Treating small fiber neuropathy by topical application of a small molecule modulator of ligand-induced GFRα/RET receptor signaling.

Small-fiber neuropathy (SFN) is a disorder of peripheral nerves commonly found in patients with diabetes mellitus, HIV infection, and those receiving chemotherapy. The complexity of disease etiology has led to a scarcity of effective treatments. Using two models of progressive SFN, we show that overexpression of glial cell line-derived neurotrophic factor (GDNF) in skin keratinocytes or topical application of XIB4035, a reported nonpeptidyl agonist of GDNF receptor α1 (GFRα1), are effective treatments for SFN. We also demonstrate that XIB4035 is not a GFRα1 agonist, but rather it enhances GFRα family receptor signaling in conjunction with ligand stimulation. Taken together, our results indicate that topical application of GFRα/RET receptor signaling modulators may be a unique therapy for SFN, and we have identified XIB4035 as a candidate therapeutic agent.

Nonneuronal cells regulate synapse formation in the vestibular sensory epithelium via erbB-dependent BDNF expression.

Recent studies indicate that molecules released by glia can induce synapse formation. However, what induces glia to produce such signals, their identity, and their in vivo relevance remain poorly understood. Here we demonstrate that supporting cells of the vestibular organ--cells that have many characteristics of glia--promote synapse formation only when induced by neuron-derived signals. Furthermore, we identify BDNF as the synaptogenic signal produced by these nonneuronal cells. Mice in which erbB signaling has been eliminated in supporting cells have vestibular dysfunction caused by failure of synapse formation between hair cells and sensory neurons. This phenotype correlates with reduced BDNF expression in supporting cells and is rescued by reexpression of BDNF in these cells. Furthermore, knockdown of BDNF expression in supporting cells postnatally phenocopies the loss of erbB signaling. These results indicate that vestibular supporting cells contribute in vivo to vestibular synapse formation and that this is mediated by reciprocal signals between sensory neurons and supporting cells involving erbB receptors and BDNF.

Dynamic patterns of neurotrophin 3 expression in the postnatal mouse inner ear.

Recent studies indicate that neurotrophin 3 (NT3) may be important for the maintenance and function of the adult inner ear, but the pattern of postnatal NT3 expression in this organ has not been characterized. We used a reporter mouse in which cells expressing NT3 also express beta-galactosidase, allowing for their histochemical visualization, to determine the pattern of NT3 expression in cochlear and vestibular organs. We analyzed animals from birth (P0) to adult (P135). At P0, NT3 was strongly expressed in supporting cells and hair cells of all vestibular and cochlear sense organs, Reissner's membrane, saccular membrane, and the dark cells adjacent to canal organs. With increasing age, staining disappeared in most cell types but remained relatively high in inner hair cells (IHCs) and to a lesser extent in IHC supporting cells. In the cochlea, by P0 there is a longitudinal gradient (apex > base) that persists into adulthood. In vestibular maculae, staining gradients are: striolar > extrastriolar regions and supporting cells > hair cells. By P135, cochlear staining is restricted to IHCs and their supporting cells, with stronger expression in the apex than the base. By the same age, in the vestibular organs, NT3 expression is weak and restricted to saccular and utricular supporting cells. These results suggest that NT3 might play a long-term role in the maintenance and functioning of the adult auditory and vestibular systems and that supporting cells are the main source of this factor in the adult.

Morphometric analysis of oligodendrocytes in the adult mouse frontal cortex.

Oligodendrocytes (OLs), the myelinating cells of the central nervous system, have specialized morphologies that subserve their function. Numerous qualitative studies suggest that OLs in different brain regions can differ in their morphological characteristics, including number of branches and internodes, internode length, etc. However, progress in identifying and characterizing the diverse types of OLs and their distribution in the brain has been made difficult by several technical constraints. Here we report a new strategy to analyze OL morphology with a high degree of quantitative power and throughput. We used confocal microscopy and three-dimensional cell tracing software to study OLs in the frontal cortex of mice expressing enhanced green fluorescent protein (eGFP) under the control of the proteolipid protein (Plp) gene promoter. Three-dimensional reconstructions were then used to analyze and quantify cell morphology, including total process length, total process surface area, total internode length, number of primary processes, number of branch points, and number of internodes. In addition, these reconstructions were subjected to Sholl analysis, which allows for the quantitative measure of OL arbor complexity. By using this approach, we identified and characterized a previously undescribed population of small OLs with a compact but complex morphology that includes numerous branching processes and a large number of short internodes. Our data suggest that other populations of OLs remain to be identified and characterized and that the tools we have developed could help in the process of characterizing them.

Loss of erbB signaling in oligodendrocytes alters myelin and dopaminergic function, a potential mechanism for neuropsychiatric disorders.

Several psychiatric disorders are associated with white matter defects, suggesting that oligodendrocyte (OL) abnormalities underlie some aspects of these diseases. Neuregulin 1 (NRG1) and its receptor, erbB4, are genetically linked with susceptibility to schizophrenia and bipolar disorder. In vitro studies suggest that NRG1-erbB signaling is important for OL development. To test whether erbB signaling contributes to psychiatric disorders by regulating the structure or function of OLs, we analyzed transgenic mice in which erbB signaling is blocked in OLs in vivo. Here we show that loss of erbB signaling leads to changes in OL number and morphology, reduced myelin thickness, and slower conduction velocity in CNS axons. Furthermore, these transgenic mice have increased levels of dopamine receptors and transporters and behavioral alterations consistent with neuropsychiatric disorders. These results indicate that defects in white matter can cause alterations in dopaminergic function and behavior relevant to neuropsychiatric disorders.

Retroviral lineage analysis of fibroblast growth factor receptor signaling in FGF2 inhibition of oligodendrocyte progenitor differentiation.

Fibroblast growth factor 2 (FGF2) inhibits oligodendrocyte progenitor cell (OPC) differentiation during development and limits remyelination following chronic demyelination. The current study examines the mechanism underlying this effect of FGF2 expression on OPC differentiation. Retroviral lineage tracing demonstrates a direct in vivo effect of FGF receptor (FGFR) signaling on OPC differentiation. Retrovirus expressing a dominant negative FGFR construct (FGFRdn) and green fluorescent protein (GFP) was injected into the dorsal columns of postnatal day 7 (P7) mice followed by perfusion at P28. Among the GFP-labeled cells, FGFRdn retrovirus generated a higher proportion of oligodendrocytes than did control infections. This result from FGFRdn expression in OPCs was similar to the result obtained in our previous study using control retrovirus in FGF2 null mice. Further, in vitro retroviral siRNA expression distinguishes the function of specific FGFR isoforms in OPC responses to FGF2. FGF2 inhibition of OPC differentiation was effectively blocked by siRNA targeted to FGFR1, but not FGFR2 or FGFR3. We propose a model of direct FGF2 activation of FGFR1 leading to inhibition of OPC differentiation. This signaling pathway may be an important regulator of oligodendrocyte generation during myelination in development and may perturb OPC generation of remyelinating oligodendrocytes in demyelinating disease.

In vivo analysis of oligodendrocyte lineage development in postnatal FGF2 null mice.

Analysis of fibroblast growth factor 2 null (FGF2-/-) and wild-type (FGF2+/+) mice was used to interpret the potential in vivo role of endogenous FGF2 on oligodendrocyte lineage cell (OLC) responses during oligodendrogenesis and myelination. In wild-type mouse spinal cord, FGF2 levels increased approximately threefold between the first and second postnatal weeks, a period corresponding with the peak of oligodendrogenesis. Absence of this developmental FGF2 elevation in FGF2-/- mice eliminated the transient overproduction of oligodendrocytes that is known to occur at the peak of oligodendrogenesis in wild-type mice. Absence of FGF2 did not affect oligodendrocyte progenitor (OP) density or proliferation, based on BrdU incorporation, and also did not alter survival, based on TUNEL analysis. To examine OLC differentiation in vivo, retrovirus encoding-enhanced green fluorescent protein (GFP) was injected into the spinal cord to heritably label endogenous cycling cells in the white matter at postnatal day 7 and then identify the generated cells at postnatal day 28. Phenotypes of cells expressing GFP were identified by morphology and immunolabeling, using CC1 for oligodendrocytes and NG2 combined with platelet-derived growth factor alpha receptor for OPs. Within the population of GFP-labeled cells, the proportion of oligodendrocytes was higher in FGF2-/- mice, indicating that endogenous FGF2 inhibited OLC differentiation in wild-type mice. Furthermore, in FGF2-/- mice fewer cells appeared to be generated from an initial retrovirus-labeled cell, consistent with more frequent differentiation into post-mitotic oligodendrocytes. This in vivo analysis demonstrates that the predominant role of endogenous FGF2 on OLCs in development is inhibition of differentiation.

PDGF and FGF2 pathways regulate distinct oligodendrocyte lineage responses in experimental demyelination with spontaneous remyelination.

Repair of myelin damage in the adult CNS requires oligodendrocyte progenitor (OP) proliferation and subsequent differentiation into remyelinating oligodendrocytes. Platelet-derived growth factor (PDGF) and fibroblast growth factor-2 (FGF2) have been predicted to act individually and/or cooperatively to generate remyelinating oligodendrocytes. Analysis of PDGF alpha receptor (PDGF alpha R) heterozygous (+/-) mice indicates that PDGF alpha R expression modulates oligodendrocyte density in non-lesioned adult CNS. Analysis of cuprizone demyelination and recovery in PDGF alpha R+/- mice, FGF2 knockout (-/-) mice, and PDGF alpha R+/- FGF2-/- mice demonstrated that: (1) OP proliferation and oligodendrocyte regeneration is impaired in PDGF alpha R heterozygotes, (2) PDGF alpha R+/- and FGF2-/- deletions do not act cooperatively to impair OP amplification, (3) oligodendrocyte differentiation is more frequent in FGF2-/- mice, and (4) FGF2 deletion in combination with the PDGF alpha R+/- genotype rescues impaired oligodendrocyte regeneration of PDGF alpha R heterozygotes. These findings demonstrate distinct roles for PDGF and FGF2 in vivo in the context of a demyelinating disease with spontaneous remyelination.

Astrocytes produce CNTF during the remyelination phase of viral-induced spinal cord demyelination to stimulate FGF-2 production.

Multiple sclerosis is characterized by multiple lesions with selective loss of myelin and oligodendrocytes, leading to deficits of sensation and movement, as well as cognitive disabilities. Consequently, a major research endeavor is to identify strategies to enhance oligodendrocyte regeneration and remyelination. FGF-2 is a potent mitogen for OPCs, and it is induced in astrocytes in animal models of demyelinating diseases in conjunction with successful remyelination. However, the factors responsible for inducing FGF-2 after demyelination in astrocytes are unknown. Here we show that CNTF mRNA and protein increase coincident with spinal cord remyelination in mice recovering from MHV-induced demyelination. We identify CNTF within astrocytes surrounding and within remyelinating lesions, and show that CNTF increases FGF-2 ligand and receptor mRNAs in spinal cord after direct application. Furthermore, we show that CNTF increases FGF-2 mRNA approximately 2.5-fold in cultured mouse spinal cord astrocytes. Altogether, these results strongly implicate CNTF as an important cytokine in demyelinating disease and as an upstream regulator of FGF-2 production in astrocytes during early remyelination.