Kindlin-2 is an essential component of intercalated discs and is required for vertebrate cardiac structure and function.

Integrins and proteins that associate with integrins are implicated in normal cardiac muscle function and development. Unc-112 is a cytoplasmic adaptor protein required for the proper establishment of integrin junctions in Caenorhabditis elegans muscle. A vertebrate homolog of unc-112, kindlin-2, is an integrin-binding protein that is expressed in cardiac muscle, but its function is unknown. We sought to understand the role of kindlin-2 in the development and function of the mouse and zebrafish heart. In the mouse, we found that kindlin-2 is highly expressed in the heart and is enriched at intercalated discs and costameres. Targeted disruption of the murine kindlin-2 gene resulted in embryonic lethality before cardiogenesis. To better assess the role of kindlin-2 in cardiac muscle development, we used morpholinos to knockdown the kindlin-2 homolog in zebrafish (z-kindlin-2), which resulted in severe abnormalities of heart development. Morphant hearts were hypoplastic and dysmorphic and exhibited significantly reduced ventricular contractility. Ultrastructural analysis of these hearts revealed disrupted intercalated disc formation and a failure in the attachment of myofibrils to membrane complexes. We conclude that kindlin-2 is an essential component of the intercalated disc, is necessary for cytoskeletal organization at sites of membrane attachment, and is required for vertebrate myocardial formation and function. These findings provide the first characterization of the in vivo functions of this novel and critical regulator of cardiogenesis.

Targeted loss of Arx results in a developmental epilepsy mouse model and recapitulates the human phenotype in heterozygous females.

Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6(CRE-IRES-GFP)(Dlx5/6(CIG)) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx(-/y);Dlx5/6(CIG) (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx(-/+);Dlx5/6(CIG)) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders.

A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death.

A growing number of human disorders have been associated with expansions of a tract of a single amino acid. Recently, polyalanine (polyA) tract expansions in the Aristaless-related homeobox (ARX) protein have been identified in a subset of patients with infantile spasms and mental retardation. How alanine expansions in ARX, or any other transcription factor, cause disease have not been determined. We generated a series of polyA expansions in Arx and expressed these in cell culture and brain slices. Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death. These inclusions are ubiquitinated and recruit Hsp70. Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions. Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation. Our data suggest expansions in one of the ARX polyA tracts results in nuclear protein aggregation and an increase in cell death; likely underlying the pathogenesis of the associated infantile spasms and mental retardation.

Characterization of the null murine sodium/myo-inositol cotransporter 1 (Smit1 or Slc5a3) phenotype: myo-inositol rescue is independent of expression of its cognate mitochondrial ribosomal protein subunit 6 (Mrps6) gene and of phosphatidylinositol levels in neonatal brain.

Ablation of the murine Slc5a3 gene results in severe myo-inositol (Ins) deficiency and congenital central apnea due to abnormal respiratory rhythmogenesis. The lethal knockout phenotype may be rescued by supplementing the maternal drinking water with 1% Ins. In order to test the hypothesis that Ins deficiency leads to inositide deficiencies, which are corrected by prenatal treatment, we measured the effects of Ins rescue on Ins, phosphatidylinositol (PtdIns) and myo-inositol polyphosphate levels in brains of E18.5 knockout fetuses. As the Slc5a3 gene structure is unique in the sodium/solute cotransporter (SLC5) family, and exon 1 is shared with the mitochondrial ribosomal protein subunit 6 (Mrps6) gene, we also sought to determine whether expression of its cognate Mrps6 gene is abnormal in knockout fetuses. The mean level of Ins was increased by 92% in brains of rescued Slc5a3 knockout fetuses (0.48 versus 0.25 nmol/mg), but was still greatly reduced in comparison to wildtype (6.97 nmol/mg). The PtdIns, InsP(5) and InsP(6) levels were normal without treatment. Mrps6 gene expression was unaffected in the E18.5 knockout fetuses. This enigmatic model is not associated with neonatal PtdIns deficiency and rescue of the phenotype may be accomplished without restoration of Ins. The biochemical mechanism that both uniformly leads to death and allows for Ins rescue remains unknown. In conclusion, in neonatal brain tissue, Mrps6 gene expression may not be contingent on function of its embedded Slc5a3 gene, while inositide deficiency may not be the mechanism of lethal apnea in null Slc5a3 mice.

Altered BMP signaling disrupts chick diencephalic development.

The diencephalon is the caudal part of the forebrain and is organized into easily identifiable clusters of neurons called nuclei. Neurons in different nuclei project to discrete brain regions. Thus precise organization of the nuclei during forebrain development is necessary to build accurate neural circuits. How diencephalic development is regulated is poorly understood. BMP signaling participates in central nervous system patterning and development at many levels along the neural axis. Based on their expression we hypothesized BMPs play a role in diencephalic development. To test this hypothesis, we electroporated constitutively active and dominant negative forms of type I BMP receptors (Bmpr1a and Bmpr1b) into the embryonic chick forebrain. Ectopic induction of BMP signaling through constitutively active forms of the type I BMP receptors perturbs the normal gene expression patterns in the diencephalon and increases apoptotic cell death. These defects lead to disorganization of the diencephalic nuclei, suggesting BMP signaling is sufficient to modify diencephalic development. Loss-of-function studies, using dominant negative forms of Bmpr1a and Bmpr1b, indicate type I BMP receptors are necessary for normal eye and craniofacial development. However, they do not appear to be required for normal diencephalic development. In summary, our data indicate that while not necessary, BMP signaling via Bmpr1a and Bmpr1b, is sufficient to modify nuclear organization in the chick diencephalon.

The proapoptotic activities of Bax and Bak limit the size of the neural stem cell pool.

The proapoptotic Bcl-2 family members Bak and Bax play central and redundant roles in the regulation of apoptosis. In this study, we investigated the effect of loss of Bax and Bak in the CNS. The adult bax-/-bak-/- mice display masses of densely staining cells in the proliferative zones of the brain. These cells are shown to be a mix of neural progenitor cells and postmitotic cells at different stages of neural and glial differentiation. Both neural progenitor cells and mature neurons derived from bax-/-bak-/- mice were resistant to various apoptotic stimuli. Despite this resistance, postmitotic mature bax-/-bak-/- neurons remain as sensitive to excitoxic death as wild-type neurons. Thus, Bax and Bak play a critical role in regulating the number of neural progenitor cells in the adult brain but are not absolutely required for the initiation of neuronal cell death after neurotoxic injury.

Neurobeachin is essential for neuromuscular synaptic transmission.

We report a random disruption in the mouse genome that resulted in lethal paralysis in homozygous newborns. The disruption blocked expression of neurobeachin, a protein containing a BEACH (beige and Chediak-Higashi) domain implicated in synaptic vesicle trafficking and an AKAP (A-kinase anchor protein) domain linked to localization of cAMP-dependent protein kinase activity. nbea-null mice demonstrated a complete block of evoked synaptic transmission at neuromuscular junctions, whereas nerve conduction, synaptic structure, and spontaneous synaptic vesicle release were completely normal. These findings support an essential role for neurobeachin in evoked neurotransmitter release at neuromuscular junctions and suggest that it plays an important role in synaptic transmission.