LRP8 (low-density lipoprotein-related receptor 8, formerly known as APOER2). There are several important levels of crosstalk between these two cytoskeletal systems to establish accurate cortical patterning in development. The recent understanding of the protein networks that govern neuronal migration by regulating cytoskeletal dynamics, from human and mouse genetics as well as molecular and cellular analyses, provides new insights on neuronal migration disorders and may help us devise novel therapeutic strategies for such brain malformations.

Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration.

Coordinated migration of newly born neurons to their prospective target laminae is a prerequisite for neural circuit assembly in the developing brain. The evolutionarily conserved LIS1/NDEL1 complex is essential for neuronal migration in the mammalian cerebral cortex. The cytoplasmic nature of LIS1 and NDEL1 proteins suggest that they regulate neuronal migration cell autonomously. Here, we extend mosaic analysis with double markers (MADM) to mouse chromosome 11 where Lis1, Ndel1, and 14-3-3ɛ (encoding a LIS1/NDEL1 signaling partner) are located. Analyses of sparse and uniquely labeled mutant cells in mosaic animals reveal distinct cell-autonomous functions for these three genes. Lis1 regulates neuronal migration efficiency in a dose-dependent manner, while Ndel1 is essential for a specific, previously uncharacterized, late step of neuronal migration: entry into the target lamina. Comparisons with previous genetic perturbations of Lis1 and Ndel1 also suggest a surprising degree of cell-nonautonomous function for these proteins in regulating neuronal migration.

The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection.

UNLABELLED: Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular transformation are poorly understood. We hypothesized that the guanosine triphosphatase activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play a role in the transformation process. Here we report that NS4B can transform NIH-3T3 cells, leading to tumor formation in vivo. This transformation was independent of co-transfection with activated Ha-ras. Detailed analyses of NS4B mutants revealed that this transforming activity could be progressively inhibited and completely abrogated by increasing genetic impairment of the NS4B nucleotide binding motif. CONCLUSION: NS4B has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is indeed mediated by its NBM. Moreover, our results suggest that pharmacological inhibition of the latter might inhibit not only HCV replication but also the associated HCC.

Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression.

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARgamma (peroxisome proliferator-activated receptor gamma) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARgamma gene is a novel target gene of LXR, since the PPARgamma promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRalpha. Moreover, activated LXRalpha exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARgamma, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRalpha by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.

Adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element-binding protein 1c regulates mouse adiponectin expression.

Adiponectin is exclusively expressed in differentiated adipocytes and plays an important role in regulating energy homeostasis, including the glucose and lipid metabolism associated with increased insulin sensitivity. However, the control of adiponectin gene expression in adipocytes is poorly understood. We show here that levels of adiponectin mRNA and protein are reduced in the white adipose tissue of ob/ob and db/db mice and that there is a concomitant reduction of the adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein 1c (SREBP1c) transcription factor. To determine whether ADD1/SREBP1c regulates adiponectin gene expression, we isolated and characterized the mouse adiponectin promoter. Analysis of the adiponectin promoter revealed putative binding sites for the adipogenic transcription factors ADD1/SREBP1c, peroxisomal proliferator-activated receptor gamma and CCAAT enhancer-binding proteins. DNase I footprinting and chromatin immunoprecipitation analyses revealed that ADD1/SREBP1c binds in vitro and in vivo to the proximal promoter containing sterol regulatory element (SRE) motifs. A luciferase reporter containing the promoter was transactivated by ADD1/SREBP1c, and introduction of SRE mutations into the construct abolished transactivation. Adenoviral overexpression of ADD1/SREBP1c also elevated adiponectin mRNA and protein levels in 3T3-L1 adipocytes. Furthermore, insulin stimulated adiponectin mRNA expression in adipocytes and augmented transactivation of the adiponectin promoter by ADD1/SREBP1c. Taken together, these data indicate that ADD1/SREBP1c controls adiponectin gene expression in differentiated adipocytes.