Valproic acid is protective in cellular and worm models of oculopharyngeal muscular dystrophy.

OBJECTIVE: To explore valproic acid (VPA) as a potentially beneficial drug in cellular and worm models of oculopharyngeal muscular dystrophy (OPMD). METHODS: Using a combination of live cell imaging and biochemical measures, we evaluated the potential protective effect of VPA in a stable C2C12 muscle cell model of OPMD, in lymphoblastoid cell lines derived from patients with OPMD and in a transgenic Caenorhabditis elegans OPMD model expressing human mutant PABPN1. RESULTS: We demonstrated that VPA protects against the toxicity of mutant PABPN1. Of note, we found that VPA confers its long-term protective effects on C2C12 cell survival, proliferation, and differentiation by increasing the acetylated level of histones. Furthermore, VPA enhances the level of histone acetylation in lymphoblastoid cell lines derived from patients with OPMD. Moreover, treatment of nematodes with moderate concentrations of VPA significantly improved the motility of the PABPN-13 Alanines worms. CONCLUSIONS: Our results suggest that VPA helps to counteract OPMD-related phenotypes in the cellular and C elegans disease models.

KCC3 axonopathy: neuropathological features in the central and peripheral nervous system.

Hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC) is an autosomal recessive disease of the central and peripheral nervous system that presents as early-onset polyneuropathy. Patients are hypotonic and areflexic from birth, with abnormal facial features and atrophic muscles. Progressive peripheral neuropathy eventually confines them to a wheelchair in the second decade of life, and death occurs by the fourth decade. We here define the neuropathologic features of the disease in autopsy tissues from eight cases. Both developmental and neurodegenerative features were found. Hypoplasia or absence of the major telencephalic commissures and a hypoplasia of corticospinal tracts to half the normal size, were the major neurodevelopmental defects we observed. Despite being a neurodegenerative disease, preservation of brain weight and a conspicuous absence of neuronal or glial cell death were signal features of this disease. Small tumor-like overgrowths of axons, termed axonomas, were found in the central and peripheral nervous system, indicating attempted axonal regeneration. We conclude that the neurodegenerative deficits in HMSN/ACC are primarily caused by an axonopathy superimposed upon abnormal development, affecting peripheral but also central nervous system axons, all ultimately because of a genetic defect in the axonal cotransporter KCC3.

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.

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.

Comparative analysis of the expression profile of Wnk1 and Wnk1/Hsn2 splice variants in developing and adult mouse tissues.

The With No lysine (K) family of serine/threonine kinase (WNK) defines a small family of kinases with significant roles in ion homeostasis. WNK1 has been shown to have different isoforms due to what seems to be largely tissue specific splicing. Here, we used two distinct in situ hybridization riboprobes on developing and adult mouse tissues to make a comparative analysis of Wnk1 and its sensory associated splice isoform, Wnk1/Hsn2. The hybridization signals in developing mouse tissues, which were prepared at embryonic day e10.5 and e12.5, revealed a homogenous expression profile with both probes. At e15.5 and in the newborn mouse, the two probes revealed different expression profiles with prominent signals in nervous system tissues and also other tissues such as kidney, thymus and testis. In adult mouse tissues, the two expression profiles appeared even more restricted to the nervous tissues, kidney, thymus and testis, with no detectable signal in the other tissues. Throughout the nervous system, sensory tissues, as well as in Cornu Ammonis 1 (CA1), CA2 and CA3 areas of the hippocampus, were strongly labeled with both probes. Hybridization signals were also strongly detected in Schwann and supporting satellite cells. Our results show that the expression profiles of Wnk1 isoforms change during the development, and that the expression of the Wnk1 splice variant containing the Hsn2 exon is prominent during developing and in adult mouse tissues, suggesting its important role in the development and maintenance of the nervous system.

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.

Expanded ATXN3 frameshifting events are toxic in Drosophila and mammalian neuron models.

Spinocerebellar ataxia type 3 is caused by the expansion of the coding CAG repeat in the ATXN3 gene. Interestingly, a -1 bp frameshift occurring within an (exp)CAG repeat would henceforth lead to translation from a GCA frame, generating polyalanine stretches instead of polyglutamine. Our results show that transgenic expression of (exp)CAG ATXN3 led to -1 frameshifting events, which have deleterious effects in Drosophila and mammalian neurons. Conversely, transgenic expression of polyglutamine-encoding (exp)CAA ATXN3 was not toxic. Furthermore, (exp)CAG ATXN3 mRNA does not contribute per se to the toxicity observed in our models. Our observations indicate that expanded polyglutamine tracts in Drosophila and mouse neurons are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with (exp)CAG repeats.

Transit defect of potassium-chloride Co-transporter 3 is a major pathogenic mechanism in hereditary motor and sensory neuropathy with agenesis of the corpus callosum.

Missense and protein-truncating mutations of the human potassium-chloride co-transporter 3 gene (KCC3) cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe neurodegenerative disease characterized by axonal dysfunction and neurodevelopmental defects. We previously reported that KCC3-truncating mutations disrupt brain-type creatine kinase-dependent activation of the co-transporter through the loss of its last 140 amino acids. Here, we report a novel and more distal HMSN/ACC-truncating mutation (3402C → T; R1134X) that eliminates only the last 17 residues of the protein. This small truncation disrupts the interaction with brain-type creatine kinase in mammalian cells but also affects plasma membrane localization of the mutant transporter. Although it is not truncated, the previously reported HMSN/ACC-causing 619C → T (R207C) missense mutation also leads to KCC3 loss of function in Xenopus oocyte flux assay. Immunodetection in Xenopus oocytes and in mammalian cultured cells revealed a decreased amount of R207C at the plasma membrane, with significant retention of the mutant proteins in the endoplasmic reticulum. In mammalian cells, curcumin partially corrected these mutant protein mislocalizations, with more protein reaching the plasma membrane. These findings suggest that mis-trafficking of mutant protein is an important pathophysiological feature of HMSN/ACC causative KCC3 mutations.

Cellular expression of the K+-Cl- cotransporter KCC3 in the central nervous system of mouse.

Potassium/Chloride cotransporters are transmembrane proteins that regulate cell volume and control neuronal activity by transporting K(+) and Cl(-) ions across the plasma membrane. Potassium/Chloride cotransporter 3 (KCC3) mutations are responsible for hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe sensory and motor neuropathy. Two major splice variants, KCC3a and KCC3b, were shown to be expressed in adult mouse tissues. Although KCC3a is mainly expressed in the central nervous system (CNS), its specific cellular expression patterns have not been determined. Here, we used an approach combining in situ hybridization and immunohistochemical techniques to determine the cellular expression of KCC3 in the mouse CNS and showed that KCC3 is mainly expressed in neurons, including a subpopulation of interneurons. Finally, we showed that some non-neuronal cells, such as radial glial-like cells in the spinal cord, also express KCC3.

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.

Soluble expanded PABPN1 promotes cell death in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by the expansion of a polyalanine repeat (GCG)(8-13) in exon 1 of the PABPN1 gene. Skeletal muscle fibers nuclei from OPMD patients contain insoluble polyalanine expanded PABPN1 (expPABPN1) nuclear aggregates that sequester different cellular components. Whether these aggregates are pathogenic, or the consequence of a molecular defense mechanism, remains controversial in the field of neurodegenerative disorders and OPMD. Our cellular model shows that interfering with the formation of expPABPN1-induced large nuclear aggregates increases the availability of nuclear expPABPN1 and significantly exacerbates cell death. Live microscopy reveals that cells harboring an increased amount of the soluble forms of expPABPN1 are significantly more prone to toxicity than those with nuclear aggregates. This is the first report directly indicating that nuclear aggregation in OPMD may reflect an active process by which cells sequester and inactivate the soluble toxic form of expPABPN1.

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.

Transgenic expression of an expanded (GCG)13 repeat PABPN1 leads to weakness and coordination defects in mice.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder caused by a (GCG)n trinucleotide repeat expansion in the poly(A) binding protein nuclear-1 (PABPN1) gene, which in turn leads to an expanded polyalanine tract in the protein. We generated transgenic mice expressing either the wild type or the expanded form of human PABPN1, and transgenic animals with the expanded form showed clear signs of abnormal limb clasping, muscle weakness, coordination deficits, and peripheral nerves alterations. Analysis of mitotic and postmitotic tissues in those transgenic animals revealed ubiquitinated PABPN1-positive intranuclear inclusions (INIs) in neuronal cells. This latter observation led us to test and confirm the presence of similar INIs in postmortem brain sections from an OPMD patient. Our results indicate that expanded PABPN1, presumably via the toxic effects of its polyalanine tract, can lead to inclusion formation and neurodegeneration in both the mouse and the human.

HnRNP A1 and A/B interaction with PABPN1 in oculopharyngeal muscular dystrophy.

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive ptosis, dysphagia and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)6 repeat to (GCG) in the PABPN1 gene. The mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of PABPN1. The mutated PABPN1 (mPABPN1) induces the formation of intranuclear filamentous inclusions that sequester poly(A) RNA and are associated with cell death. METHODS: Human fetal brain cDNA library was used to look for PABPNI binding proteins using yeast two-hybrid screen. The protein interaction was confirmed by GST pull-down and co-immunoprecipitation assays. Oculopharyngeal muscular dystrophy cellular model and OPMD patient muscle tissue were used to check whether the PABPN1 binding proteins were involved in the formation of OPMD intranuclear inclusions. RESULTS: We identify two PABPNI interacting proteins, hnRNP A1 and hnRNP A/B. When co-expressed with mPABPN1 in COS-7 cells, predominantly nuclear protein hnRNP A1 and A/B co-localize with mPABPN1 in the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. CONCLUSIONS: The hnRNP proteins are involved in mRNA processing and mRNA nucleocytoplasmic export, sequestering of hnRNPs in OPMD intranuclear aggregates supports the view that OPMD intranuclear inclusions are "poly(A) RNA traps", which would interfere with RNA export, and cause muscle cell death.

Involvement of the ubiquitin-proteasome pathway and molecular chaperones in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant muscular dystrophy that results from small expansions of a polyalanine tract in the PABPN1 gene. Intranuclear inclusions are the pathological hallmark of OPMD. The mechanism by which protein aggregation in OPMD might relate to a toxic gain-of-function has so far remained elusive. Whether protein aggregates themselves are pathogenic or are the consequence of an unidentified underlying molecular mechanism is still unclear. Here, we report that protein aggregation in a cell model of OPMD directly impaires the function of the ubiquitin-proteasome pathway (UPP) as well as molecular chaperone functions. The proteasome inhibitor lactacystin causes significant increase of protein aggregation and toxicity. Moreover, overexpression of molecular chaperones (HSP40 and HSP70) suppressed protein aggregation and toxicity. We also provide evidence that mPABPN1-ala17 protein aggregation proportionally correlates with toxicity. Furthermore, we show that co-expression of chaperones in our OPMD cell model increases the solubility of mPABPN1-ala17 and transfected cell survival rate. Our studies suggest that molecular regulators of polyalanine protein solubility and degradation may provide insights into new mechanisms in OPMD pathogenesis. Further analysis of the cellular and molecular mechanisms by which UPP and molecular chaperones influence the degradation of misfolded proteins could provide novel concepts and targets for the treatment and understanding of the pathogenesis of OPMD and neurodegenerative diseases.