Laurence Richard

INF2 Mutations in Charcot–Marie–Tooth Disease with Glomerulopathy

BACKGROUND Charcot-Marie-Tooth neuropathy has been reported to be associated with renal diseases, mostly focal segmental glomerulosclerosis (FSGS). However, the common mechanisms underlying the neuropathy and FSGS remain unknown. Mutations in INF2 were recently identified in patients with autosomal dominant FSGS. INF2 encodes a formin protein that interacts with the Rho-GTPase CDC42 and myelin and lymphocyte protein (MAL) that are implicated in essential steps of myelination and myelin maintenance. We therefore hypothesized that INF2 may be responsible for cases of Charcot-Marie-Tooth neuropathy associated with FSGS. METHODS We performed direct genotyping of INF2 in 16 index patients with Charcot-Marie-Tooth neuropathy and FSGS who did not have a mutation in PMP22 or MPZ, encoding peripheral myelin protein 22 and myelin protein zero, respectively. Histologic and functional studies were also conducted. RESULTS We identified nine new heterozygous mutations in 12 of the 16 index patients (75%), all located in exons 2 and 3, encoding the diaphanous-inhibitory domain of INF2. Patients presented with an intermediate form of Charcot-Marie-Tooth neuropathy as well as a glomerulopathy with FSGS on kidney biopsy. Immunohistochemical analysis revealed strong INF2 expression in Schwann-cell cytoplasm and podocytes. Moreover, we demonstrated that INF2 colocalizes and interacts with MAL in Schwann cells. The INF2 mutants perturbed the INF2-MAL-CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42 and mislocalization of INF2, MAL, and CDC42. CONCLUSIONS INF2 mutations appear to cause many cases of FSGS-associated Charcot-Marie-Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwann-cell function. (Funded by the Agence Nationale de la Recherche and others.).

Mitochondrial complex I deficiency in GDAP1-related autosomal dominant Charcot-Marie-Tooth disease (CMT2K)

Mutations in GDAP1, an outer mitochondrial membrane protein responsible for recessive Charcot-Marie-Tooth disease (CMT4A), have also been associated with CMT2K, a dominant form of the disease. The three CMT2K patients we studied carried a novel dominant GDAP1 mutation, C240Y (c.719G > A). Mitochondrial respiratory chain complex I activity in fibroblasts from CMT2K patients was 40% lower than in controls, whereas the tubular mitochondria were 33% larger in diameter and the mitochondrial mass was 20% greater. Thus, besides the regulatory role GDAP1 plays in mitochondrial network dynamics, it may also be involved in energy production and in the control of mitochondrial volume.

Loss-of-function mutations in WDR73 are responsible for microcephaly and steroid-resistant nephrotic syndrome: Galloway-Mowat syndrome.

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS.

Ethambutol-induced optic neuropathy linked to OPA1 mutation and mitochondrial toxicity.

Ethambutol (EMB), widely used in the treatment of tuberculosis, has been reported to cause Lebers hereditary optic neuropathy in patients carrying mitochondrial DNA mutations. We study the effect of EMB on mitochondrial metabolism in fibroblasts from controls and from a man carrying an OPA1 mutation, in whom the drug induced the development of autosomal dominant optic atrophy (ADOA). EMB produced a mitochondrial coupling defect together with a 25% reduction in complex IV activity. EMB induced the formation of vacuoles associated with decreased mitochondrial membrane potential and increased fragmentation of the mitochondrial network. Mitochondrial genetic variations may therefore be predisposing factors in EMB-induced ocular injury.

Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin

Summary While neurogenic stem cells have been identified in rodent and human skin, their manipulation and further characterization are hampered by a lack of specific markers. Here, we perform genetic tracing of the progeny of boundary cap (BC) cells, a neural-crest-derived cell population localized at peripheral nerve entry/exit points. We show that BC derivatives migrate along peripheral nerves to reach the skin, where they give rise to terminal glia associated with dermal nerve endings. Dermal BC derivatives also include cells that self-renew in sphere culture and have broad in vitro differentiation potential. Upon transplantation into adult mouse dorsal root ganglia, skin BC derivatives efficiently differentiate into various types of mature sensory neurons. Together, this work establishes the embryonic origin, pathway of migration, and in vivo neurogenic potential of a major component of skin stem-like cells. It provides genetic tools to study and manipulate this population of high interest for medical applications.

Intranervous immunoglobulin deposits: An underestimated mechanism of neuropathy

There are several pathogenic mechanisms of peripheral nerve involvement in patients with monoclonal dysglobulinemia. Intranervous proliferation of malignant cells, immunoglobulin, or amyloid deposits in the endoneurial space can only be determined by examination of nerve biopsy specimens. We present clinical, electrophysiological, and histological data from seven patients whose polyneuropathy was induced by immunoglobulin deposits in the endoneurial space. As these lesions cannot be demonstrated on clinical and electrophysiological grounds, the indication for nerve biopsy derives from careful analysis of each patient presenting with a polyneuropathy and a monoclonal dysglobulinemia. To visualize and clearly characterize these deposits, electron microscopic examination is indispensable. Immunocytochemical methods using both light and electron microscopy for ultrastructural analysis are of great value. Demonstration of endoneurial immunoglobulin deposits may have major therapeutic consequences. Indeed, identification of these deposits prompted the use of aggressive treatment, which was quite effective in five of our seven patients. Muscle Nerve 38: 904–911, 2008

Fingolimod potentiates the effects of sunitinib malate in a rat breast cancer model

Most of the antiangiogenic strategies used in oncology principally target endothelial cells through the vascular endothelial growth factor (VEGF) pathway. Multiple kinase inhibitors can secondarily reduce mural cell stabilization of the vessels by blocking platelet-derived growth factor receptor (PDGFR) activity. However, sphingosine-1-phosphate (S1P), which is also implicated in mural cell recruitment, has yet to be targeted in clinical practice. We therefore investigated the potential of a simultaneous blockade of the PDGF and S1P pathways on the chemotactic responses of vascular smooth muscle cells (VSMCs) and the resulting effects of this blockade on breast tumor growth. Due to crosstalk between the S1P and PDGF pathways, we used AG1296 and/or VPC-23019 to inhibit PDGFR-β and S1PR1/S1PR3 receptors, respectively. We showed that S1PR1 and S1PR3 are the principal receptors that mediate the S1P chemotactic signal on rat VSMCs and that they act synergistically with PDGFR-β during PDGF-B signaling. We also showed that simultaneous blockade of the PDGFR-β and S1PR1/S1PR3 signals had a synergistic effect, decreasing VSMC migration velocity toward endothelial cell and breast carcinoma cell-secreted cytokines by 65–90%. This blockade also strongly decreased the ability of VSMCs to form a three-dimensional cell network. Similar results were obtained with the combination of sunitinib malate (a VEGFR/PDGFR kinase inhibitor) and fingolimod (an S1P analog). Sunitinib malate is a clinically approved cancer treatment, whereas fingolimod is currently indicated only for treatment of multiple sclerosis. Orally administered, the combination of these drugs greatly decreased rat breast tumor growth in a syngeneic cancer model (Walker 256). This bi-therapy did not exert cumulative toxicity and histological analysis of the tumors revealed normalization of the tumor vasculature. The simultaneous blockade of these signaling pathways with sunitinib malate and fingolimod may provide an effective means of reducing tumor angiogenesis, and may improve the delivery of other chemotherapies.

Natalizumab as a Disease-Modifying Therapy in Chronic Inflammatory Demyelinating Polyneuropathy - A Report of Three Cases

Background: Several treatments are available to treat the immune-mediated chronic inflammatory demyelinating polyneuropathy (CIDP). Among these treatments, intravenous immunoglobulins, corticosteroids and plasma exchanges are validated and widely used. A few immunosuppressive drugs have been tried, but they had little efficiency. Methods: We describe three CIDP patients treated by Natalizumab (acting against cellular adhesion and T-cell migration) after a failure of the validated treatments. Results: We observed a long-term improvement in one patient, a dramatic improvement over a significant duration in another patient and stabilization in the last one. Conclusion: This open label study provides evidence for the value of Natalizumab as second-line treatment for individual patients with a high dependency on waning efficacy of first-line therapies. CIDP is characterized by heterogeneity of clinical phenotypes, electrophysiological and pathological features, and various variable courses types of evolution. The different responses to drugs of our patients are consistent with some reported cases and may reflect the spectrum of lesional mechanisms and the molecular dysfunctions in CIDP.

Neuropathologic Characterization of INF2-Related Charcot-Marie-Tooth Disease: Evidence for a Schwann Cell Actinopathy

The association of Charcot-Marie-Tooth (CMT) disease with renal dysfunction is uncommon but has long been recognized in several families. Recently, mutations in the INF2 gene, which encodes inverted formin-2, were identified in patients with focal segmental glomerulosclerosis and a dominant intermediate form of CMT (CMTDIE, OMIM #614455). We describe the pathologic lesions of nerve biopsies from 6 patients with INF2-related CMTDIE. There were 4 females and 2 males; ages were from 12 to 47 years; durations between neuropathy onset and biopsy were from 2 to 37 years. Clinical phenotypes were similar to those seen in other forms of CMT disease, but there was always an associated proteinuria (and later renal failure). Motor median nerve conduction velocities were in the range of intermediate CMT disease. Pathologic lesions suggested chronic demyelination and remyelination associated with progressive axonal loss. By electron microscopy, we observed unusual whorl-like proliferations of flattened Schwann cell cytoplasm and anomalies of unmyelinating Schwann cell cytoplasm with supernumerary elongated extensions similar to those described in CMT4C. We also observed abnormal accumulation of β-actin in the cytoplasm of Schwann cells. Our results suggest that these lesions reflect a global disorder of the actin cytoskeleton in Schwann cells and that CMTDIE is the first peripheral nerve disorder associated with a Schwann cell actinopathy.

Homozygous deletion of an EGR2 enhancer in congenital amyelinating neuropathy

The transcription factor EGR2 is expressed in Schwann cells, where it controls peripheral nerve myelination. Mutations of EGR2 have been found in patients with congenital hypomyelinating neuropathy or Charcot–Marie–Tooth disease type 1D. In a patient with congenital amyelinating neuropathy, we observed pathological abnormalities recapitulating the peripheral nervous system phenotype of homozygous Egr2‐null mice. This patient, born from consanguineous parents, showed no EGR2 immunoreactivity in Schwann cells and harbored a homozygous 10.7‐kilobase‐long deletion encompassing a myelin‐specific enhancer of EGR2. This regulatory mutation is the first genetic abnormality associated with congenital amyelinating neuropathy in humans. ANN NEUROL 2012;