Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in 'Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia'. Neuron, 21, 1315-1325, 1998; Sheen et al. in 'Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex'. Nat. Genet., 36, 69-76, 2004). Recent studies have shown that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in 'MEKK4 signaling regulates filamin expression and neuronal migration'. Neuron, 52, 789-801, 2006). Here we show that neurons in post-mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attachment protein alpha (alpha-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to periventricular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafficking and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafficking of transport vesicles. Our current findings suggest that PH formation arises from a final common pathway involving disruption of vesicle trafficking, leading to impaired cell adhesion and loss of neuroependymal integrity.

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a newly identified coronavirus (CoV), SARS-CoV. The spike (S) glycoprotein of CoV is the major structural protein responsible for induction of host immune response and virus neutralization by antibodies. Hence, knowledge of neutralization determinants on the S protein is helpful for designing protective vaccines. To analyze the antigenic structure of the SARS-CoV S2 domain, the carboxyl-terminal half of the S protein, we first used sera from convalescent SARS patients to test the antigenicity of 12 overlapping fragments spanning the entire S2 and identified two antigenic determinants (Leu 803 to Ala 828 and Pro 1061 to Ser 1093). To determine whether neutralizing antibodies can be elicited by these two determinants, we immunized animals and found that both of them could induce the S2-specific antisera. In some animals, however, only one determinant (Leu 803 to Ala 828) was able to induce the antisera with the binding ability to the native S protein and the neutralizing activity to the SARS-CoV pseudovirus. This determinant is highly conserved across different SARS-CoV isolates. Identification of a conserved antigenic determinant on the S2 domain of the SARS-CoV S protein, which has the potential for inducing neutralizing antibodies, has implications in the development of effective vaccines against SARS-CoV.

NAbs to SARS-CoV are broadly elicited in patients with SARS and, according to their kinetics, may correlate with viral load during the early stages of the disease. These results suggest that it is possible to develop effective vaccines against SARS and that NAbs provide a potential strategy for treating patients with SARS.

Filamin B (FLNB) is a cytoplasmic protein that regulates the cytoskeletal network by cross-linking actin, linking cell membrane to the cytoskeleton and regulating intracellular signaling pathways responsible for skeletal development (Stossel, T.P., Condeelis, J., Cooley, L., Hartwig, J.H., Noegel, A., Schleicher, M. and Shapiro, S.S. (2001) Filamins as integrators of cell mechanics and signalling. Nat. Rev. Mol. Cell Biol., 2, 138-145). Mutations in FLNB cause human skeletal disorders [boomerang dysplasia, spondylocarpotarsal (SCT), Larsen, and atelosteogenesis I/III syndromes], which are characterized by disrupted vertebral segmentation, joint formation and endochondral ossification [Krakow, D., Robertson, S.P., King, L.M., Morgan, T., Sebald, E.T., Bertolotto, C., Wachsmann-Hogiu, S., Acuna, D., Shapiro, S.S., Takafuta, T. et al. (2004) Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis. Nat. Genet., 36, 405-410; Bicknell, L.S., Morgan, T., Bonafe, L., Wessels, M.W., Bialer, M.G., Willems, P.J., Cohn, D.H., Krakow, D. and Robertson, S.P. (2005) Mutations in FLNB cause boomerang dysplasia. J. Med. Genet., 42, e43]. Here we show that Flnb deficient mice have shortened distal limbs with small body size, and develop fusion of the ribs and vertebrae, abnormal spinal curvatures, and dysmorphic facial/calvarial bones, similar to the human phenotype. Characterization of the mutant mice demonstrated increased apoptosis along the bone periphery of the distal appendages, consistent with reduced bone width. No changes in the initial proliferative rate of chondrocytes were observed, but the progressive differentiation of chondrocyte precursors was impaired, consistent with reduced bone length. The extracellular matrix appeared disrupted and phosphorylated beta1-integrin (a collagen receptor and Flnb binding partner) expression was diminished in the mutant growth plate. Like integrin-deficient chondrocytes, adhesion to the ECM was decreased in Flnb(-/-) chondrocytes, and inhibition of beta1-integrin in these cells led to further impairments in cell spreading. These data suggest that disruption of the ECM-beta1-integrin-Flnb pathway contributes to defects in vertebral and distal limb development, similar to those seen in the human autosomal recessive SCT due to Flnb mutations.

Cytoskeleton-associated proteins play key roles not only in regulating cell morphology and migration but also in proliferation. Mutations in the cytoskeleton-associated gene filamin A (FlnA) cause the human disorder periventricular heterotopia (PH). PH is a disorder of neural stem cell development that is characterized by disruption of progenitors along the ventricular epithelium and subsequent formation of ectopic neuronal nodules. FlnA-dependent regulation of cytoskeletal dynamics is thought to direct neural progenitor migration and proliferation. Here we show that embryonic FlnA null mice exhibited a reduction in brain size, and decline in neural progenitor numbers over time. The drop in the progenitor population was not attributable to cell death or changes in premature differentiation, but to prolonged cell cycle duration. Suppression of FlnA led to prolongation of the entire cell cycle length, principally in M-phase. FlnA loss impaired degradation of cyclin b1-related proteins, thereby delaying the onset and progression through mitosis. We found that the cdk1 kinase Wee1 bound FlnA, demonstrated increased expression levels after loss of FlnA function, and was associated with increased phosphorylation of cdk1. Phosphorylation of cdk1 inhibited activation of the anaphase promoting complex degradation system, which was responsible for cyclin b1 degradation and progression through mitosis. Collectively, our results demonstrate a molecular mechanism whereby FlnA loss impaired G2 to M phase entry, leading to cell cycle prolongation, compromised neural progenitor proliferation, and reduced brain size.