Golgi-retained Cx32 mutants interfere with gene addition therapy for CMT1X

Numerous GJB1 gene mutations cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X). GJB1 encodes connexin32 (Cx32), which forms trans-myelin gap junctions in Schwann cells. Most GJB1 mutations result in loss-of-function mechanisms, supporting the concept of gene replacement therapy. However, interactions between delivered wild type and endogenously expressed mutant Cx32 may potentially occur in the setting of gene replacement therapy. In order to screen for possible interactions of several representative CMT1X mutants with wild type Cx32 that may interfere with the functional gap junction formation, we established an in vitro screening method co-expressing in HeLa cells wild type Cx32 and one of eight different Cx32 mutants including A39P, A39V, T55I, R75W, M93V, L143P, N175D and R183S. Some of the Golgi-retained mutants hindered gap junction plaque assembly by Cx32 on the cell membrane, while co-immunoprecipitation analysis revealed a partial interaction of wild type protein with Golgi-retained mutants. Dye transfer studies confirmed that Golgi-retained R75W, M93V and N175D but not endoplasmic reticulum-retained T55I had a negative effect on wild type Cx32 function. Finally, in vivo intraneural delivery of the gene encoding the wild type Cx32 in mice bearing either the T55I or R75W mutation on Cx32 knockout background showed that virally delivered protein was correctly localized in mice expressing the endoplasmic reticulum-retained T55I whereas it did not traffic normally in mice expressing the Golgi-retained R75W. Thus, certain Golgi-retained Cx32 mutants may interfere with exogenously delivered Cx32. Screening for mutant-wild type Cx32 interactions should be considered prior to planning gene addition therapy for CMT1X

Gene replacement therapy in two Golgi-retained CMT1X mutants before and after the onset of demyelinating neuropathy

X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants

Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery

Diseases of the nervous system; Myelin biology and repair; Peripheral nervous systemEnfermedades del sistema nervioso; Biología y reparación de la mielina; Sistema nervioso periféricoMalalties del sistema nerviós; Biologia i reparació de la mielina; Sistema nerviós perifèricTo optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies.Generalitat de Catalunya, 2019FI_B2 00061, 2019FI_B2 00061, Fundació la Marató de TV3, 201607.10, Muscular Dystrophy Association, 603003

AAV9-mediated SH3TC2 gene replacement therapy targeted to Schwann cells for the treatment of CMT4C

Type 4C Charcot-Marie-Tooth (CMT4C) demyelinating neuropathy is caused by autosomal recessive SH3TC2 gene mutations. SH3TC2 is highly expressed in myelinating Schwann cells. CMT4C is a childhood-onset progressive disease without effective treatment. Here, we generated a gene therapy for CMT4C mediated by an adeno-associated viral 9 vector (AAV9) to deliver the human SH3TC2 gene in the Sh3tc2−/− mouse model of CMT4C. We used a minimal fragment of the myelin protein zero (Mpz) promoter (miniMpz), which was cloned and validated to achieve Schwann cell-targeted expression of SH3TC2. Following the demonstration of AAV9-miniMpz.SH3TC2myc vector efficacy to re-establish SH3TC2 expression in the peripheral nervous system, we performed an early as well as a delayed treatment trial in Sh3tc2−/− mice. We demonstrate both after early as well as following late treatment improvements in multiple motor performance tests and nerve conduction velocities. Moreover, treatment led to normalization of the organization of the nodes of Ranvier, which is typically deficient in CMT4C patients and Sh3tc2−/− mice, along with reduced ratios of demyelinated fibers, increased myelin thickness and reduced g-ratios at both time points of intervention. Taken together, our results provide a proof of concept for an effective and potentially translatable gene replacement therapy for CMT4C treatment

Transient bilateral abducens neuropathy with post-tetanic facilitation and acute hypokalemia associated with oxaliplatin: a case report

<p>Abstract</p> <p>Introduction</p> <p>Oxaliplatin is a cytotoxic platinum compound that is in widespread use in the treatment of gastrointestinal cancers. It has been occasionally associated with acute motor neuropathy, but the precise mechanism is uncertain. To the best of our knowledge, we report the first case of a patient demonstrating post-tetanic facilitation in the setting of transient bilateral abducens neuropathy and hypokalemia, after being infused with oxaliplatin.</p> <p>Case presentation</p> <p>A 47-year-old Indian woman with metastatic gastric cancer was receiving an oxaliplatin infusion at the initiation of her third cycle of palliative chemotherapy. She developed acute bilateral abducens neuropathy with post-tetanic facilitation alongside acute laryngopharyngodysesthesia and hypokalemia. Following supportive management, including potassium infusion and warming, her neurological signs and symptoms were spontaneously resolved. This syndrome did not recur in subsequent cycles following prolongation of infusion duration and the addition of supportive calcium and magnesium infusions.</p> <p>Conclusion</p> <p>The novel clinical observation of post-tetanic facilitation highlights a possible involvement of voltage-gated channels at the presynaptic terminals in the mechanism of acute oxaliplatin neurotoxicity.</p

Oxaliplatin neurotoxicity – no general ion channel surface-charge effect

<p>Abstract</p> <p>Background</p> <p>Oxaliplatin is a platinum-based chemotherapeutic drug. Neurotoxicity is the dose-limiting side effect. Previous investigations have reported that acute neurotoxicity could be mediated via voltage-gated ion channels. A possible mechanism for some of the effects is a modification of surface charges around the ion channel, either because of chelation of extracellular Ca²⁺, or because of binding of a charged biotransformation product of oxaliplatin to the channel. To elucidate the molecular mechanism, we investigated the effects of oxaliplatin and its chloride complex [Pt(dach)oxCl]^-on the voltage-gated Shaker K channel expressed in <it>Xenopus </it>oocytes. The recordings were made with the two-electrode and the cut-open oocyte voltage clamp techniques.</p> <p>Conclusion</p> <p>To our surprise, we did not see any effects on the current amplitudes, on the current time courses, or on the voltage dependence of the Shaker wild-type channel. Oxaliplatin is expected to bind to cysteines. Therefore, we explored if there could be a specific effect on single (E418C) and double-cysteine (R362C/F416C) mutated Shaker channels previously shown to be sensitive to cysteine-specific reagents. Neither of these channels were affected by oxaliplatin. The clear lack of effect on the Shaker K channel suggests that oxaliplatin or its monochloro complex has no general surface-charge effect on the channels, as has been suggested before, but rather a specific effect to the channels previously shown to be affected.</p

Gene replacement therapy in a model of Charcot-Marie-Tooth 4C neuropathy

Charcot-Marie-Tooth disease type 4C is the most common recessively inherited demyelinating neuropathy that results from loss of function mutations in the SH3TC2 gene. Sh3tc2-/- mice represent a well characterized disease model developing early onset progressive peripheral neuropathy with hypo- and demyelination, slowing of nerve conduction velocities and disturbed nodal architecture. The aim of this project was to develop a gene replacement therapy for treating Charcot-Marie-Tooth disease type 4C to rescue the phenotype of the Sh3tc2-/- mouse model. We generated a lentiviral vector LV-Mpz.SH3TC2.myc to drive expression of the human SH3TC2 cDNA under the control of the Mpz promoter specifically in myelinating Schwann cells. The vector was delivered into 3-week-old Sh3tc2-/- mice by lumbar intrathecal injection and gene expression was assessed 4-8 weeks after injection. Immunofluorescence analysis showed presence of myc-tagged human SH3TC2 in sciatic nerves and lumbar roots in the perinuclear cytoplasm of a subset of Schwann cells, in a dotted pattern co-localizing with physiologically interacting protein Rab11. Quantitative PCR analysis confirmed SH3TC2 mRNA expression in different peripheral nervous system tissues. A treatment trial was initiated in 3 weeks old randomized Sh3tc2-/- littermate mice which received either the full or mock (LV-Mpz.Egfp) vector. Behavioural analysis 8 weeks after injection showed improved motor performance in rotarod and foot grip tests in treated Sh3tc2-/- mice compared to mock vector-treated animals. Moreover, motor nerve conduction velocities were increased in treated Sh3tc2-/- mice. On a structural level, morphological analysis revealed significant improvement in g-ratios, myelin thickness, and ratios of demyelinated fibres in lumbar roots and sciatic nerves of treated Sh3tc2-/- mice. Finally, treated mice also showed improved nodal molecular architecture and reduction of blood neurofilament light levels, a clinically relevant biomarker for axonal injury/degeneration. This study provides a proof of principle for viral gene replacement therapy targeted to Schwann cells to treat Charcot-Marie-Tooth disease type 4C and potentially other similar demyelinating inherited neuropathies

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy

Funder: The Republic of Cyprus through the Research and Innovation Foundation Foundation (Project: CULTURE/BR-NE/0416/07)Funder: Wallenberg Scholar and the fluid biomarker measurements in the lab of HZ and AJH were supported by the UK Dementia Research Institute at UCLAbstract: Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X

The Bite of the Honeybee: 2-Heptanone Secreted from Honeybee Mandibles during a Bite Acts as a Local Anaesthetic in Insects and Mammals

Honeybees secrete 2-heptanone (2-H) from their mandibular glands when they bite. Researchers have identified several possible functions: 2-H could act as an alarm pheromone to recruit guards and soldiers, it could act as a chemical marker, or it could have some other function. The actual role of 2-H in honeybee behaviour remains unresolved. In this study, we show that 2-H acts as an anaesthetic in small arthropods, such as wax moth larva (WML) and Varroa mites, which are paralysed after a honeybee bite. We demonstrated that honeybee mandibles can penetrate the cuticle of WML, introducing less than one nanolitre of 2-H into the WML open circulatory system and causing instantaneous anaesthetization that lasts for a few minutes. The first indication that 2-H acts as a local anaesthetic was that its effect on larval response, inhibition and recovery is very similar to that of lidocaine. We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels. Although both compounds blocked the hNav1.6 and hNav1.2 channels, lidocaine was slightly more effective, 2.82 times, on hNav.6. In contrast, when the two compounds were tested using an ex vivo preparation-the isolated rat sciatic nerve-the function of the two compounds was so similar that we were able to definitively classify 2-H as a local anaesthetic. Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone. This suggests that natural selection may have favoured 2-H over other, similar compounds because of the associated fitness advantages it confers. Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine. © 2012 Papachristoforou et al