Daniela Menichella

Genetic and Physiological Evidence That Oligodendrocyte Gap Junctions Contribute to Spatial Buffering of Potassium Released during Neuronal Activity

Mice lacking the K+ channel Kir4.1 or both connexin32 (Cx32) and Cx47 exhibit myelin-associated vacuoles, raising the possibility that oligodendrocytes, and the connexins they express, contribute to recycling the K+ evolved during neuronal activity. To study this possibility, we first examined the effect of neuronal activity on the appearance of vacuoles in mice lacking both Cx32 and Cx47. The size and number of myelin vacuoles was dramatically increased when axonal activity was increased, by either a natural stimulus (eye opening) or pharmacological treatment. Conversely, myelin vacuoles were dramatically reduced when axonal activity was suppressed. Second, we used genetic complementation to test for a relationship between the function of Kir4.1 and oligodendrocyte connexins. In a Cx32-null background, haploinsufficiency of either Cx47 or Kir4.1 did not affect myelin, but double heterozygotes developed vacuoles, consistent with the idea that oligodendrocyte connexins and Kir4.1 function in a common pathway. Together, these results implicate oligodendrocytes and their connexins as having critical roles in the buffering of K+ released during neuronal activity.

Heterozygous P0 Knockout Mice Develop a Peripheral Neuropathy that Resembles Chronic Inflammatory Demyelinating Polyneuropathy (CIDP)

Demyelinating peripheral neuropathies are clinically divided into inherited and acquired types. Inherited demyelinating neuropathies are caused by mutations in genes expressed by myelinating Schwann cells, whereas acquired ones, including chronic inflammatory demyelinating polyneuropathy (CIDP), are probably caused by autoimmune mechanisms. We find that heterozygous P0 knockout (P0+/-) mice develop a neuropathy that resembles CIDP. By one year of age, P0+/- mice develop severe, asymmetric slowing of motor nerves, with temporal dispersion or conduction block, which are features of acquired demyelinating neuropathies including CIDP. Moreover, morphological analysis of affected nerves reveals severe and selective demyelination of motor fibers, focal regions of demyelination, and inflammatory cells. These data suggest that immune-mediated mechanisms may contribute to the pathogenesis of the neuropathy in P0+/- mice.

MicroRNA deficient Schwann cells display congenital hypomyelination

MicroRNAs, by modulating gene expression, have been implicated as regulators of various cellular and physiological processes, including differentiation, proliferation, and cancer. Here, we study the role of microRNAs in Schwann cell (SC) differentiation by conditional removal of the microRNA processing enzyme Dicer1. We reveal that both male and female mice lacking Dicer1 in SC (Dicer1 conditional knock-outs) display a severe neurological phenotype resembling congenital hypomyelination. Ultrastructural analyses show that many SC lacking Dicer1 are stalled in differentiation at the promyelinating state and fail to myelinate axons. Gene expression analyses reveal a failure to extinguish genes characteristic of the undifferentiated state such as Sox2, Jun, and Ccnd1. Sox2 and Jun are well characterized negative regulators of SC differentiation. Consistent with Sox2/Jun maintenance, Egr2, a master regulator of the myelinating program, is drastically downregulated and likely accounts for the myelination defect. We posit a model wherein microRNAs are critical for downregulation of antecedent programs of gene expression. In SC differentiation, this is particularly relevant in the key developmental transition from a promyelinating to myelinating SC.

Protein Zero Is Necessary for E-Cadherin-Mediated Adherens Junction Formation in Schwann Cells

Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells.

Regulation of Myelin‐Specific Gene Expression: Relevance to CMT1

ABSTRACT: Schwann cells, the myelinating cells of the peripheral nervous system, are derived from the neural crest. Once neural crest cells are committed to the Schwann cell fate, they can take on one of two phenotypes to become myelinating or nonmyelinating Schwann cells, a decision that is determined by interactions with axons. The critical step in the differentiation of myelinating Schwann cells is the establishment of a one‐to‐one relationship with axons, the so‐called “promyelinating” stage of Schwann cell development. The transition from the promyelinating to the myelinating stage of development is then accompanied by a number of significant changes in the pattern of gene expression, including the activation of a set of genes encoding myelin structural proteins and lipid biosynthetic enzymes, and the inactivation of a set of genes expressed only in immature or nonmyelinating Schwann cells. These changes are regulated mainly at the transcriptional level and also require continuous interaction between Schwann cells and their axons.

CXCR4 chemokine receptor signaling mediates pain in diabetic neuropathy.

BackgroundPainful Diabetic Neuropathy (PDN) is a debilitating syndrome present in a quarter of diabetic patients that has a substantial impact on their quality of life. Despite this significant prevalence and impact, current therapies for PDN are only partially effective. Moreover, the cellular mechanisms underlying PDN are not well understood. Neuropathic pain is caused by a variety of phenomena including sustained excitability in sensory neurons that reduces the pain threshold so that pain is produced in the absence of appropriate stimuli. Chemokine signaling has been implicated in the pathogenesis of neuropathic pain in a variety of animal models. We therefore tested the hypothesis that chemokine signaling mediates DRG neuronal hyperexcitability in association with PDN.ResultsWe demonstrated that intraperitoneal administration of the specific CXCR4 antagonist AMD3100 reversed PDN in two animal models of type II diabetes. Furthermore DRG sensory neurons acutely isolated from diabetic mice displayed enhanced SDF-1 induced calcium responses. Moreover, we demonstrated that CXCR4 receptors are expressed by a subset of DRG sensory neurons. Finally, we observed numerous CXCR4 expressing inflammatory cells infiltrating into the DRG of diabetic mice.ConclusionsThese data suggest that CXCR4/SDF-1 signaling mediates enhanced calcium influx and excitability in DRG neurons responsible for PDN. Simultaneously, CXCR4/SDF-1 signaling may coordinate inflammation in diabetic DRG that could contribute to the development of pain in diabetes. Therefore, targeting CXCR4 chemokine receptors may represent a novel intervention for treating PDN.

Modulation of cell-mediated immunity prolongs adenovirus-mediated transgene expression in sciatic nerve.

In a previous report, we demonstrated that a first-generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenovirus-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral transgene expression over time. In our current work we have optimized adenoviral vector-mediated transgene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old rats, decreases thereafter, and there is minimal associated tissue injury. In contrast, few lacZ-expressing Schwann cells are found in nerve of adult animals 10 days after injection, probably owing to immune clearance of virus-infected cells. Consistent with this notion, high levels of LacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult beta2-microglobulin-deficient mice (beta2M4-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenovirus-infected Schwann cells cocultured with axons in vitro, in the absence of a host immune response, ensheathe axons and express lacZ for at least 8 weeks. These data thus demonstrate that lacZ transgene expression of first-generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Overcoming Cellular Immunity to Prolong Adenoviral-Mediated Gene Expression in Sciatic Nerve

ABSTRACT: In a previous report, we demonstrated that a first generation (E1‐ and E3‐deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenoviral‐mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral gene expression over time. In our current work we have optimized adenoviral‐mediated gene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2‐week‐old animals, decreases thereafter, and there is minimal associated tissue injury. In contrast, very few adenoviral‐infected Schwann cells are found in nerves of adult animals 10 days after injection, probably due to immune clearance of viral‐infected cells. Consistent with this notion, high levels of lacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult β2 microglobulin‐deficient mice (β2 M −/−), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenoviral‐infected Schwann cells co‐cultured with axons in vitro, in the absence of a host immune response, ensheath axons and express lacZ for at least 8 weeks. These data thus demonstrate that expression of first generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell‐mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Chemogenetic Inhibition of Pain Neurons in a Mouse Model of Osteoarthritis

To determine the ability of drugs that activate inhibitory G protein–coupled receptors (GPCRs) expressed in peripheral voltage‐gated sodium channel 1.8 (NaV1.8)–positive sensory neurons to control osteoarthritis (OA)–associated pain.

Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice

Background Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown. Purpose Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal. Results We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro. Conclusions These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.