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

FoxO1a-Cyclic GMP-Dependent Kinase I Interactions Orchestrate Myoblast Fusion

The regulatory circuits that orchestrate mammalian myoblast cell fusion during myogenesis are poorly understood. The transcriptional activity of FoxO1a directly regulates this process, yet the molecular mechanisms governing FoxO1a activity during muscle cell differentiation remain unknown. Here we show an autoregulatory loop in which FoxO1a directly activates transcription of the cyclic GMP-dependent protein kinase I (cGKI) gene and where the ensuing cGKI activity phosphorylates FoxO1a and abolishes its DNA binding activity. These findings establish the FoxO1a-to-cGKI pathway as a novel feedback loop that allows the precise tuning of myoblast fusion. Interestingly, this pathway appears to operate independently of muscle cell differentiation programs directed by myogenic transcription factors

Potassium-chloride cotransporter 3 interacts with Vav2 to synchronize the cell volume decrease response with cell protrusion dynamics.

Loss-of-function of the potassium-chloride cotransporter 3 (KCC3) causes hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), a severe neurodegenerative disease associated with defective midline crossing of commissural axons in the brain. Conversely, KCC3 over-expression in breast, ovarian and cervical cancer is associated with enhanced tumor cell malignancy and invasiveness. We identified a highly conserved proline-rich sequence within the C-terminus of the cotransporter which when mutated leads to loss of the KCC3-dependent regulatory volume decrease (RVD) response in Xenopus Laevis oocytes. Using SH3 domain arrays, we found that this poly-proline motif is a binding site for SH3-domain containing proteins in vitro. This approach identified the guanine nucleotide exchange factor (GEF) Vav2 as a candidate partner for KCC3. KCC3/Vav2 physical interaction was confirmed using GST-pull down assays and immuno-based experiments. In cultured cervical cancer cells, KCC3 co-localized with the active form of Vav2 in swelling-induced actin-rich protruding sites and within lamellipodia of spreading and migrating cells. These data provide evidence of a molecular and functional link between the potassium-chloride co-transporters and the Rho GTPase-dependent actin remodeling machinery in RVD, cell spreading and cell protrusion dynamics, thus providing new insights into KCC3's involvement in cancer cell malignancy and in corpus callosum agenesis in HMSN/ACC

KCC3 colocalizes with the active form of Vav2 in actin-rich membrane protrusions induced by hypotonic conditions.

<p>(<b>A</b>) Distribution of wild-type and mutant KCC3 forms in transiently transfected HeLa cells. Note the aberrant distribution of the KCC3 mutant forms in the cytoplasm. (<b>B</b>) The active form of Vav2 accumulates with wild-type KCC3 but not with KCC3^mPro or KCC3^ΔCterm at the cell periphery. Wild-type KCC3 accumulates with pVav2 in actin-rich plasma membrane protrusion under hypotonic conditions (<i>left panel</i>). These results were obtained after a 10 min treatment either in an isotonic or in a hypotonic medium. In the merged images, KCC3 reactivity is indicated in red, pY-Vav2 is indicated in green and polymerized actin detected by phalloidin is indicated in blue. Arrows indicate co-localisation; the stars (*) indicate actin-rich membrane not showing KCC3 immuno-detection; the arrowheads indicate actin-rich membrane showing KCC3 immuno-reactivity but not pVav2 co-localisation.</p

Distal truncation of KCC3 in non-French Canadian HMSN/ACC families.

Item does not contain fulltextBACKGROUND: Hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC) is a severe and progressive autosomal recessive polyneuropathy. Mutations in the potassium-chloride cotransporter 3 gene (KCC3) were identified as responsible for HMSN/ACC in the French Canadian (FC) population. In the present study, the authors were interested in finding new mutations in non-FC populations, assessing the activity of mutant proteins and refining genotype-phenotype correlations. METHODS: The authors screened KCC3 for mutations using direct sequencing in six non-FC HMSN/ACC families. They then assessed the functionality of the most common mutant protein using a flux assay in Xenopus laevis oocytes. RESULTS: The authors identified mutations in exon 22 of KCC3: a novel mutation (del + 2994-3003; E1015X) in one family, as well as a known mutation (3031C-->T; R1011X) found in five unrelated families and associated with two different haplotypes. The function of the cotransporter was abolished, although a limited amount of mutant proteins were correctly localized at the membrane. CONCLUSIONS: KCC3 mutations in exon 22 constitute a recurrent mutation site for hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), regardless of ethnic origin, and are the most common cause of HMSN/ACC in the non-French Canadian (FC) families analyzed so far. Therefore, for genetic analysis, exon 22 screening should be prioritized in non-FC populations. Finally, the R1011X mutation leads to the abrogation of KCC3's function in Xenopus laevis oocytes, likely due to impaired transit of the cotransporter

KCC3 proline-rich segment mediates binding to Vav2 SH3-domain.

<p>The proline-rich sequence interacts with a collection of SH3 domains detected after incubation of a biotinylated wild-type peptide (LLNMPG<b>PP</b>RNPEGDE) with a commercially available SH3 domain array (<i>Panomics</i>). Wild-type peptide binds to the second SH3 domain (D2) of the Vav proteins Vav2 and Vav3 but not to the SH3-domain of cortactin. To control for binding specificity of the wild-type peptide on the array, a peptide mutated for two proline residues (LLNMPG<b>QA</b>RNPEGDE) was used in parallel experiments.</p

Identification of a functional proline-rich motif in the C-terminal domain of KCC3.

<p>(<b>A</b>) Protein sequence of the KCC3 C-terminal domain. The C-terminus of KCC3 contains a distal poly-proline motif indicated in bold characters which is predicted to be a binding site for SH3 domains with a non-canonical class I recognition specificity (<i>ELM motif data base</i>). The C-terminus also contains a hydrophobic tetrad and a predicted Tyrosine residue (both underlined) involved in cation-chloride co-transporter trafficking. Predicted PDZ domains interacting motifs are shown in italic. Three HMSN/ACC non-sense mutations are indicated by a dot (.). The phosphorylated sites are indicated by a star (*). (<b>B</b>) KCC3 C-terminal domain is highly conserved in the proline-rich region. The motif is conserved at 100% between KCC3 homologs but also between KCC3 orthologs KCC1, KCC2 and KCC4. The proline stretch was mutated in order to generate mPro mutant forms of KCC3 polypeptide and protein.</p

Vav2 interacts with the C-terminal domain of KCC3.

<p>(<b>A</b>) Vav2 endogenously expressed by HeLa cells binds to the C-terminal domain of KCC3 in GST-pull down assays. Lysate  =  whole protein lysate extracted from HeLa cells; Lysate*  =  pre-cleared HeLa cell protein lysate incubated with sepharose beads; GST  =  pre-cleared protein lysate incubated with GST only; GST-WT  =  pre-cleared protein lysate incubated with chimeric protein where KCC3 C-teminus was fused to GST; GST-mPro  =  pre-cleared protein lysate incubated with chimeric protein where KCC3 C-terminus was mutated for two prolines (PP→QA) and fused to GST. (<b>B</b>) KCC3 and Vav2 are endogenously expressed in HeLa cells. Transient over-expression of KCC3 in HeLa cells is associated with abundant accumulation of the wild-type protein (but not the proline mutated form) in cellular protrusions. (<b>C</b>) Vav2 and KCC3 co-immunoprecipitate together in HeLa cells. Lysate  =  whole protein lysate extracted from HeLa cells; Mock  =  protein lysate extracted from HeLa cells transfected with the control empty vector; KCC3, KCC3^mPro and KCC3^ΔCterm  =  protein lysate from HeLa cells transfected with the indicated KCC3 form.</p

Evaluation of wild-type and mutant KCC3^ΔCterm, KCC3^mPro functions in <i>Xenopus</i> oocyte flux assays.

<p>(<b>A</b>) Wild-type KCC3 and mutant KCC3^ΔCterm, KCC3^mPro transit to the plasma membrane in <i>Xenopus Laevis</i> oocytes. Immunofluorescence staining of KCC3 using a specific antibody shows all generated forms at the plasma membrane of the injected oocytes. Control oocytes were injected with water instead of the wild-type and mutant KCC3 cRNAs. (<b>B</b>) Directed mutagenesis of the proline motif leads to impaired function of KCC3 in <i>Xenopus Laevis</i> oocyte flux assay. ⁸⁶Rb⁺ flux of wild-type and mutant KCC3 is measured under hypotonic conditions in presence or absence of KCCs inhibitor, furosemide.</p