Mutations in GDAP1 lead to severe forms of the peripheral motor and sensory neuropathy, Charcot-Marie-Tooth disease (CMT), which is characterized by heterogeneous phenotypes, including pronounced axonal damage and demyelination. We show that neurons and Schwann cells express ganglioside-induced differentiation associated protein 1 (GDAP1), which suggest that both cell types may contribute to the mixed features of the disease. GDAP1 is located in the mitochondrial outer membrane and regulates the mitochondrial network. Overexpression of GDAP1 induces fragmentation of mitochondria without inducing apoptosis, affecting overall mitochondrial activity, or interfering with mitochondrial fusion. The mitochondrial fusion proteins, mitofusin 1 and 2 and Drp1(K38A), can counterbalance the GDAP1-dependent fission. GDAP1-specific knockdown by RNA interference results in a tubular mitochondrial morphology. GDAP1 truncations that are found in patients who have CMT are not targeted to mitochondria and have lost mitochondrial fragmentation activity. The latter activity also is reduced strongly for disease-associated GDAP1 point mutations. Our data indicate that an exquisitely tight control of mitochondrial dynamics, regulated by GDAP1, is crucial for the proper function of myelinated peripheral nerves.

Idiopathic epilepsies (IEs) are a group of disorders characterized by recurrent seizures in the absence of detectable brain lesions or metabolic abnormalities. IEs include common disorders with a complex mode of inheritance and rare Mendelian traits suggesting the occurrence of several alleles with variable penetrance. We previously described a large family with a recessive form of idiopathic epilepsy, named familial infantile myoclonic epilepsy (FIME), and mapped the disease locus on chromosome 16p13.3 by linkage analysis. In the present study, we found that two compound heterozygous missense mutations (D147H and A509V) in TBC1D24, a gene of unknown function, are responsible for FIME. In situ hybridization analysis revealed that Tbc1d24 is mainly expressed at the level of the cerebral cortex and the hippocampus. By coimmunoprecipitation assay we found that TBC1D24 binds ARF6, a Ras-related family of small GTPases regulating exo-endocytosis dynamics. The main recognized function of ARF6 in the nervous system is the regulation of dendritic branching, spine formation, and axonal extension. TBC1D24 overexpression resulted in a significant increase in neurite length and arborization and the FIME mutations significantly reverted this phenotype. In this study we identified a gene mutation involved in autosomal-recessive idiopathic epilepsy, unveiled the involvement of ARF6-dependent molecular pathway in brain hyperexcitability and seizures, and confirmed the emerging role of subtle cytoarchitectural alterations in the etiology of this group of common epileptic disorders.

Mutations in the gene MPZ, encoding myelin protein zero (MPZ), cause inherited neuropathies collectively called Charcot-Marie-Tooth type 1B (CMT1B). Based on the age of onset, clinical and pathological features, most MPZ mutations are separable into two groups: one causing a severe, early-onset, demyelinating neuropathy and a second, causing a late-onset neuropathy with prominent axonal loss. To investigate potential pathomechanisms underlying the two phenotypes, we transiently transfected HeLa cells with two late-onset (T95M, H10P) and two early-onset (H52R, S22_W28 deletion) mutations and analyzed their effects on intracellular protein trafficking, glycosylation, cell viability and intercellular adhesion. We found that the two late-onset mutations were both transported to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion. The two early-onset mutations caused two distinct abnormalities. H52R was correctly glycosylated and trafficked to the plasma membrane, but strongly affected intercellular adhesion. When co-expressed with wild-type MPZ (wtMPZ), a functional dominant negative effect was observed. Alternatively, S22_W28 deletion was retained within the cytoplasm and reduced both adhesion caused by wtMPZ and cellular viability. Since the same trafficking patterns were observed in transfected murine Schwann cells, they are not an artifact of heterologous cell expression. Our results suggest that at least some late-onset mutations cause a partial loss of function in the transfected cells, whereas multiple abnormal gain of function pathways can result in early-onset neuropathy. Further characterization of these pathways will lead to a better understanding of the pathogenesis of CMT1B and a rational basis for treating these debilitating inherited neuropathies.

Objective: Pain, temperature, and itch are conventionally thought to be exclusively transduced by the intraepidermal nerve endings. Although recent studies have shown that epidermal keratinocytes also participate in sensory transduction, the mechanism underlying keratinocyte communication with intraepidermal nerve endings remains poorly understood. We sought to demonstrate the synaptic character of the contacts between keratinocytes and sensory neurons and their involvement in sensory communication between keratinocytes and sensory neurons. Methods: Contacts were explored by morphological, molecular, and functional approaches in cocultures of epidermal keratinocytes and sensory neurons. To interrogate whether structures observed in vitro were also present in the human epidermis, in situ correlative light electron microscopy was performed on human skin biopsies. Results: Epidermal keratinocytes dialogue with sensory neurons through en passant synaptic-like contacts. These contacts have the ultrastructural features and molecular hallmarks of chemical synaptic-like contacts: narrow intercellular cleft, keratinocyte synaptic vesicles expressing synaptophysin and synaptotagmin 1, and sensory information transmitted from keratinocytes to sensory neurons through SNARE-mediated (syntaxin1) vesicle release. Interpretation: By providing selective communication between keratinocytes and sensory neurons, synaptic-like contacts are the hubs of a 2-site receptor. The permanent epidermal turnover, implying a specific en passant structure and high plasticity, may have delayed their identification, thereby contributing to the long-held concept of nerve endings passing freely between keratinocytes. The discovery of keratinocyte-sensory neuron synaptic-like contacts may call for a reassessment of basic assumptions in cutaneous sensory perception and sheds new light on the pathophysiology of pain and itch as well as the physiology of touch.

Phospholipases cleave phospholipids, major membrane constituents. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD, also known as MTL or ZmPLA1) leads to peculiar defects in sexual reproduction, notably the induction of maternal haploid embryos. Contrary to previous reports, NLD does not localize to cytosol and plasma membrane of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. After pollen tube burst, NLD localizes at the apical region of the egg apparatus. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and lipid biosensors indicated that phosphatidylinositol-4,5-bisphosphate is enriched in the endo-PM, uncovering a unique example of how membrane electrostatic properties can define a specific polar domain (i.e., endo-PM), which is critical for plant reproduction and gamete formation.

The human small heat shock proteins (HSPBs) form a family of molecular chaperones comprising ten members (HSPB1-HSPB10), whose functions span from protein quality control to cytoskeletal dynamics and cell death control. Mutations in HSPBs can lead to human disease and particularly point mutations in HSPB1 and HSPB8 are known to lead to peripheral neuropathies. Recently, a missense mutation (R7S) in yet another member of this family, HSPB3, was found to cause an axonal motor neuropathy (distal hereditary motor neuropathy type 2C, dHMN2C). Until now, HSPB3 protein localization and function in motoneurons (MNs) have not yet been characterized. Therefore, we studied the endogenous HSPB3 protein distribution in the spinal cords of chicken and mouse embryos and in the postnatal nervous system (central and peripheral) of chicken, mouse and human. We further investigated the impact of wild-type and mutated HSPB3 on MN cell death via overexpressing these genes in ovo in an avian model of MN degeneration, the limb-bud removal. Altogether, our findings represent a first step for a better understanding of the cellular and molecular mechanisms leading to dHMN2C.

“Hypomyelination and Congenital Cataract”, HCC (MIM #610532), is an autosomal recessive disorder characterized by congenital cataract and diffuse cerebral and peripheral hypomyelination. HCC is caused by deficiency of Hyccin, a protein whose biological role has not been clarified yet. Since the identification of the cell types expressing a protein of unknown function can contribute to define the physiological context in which the molecule is explicating its function, we analyzed the pattern of Hyccin expression in the central and peripheral nervous system (CNS and PNS). Using heterozygous mice expressing the b-galactosidase (LacZ) gene under control of the Hyccin gene regulatory elements, we show that the gene is primarily expressed in neuronal cells. Indeed, Hyccin-LacZ signal was identified in CA1 hippocampal pyramidal neurons, olfactory bulb, and cortical pyramidal neurons, while it did not colocalize with oligodendroglial or astrocytic markers. In the PNS, Hyccin was detectable only in axons isolated from newborn mice. In the brain, Hyccin transcript levels were higher in early postnatal development (postnatal days 2 and 10) and then declined in adult mice. In a model of active myelinogenesis, organotypic cultures of rat Schwann cells (SC)/Dorsal Root Ganglion (DRG) sensory neurons, Hyccin was detected along the neurites, while it was absent from SC. Intriguingly, the abundance of the molecule was upregulated at postnatal days 10 and 15, in the initial steps of myelinogenesis and then declined at 30 days when the process is complete. As Hyccin is primarily expressed in neurons and its mutation leads to hypomyelination in human patients, we suggest that the protein is involved in neuron-to-glia signalling to initiate or maintain myelination.

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