Fasciclin 2 engages EGFR in an auto-stimulatory loop to promote imaginal disc cell proliferation in Drosophila.

How cell to cell interactions control local tissue growth to attain a species-specific organ size is a central question in developmental biology. The Drosophila Neural Cell Adhesion Molecule, Fasciclin 2, is expressed during the development of neural and epithelial organs. Fasciclin 2 is a homophilic-interaction protein that shows moderate levels of expression in the proliferating epithelia and high levels in the differentiating non-proliferative cells of imaginal discs. Genetic interactions and mosaic analyses reveal a cell autonomous requirement of Fasciclin 2 to promote cell proliferation in imaginal discs. This function is mediated by the EGFR, and indirectly involves the JNK and Hippo signaling pathways. We further show that Fasciclin 2 physically interacts with EGFR and that, in turn, EGFR activity promotes the cell autonomous expression of Fasciclin 2 during imaginal disc growth. We propose that this auto-stimulatory loop between EGFR and Fasciclin 2 is at the core of a cell to cell interaction mechanism that controls the amount of intercalary growth in imaginal discs.

Schwann cell JUN expression worsens motor performance in an amyotrophic lateral sclerosis mouse model.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease characterized by motor neuron death and distal axonopathy. Despite its clinical severity and profound impact in the patients and their families, many questions about its pathogenesis remain still unclear, including the role of Schwann cells and axon-glial signaling in disease progression. Upon axonal injury, upregulation of JUN transcription factor promotes Schwann cell reprogramming into a repair phenotype that favors axon regrowth and neuronal survival. To study the potential role of repair Schwann cells on motoneuron survival in amyotrophic lateral sclerosis, we generated a mouse line that over-expresses JUN in the Schwann cells of the SOD1G93A mutant, a mouse model of this disease. Then, we explored disease progression by evaluating survival, motor performance and histology of peripheral nerves and spinal cord of these mice. We found that Schwann cell JUN overexpression does not prevent axon degeneration neither motor neuron death in the SOD1G93A mice. Instead, it induces a partial demyelination of medium and large size axons, worsening motor performance and resulting in more aggressive disease phenotype.

Emerging Role of HDACs in Regeneration and Ageing in the Peripheral Nervous System: Repair Schwann Cells as Pivotal Targets.

The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated axons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene expression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn2+-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.

Epigenetic induction of the Ink4a/Arf locus prevents Schwann cell overproliferation during nerve regeneration and after tumorigenic challenge.

The number of Schwann cells is fitted to axonal length in peripheral nerves. This relationship is lost when tumorigenic stimuli induce uncontrolled Schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. Schwann cells also re-enter the cell cycle following nerve injury during the process of Wallerian degeneration. In both cases proliferation is finally arrested. We show that in neurofibroma, the induction of Jmjd3 (jumonji domain containing 3, histone lysine demethylase) removes trimethyl groups on lysine-27 of histone-H3 and epigenetically activates the Ink4a/Arf-locus, forcing Schwann cells towards replicative senescence. Remarkably, blocking this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. Interestingly, our data suggest that in injured nerves, Schwann cells epigenetically activate the same locus to switch off proliferation and enter the senescence programme. Indeed, when this pathway is genetically blocked, Schwann cells fail to drop out of the cell cycle and continue to proliferate. We postulate that the Ink4a/Arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated Schwann cell generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system.

SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice.

Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.

A genetic compensatory mechanism regulated by Jun and Mef2d modulates the expression of distinct class IIa Hdacs to ensure peripheral nerve myelination and repair.

The class IIa histone deacetylases (HDACs) have pivotal roles in the development of different tissues. Of this family, Schwann cells express Hdac4, 5, and 7 but not Hdac9. Here, we show that a transcription factor regulated genetic compensatory mechanism within this family of proteins, blocks negative regulators of myelination ensuring peripheral nerve developmental myelination and remyelination after injury. Thus, when Hdac4 and 5 are knocked-out from Schwann cells in mice, a JUN-dependent mechanism induces the compensatory overexpression of Hdac7 permitting, although with a delay, the formation of the myelin sheath. When Hdac4, 5, and 7 are simultaneously removed, the myocyte-specific enhancer-factor d (MEF2D) binds to the promoter and induces the de novo expression of Hdac9, and although several melanocytic lineage genes are misexpressed and Remak bundle structure is disrupted, myelination proceeds after a long delay. Thus, our data unveil a finely tuned compensatory mechanism within the class IIa Hdac family, coordinated by distinct transcription factors, that guarantees the ability of Schwann cells to myelinate during development and remyelinate after nerve injury.

Failures of nerve regeneration caused by aging or chronic denervation are rescued by restoring Schwann cell c-Jun.

After nerve injury, myelin and Remak Schwann cells reprogram to repair cells specialized for regeneration. Normally providing strong regenerative support, these cells fail in aging animals, and during chronic denervation that results from slow axon growth. This impairs axonal regeneration and causes significant clinical problems. In mice, we find that repair cells express reduced c-Jun protein as regenerative support provided by these cells declines during aging and chronic denervation. In both cases, genetically restoring Schwann cell c-Jun levels restores regeneration to control levels. We identify potential gene candidates mediating this effect and implicate Shh in the control of Schwann cell c-Jun levels. This establishes that a common mechanism, reduced c-Jun in Schwann cells, regulates success and failure of nerve repair both during aging and chronic denervation. This provides a molecular framework for addressing important clinical problems, suggesting molecular pathways that can be targeted to promote repair in the PNS.

Sustained axon-glial signaling induces Schwann cell hyperproliferation, Remak bundle myelination, and tumorigenesis.

Type III neuregulins exposed on axon surfaces control myelination of the peripheral nervous system. It has been shown, for example, that threshold levels of type III beta1a neuregulin dictate not only the myelination fate of axons but also myelin thickness. Here we show that another neuregulin isoform, type III-beta3, plays a distinct role in myelination. Neuronal overexpression of this isoform in mice stimulates Schwann cell proliferation and dramatically enlarges peripheral nerves and ganglia-which come to resemble plexiform neurofibromas-but have no effect on myelin thickness. The nerves display other neurofibroma-like properties, such as abundant collagen fibrils and abundant dissociated Schwann cells that in some cases produce big tumors. Moreover, the organization of Remak bundles is dramatically altered; the small-caliber axons of each bundle are no longer segregated from one another within the cytoplasm of a nonmyelinating Schwann cell but instead are close packed and the whole bundle wrapped as a single unit, frequently by a compact myelin sheath. Because Schwann cell hyperproliferation and Remak bundle degeneration are early hallmarks of type I neurofibromatosis, we suggest that sustained activation of the neuregulin pathway in Remak bundles can contribute to neurofibroma development.

Novel EGR2 variant that associates with Charcot-Marie-Tooth disease when combined with lipopolysaccharide-induced TNF-α factor T49M polymorphism.

OBJECTIVE: To identify novel genetic mechanisms causing Charcot-Marie-Tooth (CMT) disease. METHODS: We performed a next-generation sequencing study of 34 genes associated with CMT in a patient with peripheral neuropathy. RESULTS: We found a non-previously described mutation in EGR2 (p.P397H). P397H mutation is located within the loop that connects zinc fingers 2 and 3, a pivotal domain for the activity of this transcription factor. Using promoter activity luciferase assays, we found that this mutation promotes decreased transcriptional activity of EGR2. In this patient, we also found a previously described nonpathogenic polymorphism in lipopolysaccharide-induced TNF-α factor (LITAF) (p.T49M). We show that the p.T49M mutation decreases the steady-state levels of the LITAF protein in Schwann cells. Loss of function of LITAF has been shown to produce deregulation in the NRG1-erbB signaling, a pivotal pathway for EGR2 expression by Schwann cells. Surprisingly, our segregation study demonstrates that p.P397H mutation in EGR2 is not sufficient to produce CMT disease. Most notably, only those patients expressing simultaneously the LITAF T49M polymorphism develop peripheral neuropathy. CONCLUSIONS: Our data support that the LITAF loss-of-function interferes with the expression of the transcriptional-deficient EGR2 P397H mutant hampering Schwann cell differentiation and suggest that in vivo both genes act in tandem to allow the proper development of myelin.

Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells.

Schwann cells respond to cyclic adenosine monophosphate (cAMP) halting proliferation and expressing myelin proteins. Here we show that cAMP signaling induces the nuclear shuttling of the class IIa histone deacetylase (HDAC)-4 in these cells, where it binds to the promoter and blocks the expression of c-Jun, a negative regulator of myelination. To do it, HDAC4 does not interfere with the transcriptional activity of MEF2. Instead, by interacting with NCoR1, it recruits HDAC3 and deacetylates histone 3 in the promoter of c-Jun, blocking gene expression. Importantly, this is enough to up-regulate Krox20 and start Schwann cell differentiation program-inducing myelin gene expression. Using conditional knockout mice, we also show that HDAC4 together with HDAC5 redundantly contribute to activate the myelin transcriptional program and the development of myelin sheath in vivo. We propose a model in which cAMP signaling shuttles class IIa HDACs into the nucleus of Schwann cells to regulate the initial steps of myelination in the peripheral nervous system.

L1CAM binds ErbB receptors through Ig-like domains coupling cell adhesion and neuregulin signalling.

During nervous system development different cell-to-cell communication mechanisms operate in parallel guiding migrating neurons and growing axons to generate complex arrays of neural circuits. How such a system works in coordination is not well understood. Cross-regulatory interactions between different signalling pathways and redundancy between them can increase precision and fidelity of guidance systems. Immunoglobulin superfamily proteins of the NCAM and L1 families couple specific substrate recognition and cell adhesion with the activation of receptor tyrosine kinases. Thus it has been shown that L1CAM-mediated cell adhesion promotes the activation of the EGFR (erbB1) from Drosophila to humans. Here we explore the specificity of the molecular interaction between L1CAM and the erbB receptor family. We show that L1CAM binds physically erbB receptors in both heterologous systems and the mammalian developing brain. Different Ig-like domains located in the extracellular part of L1CAM can support this interaction. Interestingly, binding of L1CAM to erbB enhances its response to neuregulins. During development this may synergize with the activation of erbB receptors through L1CAM homophilic interactions, conferring diffusible neuregulins specificity for cells or axons that interact with the substrate through L1CAM.

Lipid raft segregation modulates TRPM8 channel activity.

Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterol-rich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn(934) residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol- and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.

Transcriptional control of cholesterol biosynthesis in Schwann cells by axonal neuregulin 1.

A characteristic feature of many vertebrate axons is their wrapping by a lamellar stack of glially derived membranes known as the myelin sheath. Myelin is a cholesterol-rich membrane that allows for rapid saltatory nerve impulse conduction. Axonal neuregulins instruct glial cells on when and how much myelin they should produce. However, how neuregulin regulates myelin sheath development and thickness is unknown. Here we show that neuregulin receptors are activated by drops in plasma membrane cholesterol, suggesting that they can sense sterol levels. In Schwann cells neuregulin-1 increases the transcription of the 3-hydroxy-3-methylglutarylcoenzyme A reductase, the rate-limiting enzyme for cholesterol biosynthesis. Neuregulin activity is mediated by the phosphatidylinositol 3-kinase pathway and a cAMP-response element located on the reductase promoter. We propose that by activating neuregulin receptors, neurons exploit a cholesterol homeostatic mechanism forcing Schwann cells to produce new membranes for the myelin sheath. We also show that a strong phylogenetic correlation exists between myelination and cholesterol biosynthesis, and we propose that the absence of the sterol branch of the mevalonate pathway in invertebrates precluded the myelination of their nervous system.

Characterization of a neural-specific splicing form of the human neuregulin 3 gene involved in oligodendrocyte survival.

Neuregulins are a family of genes involved in key aspects of neural biology. Neuregulins 1, 2 and 3 (NRG1, NRG2 and NRG3) are expressed in the mammalian nervous system. It is well established that NRG1, with fifteen different splicing forms, is central for brain development and function. However, the biological relevance of NRG2 and NRG3 remains elusive. Here, we report the identification of a new isoform of NRG3 that is specifically expressed in the human embryonic central nervous system. Sequence alignment with the human genome suggests that this transcript is produced by alternative promoter usage. The encoded polypeptide is a type-I-glycosylated plasma membrane protein, which is shed into the extracellular space where it activates erbB4, a pivotal receptor for brain development. In addition, we show that the protein has a signal sequence that is cleaved after membrane insertion. Proteasome inhibition with Lactacystin enhances the expression of the protein, whereas impairment of ubiquitylation in the conditional mutant cell line ts20 protects the protein from degradation. These observations imply that the ubiquitin/proteasome pathway regulates biogenesis of the protein. We also show that recombinant neuregulin 3 acts as an oligodendrocyte survival factor by activating the phosphoinositide 3-kinase signalling pathway. Therefore, we report a new post-translationally regulated isoform of neuregulin 3 expressed in the developing human central nervous system with a role in oligodendrocyte survival.

The contribution of TRPM8 channels to cold sensing in mammalian neurones.

Different classes of ion channels have been implicated in sensing cold temperatures at mammalian thermoreceptor nerve endings. A major candidate is TRPM8, a non-selective cation channel of the transient receptor potential family, activated by menthol and low temperatures. We investigated the role of TRPM8 in cold sensing during transient expression in mouse cultured hippocampal neurones, a tissue that lacks endogenous expression of thermosensitive TRPs. In the absence of synaptic input, control hippocampal neurones were not excited by cooling. In contrast, all TRPM8-transfected hippocampal neurones were excited by cooling and menthol. However, in comparison to cold-sensitive trigeminal sensory neurones, hippocampal neurones exhibited much lower threshold temperatures, requiring temperatures below 27 degrees C to fire action potentials. These results directly demonstrate that expression of TRPM8 in mammalian neurones induces cold sensing, albeit at lower temperatures than native TRPM8-expressing neurones, suggesting the presence of additional modulatory mechanisms in the cold response of sensory neurones.

Oligomerization of the sensory and motor neuron-derived factor prevents protein O-glycosylation.

The sensory and motor neuron-derived factor (SMDF) is a neuregulin that promotes Schwann cell proliferation and differentiation. Hence, understanding axon myelination is important to unveil the mechanisms involved in SMDF biogenesis, membrane delivery, and compartmentalization. SMDF is a type II membrane protein expressed as two distinct polypeptides of approximately 40 and 83 kDa. Whether the 83-kDa polypeptide results from posttranslational modifications of the protein monomers or protein dimerization remains unknown. Here we have addressed this question and shown that the 83-kDa polypeptide is an O-glycosylated form of the protein. Deletion of the N-terminal domain fully abrogates the SMDF O-glycosylation, indicating that incorporation of O-glycans occurs in the intracellular domain of the protein. Notably, O-glycosylated forms are excluded from partitioning into lipid raft microdomains. In addition, we found that heterologously expressed SMDF monomers interact in intact living cells as evidenced from fluorescence resonance energy transfer of cyan fluorescent protein/yellow fluorescent protein.SMDF fusion proteins. A stepwise deletion approach demonstrated that SMDF self-association is primarily determined by its transmembrane segment. Notably, biochemical analysis revealed that SMDF multimers are exclusively composed of the 40-kDa polypeptide. Collectively, these findings indicate that the 40-kDa form corresponds to unmodified SMDF, which may be present as multimers, whereas the 83-kDa polypeptide is a monomeric O-glycosylated form of the protein. Furthermore, our observations imply a role for oligomerization as a potential modulator of the distribution in membrane domains and O-glycosylation of the protein.

Molecular determinants of the sensory and motor neuron-derived factor insertion into plasma membrane.

The sensory and motor neuron-derived factor (SMDF) is a type III neuregulin that regulates development and proliferation of Schwann cells. Although SMDF has been shown to be a type II protein, the molecular determinants of membrane biogenesis, insertion, and topology remain elusive. Here we used heterologous expression of a yellow fluorescent protein-SMDF fusion protein along with a stepwise deletion strategy to show that the apolar/uncharged segment (Ile(76)-Val(100)) acts as an internal, uncleaved membrane insertion signal that defines the topology of the protein. Unexpectedly, removal of the transmembrane segment (TM) did not eliminate completely membrane association of C-terminal fragments. TM-deleted fusion proteins, bearing the amino acid segment (Ser(283)-Glu(296)) located downstream to the epidermal growth factor-like motif, strongly interacted with plasma membrane fractions. However, synthetic peptides patterned after this segment did not insert into artificial lipid vesicles, suggesting that membrane interaction of the SMDF C terminus may be the result of a post-translational modification. Subcellular localization studies demonstrated that the 40-kDa form, but not the 83-kDa form, of SMDF was segregated into lipid rafts. Deletion of the N-terminal TM did not affect the interaction of the protein with these lipid microdomains. In contrast, association with membrane rafts was abolished completely by truncation of the protein C terminus. Collectively, these findings are consistent with a topological model for SMDF in which the protein associates with the plasma membrane through both the TM and the C-terminal end domains resembling the topology of other type III neuregulins. The TM defines its characteristic type II membrane topology, whereas the C terminus is a newly recognized anchoring motif that determines its compartmentalization into lipid rafts. The differential localization of the 40- and 83-kDa forms of the neuregulin into rafts and non-raft domains implies a central role in the protein biological activity.

GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity.

The identification of osmo/mechanosensory proteins in mammalian sensory neurons is still elusive. We have used an expression cloning approach to screen a human dorsal root ganglion cDNA library to look for proteins that respond to hypotonicity by raising the intracellular Ca(2+) concentration ([Ca(2+)](i)). We report the unexpected identification of GAP43 (also known as neuromodulin or B50), a membrane-anchored neuronal protein implicated in axonal growth and synaptic plasticity, as an osmosensory protein that augments [Ca(2+)](i) in response to hypotonicity. Palmitoylation of GAP43 plays an important role in the protein osmosensitivity. Depletion of intracellular stores or inhibition of phospholipase C (PLC) activity abrogates hypotonicity-evoked, GAP43-mediated [Ca(2+)](i) elevations. Notably, hypotonicity promoted the selective association of GAP43 with the PLC-delta(1) isoform, and a concomitant increase in inositol-1,4,5-trisphosphate (IP(3)) formation. Collectively, these findings indicate that hypo-osmotic activation of GAP43 induces Ca(2+) release from IP(3)-sensitive intracellular stores. The osmosensitivity of GAP43 furnishes a mechanistic framework that links axon elongation with phospho inositide metabolism, spontaneous triggering of cytosolic Ca(2+) transients and the regulation of actin dynamics and motility at the growth cone in response to temporal and local mechanical forces.