Rare pathogenic variants in WNK3 cause X-linked intellectual disability.

PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had ID with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked ID with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.

Recessive Inheritance of Congenital Hydrocephalus With Other Structural Brain Abnormalities Caused by Compound Heterozygous Mutations in ATP1A3.

BACKGROUND: ATP1A3 encodes the α3 subunit of the Na+/K+ ATPase, a fundamental ion-transporting enzyme. Primarily expressed in neurons, ATP1A3 is mutated in several autosomal dominant neurological diseases. To our knowledge, damaging recessive genotypes in ATP1A3 have never been associated with any human disease. Atp1a3 deficiency in zebrafish results in hydrocephalus; however, no known association exists between ATP1A3 and human congenital hydrocephalus (CH). METHODS: We utilized whole-exome sequencing (WES), bioinformatics, and computational modeling to identify and characterize novel ATP1A3 mutations in a patient with CH. We performed immunohistochemical studies using mouse embryonic brain tissues to characterize Atp1a3 expression during brain development. RESULTS: We identified two germline mutations in ATP1A3 (p. Arg19Cys and p.Arg463Cys), each of which was inherited from one of the patient's unaffected parents, in a single patient with severe obstructive CH due to aqueductal stenosis, along with open schizencephaly, type 1 Chiari malformation, and dysgenesis of the corpus callosum. Both mutations are predicted to be highly deleterious and impair protein stability. Immunohistochemical studies demonstrate robust Atp1a3 expression in neural stem cells (NSCs), differentiated neurons, and choroid plexus of the mouse embryonic brain. CONCLUSION: These data provide the first evidence of a recessive human phenotype associated with mutations in ATP1A3, and implicate impaired Na+/K+ ATPase function in the pathogenesis of CH.

Protocadherin-18b interacts with Nap1 to control motor axon growth and arborization in zebrafish.

The proper assembly of neural circuits during development requires the precise control of axon outgrowth, guidance, and arborization. Although the protocadherin family of cell surface receptors is widely hypothesized to participate in neural circuit assembly, their specific roles in neuronal development remain largely unknown. Here we demonstrate that zebrafish pcdh18b is involved in regulating axon arborization in primary motoneurons. Although axon outgrowth and elongation appear normal, antisense morpholino knockdown of pcdh18b results in dose-dependent axon branching defects in caudal primary motoneurons. Cell transplantation experiments show that this effect is cell autonomous. Pcdh18b interacts with Nap1, a core component of the WAVE complex, through its intracellular domain, suggesting a role in the control of actin assembly. Like that of Pcdh18b, depletion of Nap1 results in reduced branching of motor axons. Time-lapse imaging and quantitative analysis of axon dynamics indicate that both Pcdh18b and Nap1 regulate axon arborization by affecting the density of filopodia along the shaft of the extending axon.

Small molecule suppressors of Drosophila kinesin deficiency rescue motor axon development in a zebrafish model of spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is the most common inherited motor neuropathy and the leading hereditary cause of infant mortality. Currently there is no effective treatment for the disease, reflecting a need for pharmacologic interventions that restore performance of dysfunctional motor neurons or suppress the consequences of their dysfunction. In a series of assays relevant to motor neuron biology, we explored the activities of a collection of tetrahydroindoles that were reported to alter the metabolism of amyloid precursor protein (APP). In Drosophila larvae the compounds suppressed aberrant larval locomotion due to mutations in the Khc and Klc genes, which respectively encode the heavy and light chains of kinesin-1. A representative compound of this class also suppressed the appearance of axonal swellings (alternatively termed axonal spheroids or neuritic beads) in the segmental nerves of the kinesin-deficient Drosophila larvae. Given the importance of kinesin-dependent transport for extension and maintenance of axons and their growth cones, three members of the class were tested for neurotrophic effects on isolated rat spinal motor neurons. Each compound stimulated neurite outgrowth. In addition, consistent with SMA being an axonopathy of motor neurons, the three axonotrophic compounds rescued motor axon development in a zebrafish model of SMA. The results introduce a collection of small molecules as pharmacologic suppressors of SMA-associated phenotypes and nominate specific members of the collection for development as candidate SMA therapeutics. More generally, the results reinforce the perception of SMA as an axonopathy and suggest novel approaches to treating the disease.

Temporal requirement for SMN in motoneuron development.

Proper function of the motor unit is dependent upon the correct development of dendrites and axons. The infant/childhood onset motoneuron disease spinal muscular atrophy (SMA), caused by low levels of the survival motor neuron (SMN) protein, is characterized by muscle denervation and paralysis. Although different SMA models have shown neuromuscular junction defects and/or motor axon defects, a comprehensive analysis of motoneuron development in vivo under conditions of low SMN will give insight into why the motor unit becomes dysfunctional. We have generated genetic mutants in zebrafish expressing low levels of SMN from the earliest stages of development. Analysis of motoneurons in these mutants revealed motor axons were often shorter and had fewer branches. We also found that motoneurons had significantly fewer dendritic branches and those present were shorter. Analysis of motor axon filopodial dynamics in live embryos revealed that mutants had fewer filopodia and their average half-life was shorter. To determine when SMN was needed to rescue motoneuron development, SMN was conditionally induced in smn mutants during embryonic stages. Only when SMN was added back soon after motoneurons were born, could later motor axon development be rescued. Importantly, analysis of motor behavior revealed that animals with motor axon defects had significant deficits in motor output. We also show that SMN is required earlier for motoneuron development than for survival. These data support that SMN is needed early in development of motoneuron dendrites and axons to develop normally and that this is essential for proper connectivity and movement.

An SMN-dependent U12 splicing event essential for motor circuit function.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA.

Survival motor neuron affects plastin 3 protein levels leading to motor defects.

The actin-binding protein plastin 3 (PLS3) has been identified as a modifier of the human motoneuron disease spinal muscular atrophy (SMA). SMA is caused by decreased levels of the survival motor neuron protein (SMN) and in its most severe form causes death in infants and young children. To understand the mechanism of PLS3 in SMA, we have analyzed pls3 RNA and protein in zebrafish smn mutants. We show that Pls3 protein levels are severely decreased in smn(-/-) mutants without a reduction in pls3 mRNA levels. Moreover, we show that both pls3 mRNA and protein stability are unaffected when Smn is reduced. This indicates that SMN affects PLS3 protein production. We had previously shown that, in smn mutants, the presynaptic protein SV2 is decreased at neuromuscular junctions. Transgenically driving human PLS3 in motoneurons rescues the decrease in SV2 expression. To determine whether PLS3 could also rescue function, we performed behavioral analysis on smn mutants and found that they had a significant decrease in spontaneous swimming and turning. Driving PLS3 transgenically in motoneurons rescued both of these defects. These data show that PLS3 protein levels are dependent on SMN and that PLS3 is able to rescue the neuromuscular defects and corresponding movement phenotypes caused by low levels of Smn suggesting that decreased PLS3 contributes to SMA motor phenotypes.

Zebrafish mnx1 controls cell fate choice in the developing endocrine pancreas.

The vertebrate endocrine pancreas has the crucial function of maintaining blood sugar homeostasis. This role is dependent upon the development and maintenance of pancreatic islets comprising appropriate ratios of hormone-producing cells. In all vertebrate models studied, an initial precursor population of Pdx1-expressing endoderm cells gives rise to separate endocrine and exocrine cell lineages. Within the endocrine progenitor pool a variety of transcription factors influence cell fate decisions, such that hormone-producing differentiated cell types ultimately arise, including the insulin-producing beta cells and the antagonistically acting glucagon-producing alpha cells. In previous work, we established that the development of all pancreatic lineages requires retinoic acid (RA) signaling. We have used the zebrafish to uncover genes that function downstream of RA signaling, and here we identify mnx1 (hb9) as an RA-regulated endoderm transcription factor-encoding gene. By combining manipulation of gene function, cell transplantation approaches and transgenic reporter analysis we establish that Mnx1 functions downstream of RA within the endoderm to control cell fate decisions in the endocrine pancreas progenitor lineage. We confirm that Mnx1-deficient zebrafish lack beta cells, and, importantly, we make the novel observation that they concomitantly gain alpha cells. In Mnx1-deficient embryos, precursor cells that are normally destined to differentiate as beta cells instead take on an alpha cell fate. Our findings suggest that Mnx1 functions to promote beta and suppress alpha cell fates.

Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits.

Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of ß-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation, suggesting a crucial role in the development of motor axons and neuromuscular junctions. Cpg15 mRNA previously has been shown to localize into axonal processes. Here we show that SMN deficiency reduces cpg15 mRNA levels in neurons, and, more importantly, cpg15 overexpression partially rescues the SMN-deficiency phenotype in zebrafish. Our results provide insight into the function of SMN protein in axons and also identify potential targets for the study of mechanisms that lead to the SMA pathology and related neuromuscular diseases.

Generation and Characterization of a genetic zebrafish model of SMA carrying the human SMN2 gene.

BACKGROUND: Animal models of human diseases are essential as they allow analysis of the disease process at the cellular level and can advance therapeutics by serving as a tool for drug screening and target validation. Here we report the development of a complete genetic model of spinal muscular atrophy (SMA) in the vertebrate zebrafish to complement existing zebrafish, mouse, and invertebrate models and show its utility for testing compounds that alter SMN2 splicing. RESULTS: The human motoneuron disease SMA is caused by low levels, as opposed to a complete absence, of the survival motor neuron protein (SMN). To generate a true model of SMA in zebrafish, we have generated a transgenic zebrafish expressing the human SMN2 gene (hSMN2), which produces only a low amount of full-length SMN, and crossed this onto the smn-/- background. We show that human SMN2 is spliced in zebrafish as it is in humans and makes low levels of SMN protein. Moreover, we show that an antisense oligonucleotide that enhances correct hSMN2 splicing increases full-length hSMN RNA in this model. When we placed this transgene on the smn mutant background it rescued the neuromuscular presynaptic SV2 defect that occurs in smn mutants and increased their survival. CONCLUSIONS: We have generated a transgenic fish carrying the human hSMN2 gene. This gene is spliced in fish as it is in humans and mice suggesting a conserved splicing mechanism in these vertebrates. Moreover, antisense targeting of an intronic splicing silencer site increased the amount of full length SMN generated from this transgene. Having this transgene on the smn mutant fish rescued the presynaptic defect and increased survival. This model of zebrafish SMA has all of the components of human SMA and can thus be used to understand motoneuron dysfunction in SMA, can be used as an vivo test for drugs or antisense approaches that increase full-length SMN, and can be developed for drug screening.