KCC3 loss-of-function contributes to Andermann syndrome by inducing activity-dependent neuromuscular junction defects.

Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.

RNF213 Is Associated with Intracranial Aneurysms in the French-Canadian Population.

Intracranial aneurysms (IAs) are the result of focal weakness in the artery wall and have a complex genetic makeup. To date, genome-wide association and sequencing studies have had limited success in identifying IA risk factors. Distinct populations, such as the French-Canadian (FC) population, have increased IA prevalence. In our study, we used exome sequencing to prioritize risk variants in a discovery cohort of six FC families affected by IA, and the analysis revealed an increased variation burden for ring finger protein 213 (RNF213). We resequenced RNF213 in a larger FC validation cohort, and association tests on further identified variants supported our findings (SKAT-O, p = 0.006). RNF213 belongs to the AAA+ protein family, and two variants (p.Arg2438Cys and p.Ala2826Thr) unique to affected FC individuals were found to have increased ATPase activity, which could lead to increased risk of IA by elevating angiogenic activities. Common SNPs in RNF213 were also extracted from the NeuroX SNP-chip genotype data, comprising 257 FC IA-affected and 1,988 control individuals. We discovered that the non-ancestral allele of rs6565666 was significantly associated with the affected individuals (p = 0.03), and it appeared as though the frequency of the risk allele had changed through genetic drift. Although RNF213 is a risk factor for moyamoya disease in East Asians, we demonstrated that it might also be a risk factor for IA in the FC population. It therefore appears that the function of RNF213 can be differently altered to predispose distinct populations to dissimilar neurovascular conditions, highlighting the importance of a population's background in genetic studies of heterogeneous disease.

Inhibition of the kinase WNK1/HSN2 ameliorates neuropathic pain by restoring GABA inhibition.

HSN2is a nervous system predominant exon of the gene encoding the kinase WNK1 and is mutated in an autosomal recessive, inherited form of congenital pain insensitivity. The HSN2-containing splice variant is referred to as WNK1/HSN2. We created a knockout mouse specifically lacking theHsn2exon ofWnk1 Although these mice had normal spinal neuron and peripheral sensory neuron morphology and distribution, the mice were less susceptible to hypersensitivity to cold and mechanical stimuli after peripheral nerve injury. In contrast, thermal and mechanical nociceptive responses were similar to control mice in an inflammation-induced pain model. In the nerve injury model of neuropathic pain, WNK1/HSN2 contributed to a maladaptive decrease in the activity of the K(+)-Cl(-)cotransporter KCC2 by increasing its inhibitory phosphorylation at Thr(906)and Thr(1007), resulting in an associated loss of GABA (γ-aminobutyric acid)-mediated inhibition of spinal pain-transmitting nerves. Electrophysiological analysis showed that WNK1/HSN2 shifted the concentration of Cl(-)such that GABA signaling resulted in a less hyperpolarized state (increased neuronal activity) rather than a more hyperpolarized state (decreased neuronal activity) in mouse spinal nerves. Pharmacologically antagonizing WNK activity reduced cold allodynia and mechanical hyperalgesia, decreased KCC2 Thr(906)and Thr(1007)phosphorylation, and restored GABA-mediated inhibition (hyperpolarization) of injured spinal cord lamina II neurons. These data provide mechanistic insight into, and a compelling therapeutic target for treating, neuropathic pain after nerve injury.

Exome sequencing identifies recessive CDK5RAP2 variants in patients with isolated agenesis of corpus callosum.

Agenesis of the corpus callosum (ACC) is a common brain malformation which can be observed either as an isolated condition or as part of numerous congenital syndromes. Therefore, cognitive and neurological involvements in patients with ACC are variable, from mild linguistic and behavioral impairments to more severe neurological deficits. To date, the underlying genetic causes of isolated ACC remains elusive and causative genes have yet to be identified. We performed exome sequencing on three acallosal siblings from the same non-consanguineous family and identified compound heterozygous variants, p.[Gly94Arg];[Asn1232Ser], in the protein encoded by the CDK5RAP2 gene, also known as MCPH3, a gene previously reported to cause autosomal recessive primary microcephaly. Our findings suggest a novel role for this gene in the pathogenesis of isolated ACC.

Deleterious mutations in the essential mRNA metabolism factor, hGle1, in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons. Causative mutations in the global RNA-processing proteins TDP-43 and FUS among others, as well as their aggregation in ALS patients, have identified defects in RNA metabolism as an important feature in this disease. Lethal congenital contracture syndrome 1 and lethal arthrogryposis with anterior horn cell disease are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1. In this study, we carried out the first screening of GLE1 in ALS patients (173 familial and 760 sporadic) and identified 2 deleterious mutations (1 splice site and 1 nonsense mutation) and 1 missense mutation. Functional analysis of the deleterious mutants revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells, both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA. These results suggest a haploinsufficiency mechanism and point to a causative role for GLE1 mutations in ALS patients. This further supports the involvement of global defects in RNA metabolism in ALS.

Exome sequencing identifies FUS mutations as a cause of essential tremor.

Essential tremor (ET) is a common neurodegenerative disorder that is characterized by a postural or motion tremor. Despite a strong genetic basis, a gene with rare pathogenic mutations that cause ET has not yet been reported. We used exome sequencing to implement a simple approach to control for misdiagnosis of ET, as well as phenocopies involving sporadic and senile ET cases. We studied a large ET-affected family and identified a FUS p.Gln290(∗) mutation as the cause of ET in this family. Further screening of 270 ET cases identified two additional rare missense FUS variants. Functional considerations suggest that the pathogenic effects of ET-specific FUS mutations are different from the effects observed when FUS is mutated in amyotrophic lateral sclerosis cases; we have shown that the ET FUS nonsense mutation is degraded by the nonsense-mediated-decay pathway, whereas amyotrophic lateral sclerosis FUS mutant transcripts are not.

Loss of neuronal potassium/chloride cotransporter 3 (KCC3) is responsible for the degenerative phenotype in a conditional mouse model of hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum.

Disruption of the potassium/chloride cotransporter 3 (KCC3), encoded by the SLC12A6 gene, causes hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC), a neurodevelopmental and neurodegenerative disorder affecting both the peripheral nervous system and CNS. However, the precise role of KCC3 in the maintenance of ion homeostasis in the nervous system and the pathogenic mechanisms leading to HMSN/ACC remain unclear. We established two Slc12a6 Cre/LoxP transgenic mouse lines expressing C-terminal truncated KCC3 in either a neuron-specific or ubiquitous fashion. Our results suggest that neuronal KCC3 expression is crucial for axon volume control. We also demonstrate that the neuropathic features of HMSN/ACC are predominantly due to a neuronal KCC3 deficit, while the auditory impairment is due to loss of non-neuronal KCC3 expression. Furthermore, we demonstrate that KCC3 plays an essential role in inflammatory pain pathways. Finally, we observed hypoplasia of the corpus callosum in both mouse mutants and a marked decrease in axonal tracts serving the auditory cortex in only the general deletion mutant. Together, these results establish KCC3 as an important player in both central and peripheral nervous system maintenance.

KIF1A, an axonal transporter of synaptic vesicles, is mutated in hereditary sensory and autonomic neuropathy type 2.

Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system.

Mutations in DCC cause congenital mirror movements.

Mirror movements are involuntary contralateral movements that mirror voluntary ones and are often associated with defects in midline crossing of the developing central nervous system. We studied two large families, one French Canadian and one Iranian, in which isolated congenital mirror movements were inherited as an autosomal dominant trait. We found that affected individuals carried protein-truncating mutations in DCC (deleted in colorectal carcinoma), a gene on chromosome 18q21.2 that encodes a receptor for netrin-1, a diffusible protein that helps guide axon growth across the midline. Functional analysis of the mutant DCC protein from the French Canadian family revealed a defect in netrin-1 binding. Thus, DCC has an important role in lateralization of the human nervous system.

HMSN/ACC truncation mutations disrupt brain-type creatine kinase-dependant activation of K+/Cl- co-transporter 3.

The potassium-chloride co-transporter 3 (KCC3) is mutated in hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC); however, the molecular mechanisms of HMSN/ACC pathogenesis and the exact role of KCC3 in the development of the nervous system remain poorly understood. The functional regulation of this transporter by protein partners is also largely unknown. Using a yeast two-hybrid approach, we discovered that the C-terminal domain (CTD) of KCC3, which is lost in most HMSN/ACC-causing mutations, directly interacts with brain-specific creatine kinase (CK-B), an ATP-generating enzyme that is also a partner of KCC2. The interaction of KCC3 with CK-B was further confirmed by in vitro glutathione S-transferase pull-down assay, followed by sequencing of the pulled-down complexes. In transfected cultured cells, immunofluorescence labeling showed that CK-B co-localizes with wild-type KCC3, whereas the kinase fails to interact with the inactive truncated KCC3. Finally, CK-B's inhibition by DNFB results in reduction of activity of KCC3 in functional assays using Xenopus laevis oocytes. This physical and functional association between the co-transporter and CK-B is, therefore, the first protein-protein interaction identified to be potentially involved in the pathophysiology of HMSN/ACC.

Mutations in the nervous system--specific HSN2 exon of WNK1 cause hereditary sensory neuropathy type II.

Hereditary sensory and autonomic neuropathy type II (HSANII) is an early-onset autosomal recessive disorder characterized by loss of perception to pain, touch, and heat due to a loss of peripheral sensory nerves. Mutations in hereditary sensory neuropathy type II (HSN2), a single-exon ORF originally identified in affected families in Quebec and Newfoundland, Canada, were found to cause HSANII. We report here that HSN2 is a nervous system-specific exon of the with-no-lysine(K)-1 (WNK1) gene. WNK1 mutations have previously been reported to cause pseudohypoaldosteronism type II but have not been studied in the nervous system. Given the high degree of conservation of WNK1 between mice and humans, we characterized the structure and expression patterns of this isoform in mice. Immunodetections indicated that this Wnk1/Hsn2 isoform was expressed in sensory components of the peripheral nervous system and CNS associated with relaying sensory and nociceptive signals, including satellite cells, Schwann cells, and sensory neurons. We also demonstrate that the novel protein product of Wnk1/Hsn2 was more abundant in sensory neurons than motor neurons. The characteristics of WNK1/HSN2 point to a possible role for this gene in the peripheral sensory perception deficits characterizing HSANII.