Expression of MCT1 and MCT4 in a patient with mitochondrial myopathy

Mitochondrial myopathies (MM) are characterized by alterations in oxidative phosphorylation. The resultant increase in glycolytic flux produces a variable lactic acidosis. Intracellular acidification can induce both metabolic and, in the case of skeletal muscle, contractile dysfunction. Skeletal muscle lactate transporters have recently been identified which include both monocarboxylate transporter 1 (MCT1) and 4 (MCT4). Lactate import into oxidative skeletal muscle appears to be catalyzed by MCT1, whereas its extrusion from glycolytic fibers may be mediated by MCT4. We describe the expression of these isoforms in a patient with MM as compared to controls (n = 5). MCT4 content was 86% (>3 SD) higher in the patient with MM, whereas MCT1 content was less markedly elevated (47%), as compared to controls. These findings support previous work suggesting that the major role of MCT4 is to defend intracellular pH by extruding lactate and H(+) to the interstitium. The role of MCT1 in MM is less clear.     

Cadm3 (Necl‐1) interferes with the activation of the PI3 kinase/Akt signaling cascade and inhibits Schwann cell myelination in vitro

Axo‐glial interactions are critical for myelination and the domain organization of myelinated fibers. Cell adhesion molecules belonging to the Cadm family, and in particular Cadm3 (axonal) and its heterophilic binding partner Cadm4 (Schwann cell), mediate these interactions along the internode. Using targeted shRNA‐mediated knockdown, we show that the removal of axonal Cadm3 promotes Schwann cell myelination in the in vitro DRG neuron/Schwann cell myelinating system. Conversely, over‐expressing Cadm3 on the surface of DRG neuron axons results in an almost complete inability by Schwann cells to form myelin segments. Axons of superior cervical ganglion (SCG) neurons, which do not normally support the formation of myelin segments by Schwann cells, express higher levels of Cadm3 compared to DRG neurons. Knocking down Cadm3 in SCG neurons promotes myelination. Finally, the extracellular domain of Cadm3 interferes in a dose‐dependent manner with the activation of ErbB3 and of the pro‐myelinating PI3K/Akt pathway, but does not interfere with the activation of the Mek/Erk1/2 pathway. While not in direct contradiction, these in vitro results shed lights on the apparent lack of phenotype that was reported from in vivo studies of Cadm3^−/− mice. Our results suggest that Cadm3 may act as a negative regulator of PNS myelination, potentially through the selective regulation of the signaling cascades activated in Schwann cells by axonal contact, and in particular by type III Nrg‐1. Further analyses of peripheral nerves in the Cadm^−/− mice will be needed to determine the exact role of axonal Cadm3 in PNS myelination. GLIA 2016;64:2247–2262     

Notch1 signaling influences v2 interneuron and motor neuron development in the spinal cord

The Notch signaling pathway plays a variety of roles in cell fate decisions during development. Previous studies have shown that reduced Notch signaling results in premature differentiation of neural progenitor cells, while increased Notch activities promote apoptotic death of neural progenitor cells in the developing brain. Whether Notch signaling is involved in the specification of neuronal subtypes is unclear. Here we examine the role of Notch1 in the development of neuronal subtypes in the spinal cord using conditional knockout (cKO) mice lacking Notch1 specifically in neural progenitor cells. Notch1 inactivation results in accelerated neuronal differentiation in the ventral spinal cord and gradual disappearance of the ventral central canal. These changes are accompanied by reduced expression of Hes1 and Hes5 and increased expression of Mash1 and Neurogenin 1 and 2. Using markers (Nkx2.2, Nkx6.1, Olig2, Pax6 and Dbx1) for one or multiple progenitor cell types, we found reductions of all subtypes of progenitor cells in the ventral spinal cord of Notch1 cKO mice. Similarly, using markers (Islet1/2, Lim3, Sim1, Chox10, En1 and Evx1/2) specific for motor neurons and distinct classes of interneurons, we found increases in the number of V0-2 interneurons in the ventral spinal cord of Notch1 cKO mice. Specifically, the number of Lim3+/Chox10+ V2 interneurons is markedly increased while the number of Lim3+/Islet+motor neurons is decreased in the Notch1 cKO spinal cord, suggesting that V2 interneurons are generated at the expense of motor neurons in the absence of Notch1. These results provide support for a role of Notch1 in neuronal subtype specification in the ventral spinal cord.     

Canadian Association of Neuroscience review: axonal regeneration in the peripheral and central nervous systems--current issues and advances

Injured nerves regenerate their axons in the peripheral (PNS) but not the central nervous system (CNS). The contrasting capacities have been attributed to the growth permissive Schwann cells in the PNS and the growth inhibitory environment of the oligodendrocytes in the CNS. In the current review, we first contrast the robust regenerative response of injured PNS neurons with the weak response of the CNS neurons, and the capacity of Schwann cells and not the oligodendrocytes to support axonal regeneration. We then consider the factors that limit axonal regeneration in both the PNS and CNS. Limiting factors in the PNS include slow regeneration of axons across the injury site, progressive decline in the regenerative capacity of axotomized neurons (chronic axotomy) and progressive failure of denervated Schwann cells to support axonal regeneration (chronic denervation). In the CNS on the other hand, it is the poor regenerative response of neurons, the inhibitory proteins that are expressed by oligodendrocytes and act via a common receptor on CNS neurons, and the formation of the glial scar that prevent axonal regeneration in the CNS. Strategies to overcome these limitations in the PNS are considered in detail and contrasted with strategies in the CNS.     

Proteolipid protein cannot replace P0 protein as the major structural protein of peripheral nervous system myelin

The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when proteolipid protein (PLP) replaced P₀ protein as the most abundant protein of CNS myelin. However, PLP did not replace P₀ in peripheral nervous system (PNS) myelin. To investigate the possible consequences of a PLP to P₀ shift in PNS myelin, we engineered mice to express PLP instead of P₀ in PNS myelin (PLP‐PNS mice). PLP‐PNS mice had severe neurological disabilities and died between 3 and 6 months of age. Schwann cells in sciatic nerves from PLP‐PNS mice sorted axons into one‐to‐one relationships but failed to form myelin internodes. Mice with equal amounts of P₀ and PLP had normal PNS myelination and lifespans similar to wild‐type (WT) mice. When PLP was overexpressed with one copy of the P₀ gene, sciatic nerves were hypomyelinated; mice displayed motor deficits, but had normal lifespans. These data support the hypothesis that while PLP can co‐exist with P₀ in PNS myelin, PLP cannot replace P₀ as the major structural protein of PNS myelin. GLIA 2015;63:66–77     

Embryonic expression of epithelial membrane protein 1 in early neurons.

Epithelial membrane protein 1 (EMP1) is a member of the peripheral myelin protein 22 (PMP22) family. This family is best known for the crucial contribution of PMP22 to the development and maintenance of the peripheral nervous system (PNS). PMP22 is widely expressed, with highest levels in myelinating Schwann cells, and mutations affecting the PMP22 gene lead to PNS-restricted neuropathies. We have investigated the spatio-temporal distribution of EMP1 and compared it to that of PMP22. We found that EMP1 and PMP22 mRNA are most conspicuously expressed in the prenatal mouse brain during neurogenesis. In the developing forebrain, we localized EMP1 mRNA and protein to the first set of neurons that are generated and leave the ventricular zone to form the preplate. Later in development, EMP1 was found in derivatives of the preplate, the marginal zone and the subplate. Reduced expression was observed in the newly generated cortical plate neurons. In other parts of the developing CNS and PNS, EMP1 was also detected in early neurons and along the initial fiber tracts. Furthermore, EMP1 was highly expressed by immature neurons in embryonal dorsal root ganglia-explant cultures and in neuroectodermal differentiated P19 cells. While PMP22 functions mainly in Schwann cell growth and differentiation, the spatio-temporal localization of EMP1 suggests a role in neuronal differentiation and neurite outgrowth.     

Reduction in delayed mortality and subtle improvement in retrograde memory performance in pilocarpine-treated mice with conditional neuronal deletion of cyclooxygenase-2 gene

Purpose: Pilocarpine induces prolonged status epilepticus (SE) in rodents that results in neurodegeneration and cognitive deficits, both commonly observed to be associated with human temporal lobe epilepsy. The multifunctional neuronal modulator, cyclooxygenase‐2 (PTGS2 or COX‐2), is rapidly induced after SE, mainly in principal neurons of the hippocampal formation and cortex. We used mice in which COX‐2 is conditionally ablated in principal forebrain neurons to investigate the involvement of neuron‐derived COX‐2 in delayed mortality and performance in the Barnes maze. Methods: Using the COX‐2 conditional knockout mouse (nCOX‐2 cKO) and their littermate wild‐type controls, we compared motor behavior and performance in the Barnes maze before and 3 weeks after the induction of SE by pilocarpine. Mortality rate was also measured during SE and in the week following SE. Key Findings: nCOX‐2 cKO mice showed less delayed mortality than wild‐type mice in the week after SE. Although motor behavior and most cognitive measures were not different in the nCOX‐2 cKO, upon reexposure to the maze 3 weeks after pilocarpine, the latency to find the previously learned target hole was significantly shorter in the nCOX‐2 cKO than their wild‐type littermate controls. By this measure pilocarpine‐treated nCOX‐2 cKO mice were identical to mice that had not experienced SE. Significance: Results point to a role for neuronal COX‐2 in delayed mortality in mice during the week following SE and suggest that neuronal COX‐2 contributes to selected cognitive deficits observed after SE. Taking into consideration our previous findings that neurodegeneration and neuroinflammation after SE are reduced in the nCOX‐2 cKO, and opening of the blood–brain barrier after pilocarpine is prevented, we conclude that neuronal COX‐2 induction is an early step in many of the deleterious consequences of SE.     

Corneal epithelial cells function as surrogate Schwann cells for their sensory nerves

The eye is innervated by neurons derived from both the central nervous system and peripheral nervous system (PNS). While much is known about retinal neurobiology and phototransduction, less attention has been paid to the innervation of the eye by the PNS and the roles it plays in maintaining a functioning visual system. The ophthalmic branch of the trigeminal ganglion contains somas of neurons that innervate the cornea. These nerves provide sensory functions for the cornea and are referred to as intraepithelial corneal nerves (ICNs) consisting of subbasal nerves and their associated intraepithelial nerve terminals. ICNs project for several millimeters within the corneal epithelium without Schwann cell support. Here, we present evidence for the hypothesis that corneal epithelial cells function as glial cells to support the ICNs. Much of the data supporting this hypothesis is derived from studies of corneal development and the reinnervation of the ICNs in the rodent and rabbit cornea after superficial wounds. Corneal epithelial cells activate in response to injury via mechanisms similar to those induced in Schwann cells during Wallerian Degeneration. Corneal epithelial cells phagocytize distal axon fragments within hours of ICN crush wounds. During aging, the proteins, lipids, and mitochondria within the ICNs become damaged in a process exacerbated by UV light. We propose that ICNs shed their aged and damaged termini and continuously elongate to maintain their density. Available evidence points to new unexpected roles for corneal epithelial cells functioning as surrogate Schwann cells for the ICNs during homeostasis and in response to injury. GLIA 2017;65:851–863     

Adhesion molecule L1 overexpressed under the control of the neuronal Thy-1 promoter improves myelination after peripheral nerve injury in adult mice

L1 is an adhesion molecule favorably influencing the functional and anatomical recoveries after central nervous system (CNS) injuries. Its roles in peripheral nervous system (PNS) regeneration are less well understood. Studies using knockout mice have surprisingly revealed that L1 has a negative impact on functional nerve regeneration by inhibiting Schwann cell proliferation. To further elucidate the roles of L1 in PNS regeneration, here we used a novel transgenic mouse overexpressing L1 in neurons, but not in PNS or CNS glial cells, under the control of a neuron-specific Thy-1 promoter. Without nerve injury, the transgene expression, as compared to wild-type mice, had no effect on femoral nerve function, numbers of quadriceps motoneurons and myelinated axons in the femoral nerve but resulted in slightly reduced myelination in the sensory saphenous nerve and increased neurofilament density in myelinated axons of the quadriceps motor nerve branch. After femoral nerve injury, L1 overexpression had no impact on the time course and degree of functional recovery. Unaffected were also numbers of regenerated quadriceps motoneurons, precision of muscle reinnervation, axon numbers and internodal lengths in the regenerated nerves. Despite the lack of functional effects, myelination in the motor and sensory femoral nerve branches was significantly improved and loss of perisomatic inhibitory terminals on motoneurons was attenuated in the transgenic mice. Our results indicate that L1 is a regulator of myelination in the injured PNS and warrant studies aiming to improve function in demyelinating PNS and CNS disorders using exogenous L1.     

Ultrastructural characterization of the mesostriatal dopamine innervation in mice, including two mouse lines of conditional VGLUT2 knockout in dopamine neurons

Despite the increasing use of genetically modified mice to investigate the dopamine (DA) system, little is known about the ultrastructural features of the striatal DA innervation in the mouse. This issue is particularly relevant in view of recent evidence for expression of the vesicular glutamate transporter 2 (VGLUT2) by a subset of mesencephalic DA neurons in mouse as well as rat. We used immuno-electron microscopy to characterize tyrosine hydroxylase (TH)-labeled terminals in the core and shell of nucleus accumbens and the neostriatum of two mouse lines in which the Vglut2 gene was selectively disrupted in DA neurons (cKO), their control littermates, and C57BL/6/J wild-type mice, aged P15 or adult. The three regions were also examined in cKO mice and their controls of both ages after dual TH-VGLUT2 immunolabeling. Irrespective of the region, age and genotype, the TH-immunoreactive varicosities appeared similar in size, vesicular content, percentage with mitochondria, and exceedingly low frequency of synaptic membrane specialization. No dually labeled axon terminals were found at either age in control or in cKO mice. Unless TH and VGLUT2 are segregated in different axon terminals of the same neurons, these results favor the view that the glutamatergic cophenotype of mesencephalic DA neurons is more important during the early development of these neurons than for the establishment of their scarce synaptic connectivity. They also suggest that, in mouse even more than rat, the mesostriatal DA system operates mainly through non-targeted release of DA, diffuse transmission and the maintenance of an ambient DA level.     

Vascular innervation in human skeletal muscle with and without neuromuscular disease

Summary A quantitative ultrastructural study has been made of the innervation of 461 arterioles in 114 skeletal muscle biopsies of patients with or without neuromuscular disease excluding diabetes and autonomic neuropathy. In 18 controls the number of nerves and Schwann cells around each vessel was related to the size of the vessel, whether the vessel was within a muscle fascicle or between muscle fascicles. The innervation of arterioles increased with increased diastolic blood pressure. There was no statistically significant change in innervation with increased systolic blood pressure or with age, from 4 to 85 years. In 96 cases of neuromuscular disease and especially in motor neurone disease, axonal varicosities in cross section tended to be larger, more often contained no vesicles or only a few and had altered satellite cell cover depending on the location of the arteriole. Whilst the numerical density of Schwann cells did not change with disease, fewer varicosities were identified within Schwann cells in motor neurone disease, metabolic myopathy and neuropathy and myopathy due to toxins or vascular disease. Preterminal axons in nerve fascicles adjacent to arterioles were lost in polymyositis and muscle disease due to toxins or vascular disease. In polymyositis, metabolic myopathy and motor neurone disease there was some evidence of compensatory nerve sprouting, either in the nerve fascicles or in the adventitia of the arterioles. These structural changes may be related to the changes in blood flow or vascular reactivity described by others in motor neurone disease, polymyositis and metabolic myopathy. It is concluded that the ultrastructure of the vascular innervation of human skeletal muscle is similar to that in other mammals [12, 19] and is changed more with increased diastolic blood pressure and neuromuscular disease than with age.     

Inactivation of ATRX in forebrain excitatory neurons affects hippocampal synaptic plasticity

α‐Thalassemia X‐linked intellectual disability (ATR‐X) syndrome is a neurodevelopmental disorder caused by mutations in the ATRX gene that encodes a SNF2‐type chromatin‐remodeling protein. The ATRX protein regulates chromatin structure and gene expression in the developing mouse brain and early inactivation leads to DNA replication stress, extensive cell death, and microcephaly. However, the outcome of Atrx loss of function postnatally in neurons is less well understood. We recently reported that conditional inactivation of Atrx in postnatal forebrain excitatory neurons (ATRX‐cKO) causes deficits in long‐term hippocampus‐dependent spatial memory. Thus, we hypothesized that ATRX‐cKO mice will display impaired hippocampal synaptic transmission and plasticity. In the present study, evoked field potentials and current source density analysis were recorded from a multichannel electrode in male, urethane‐anesthetized mice. Three major excitatory synapses, the Schaffer collaterals to basal dendrites and proximal apical dendrites, and the temporoammonic path to distal apical dendrites on hippocampal CA1 pyramidal cells were assessed by their baseline synaptic transmission, including paired‐pulse facilitation (PPF) at 50‐ms interpulse interval, and by their long‐term potentiation (LTP) induced by theta‐frequency burst stimulation. Baseline single‐pulse excitatory response at each synapse did not differ between ATRX‐cKO and control mice, but baseline PPF was reduced at the CA1 basal dendritic synapse in ATRX‐cKO mice. While basal dendritic LTP of the first‐pulse excitatory response was not affected in ATRX‐cKO mice, proximal and distal apical dendritic LTP were marginally and significantly reduced, respectively. These results suggest that ATRX is required in excitatory neurons of the forebrain to achieve normal hippocampal LTP and PPF at the CA1 apical and basal dendritic synapses, respectively. Such alterations in hippocampal synaptic transmission and plasticity could explain the long‐term spatial memory deficits in ATRX‐cKO mice and provide insight into the physiological mechanisms underlying intellectual disability in ATR‐X syndrome patients.     

Transforming growth factor β as a neuronoglial signal during peripheral nervous system response to injury

In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) displays an important regenerative ability which is dependent, at least in part, on Schwann cell properties. The mechanisms which stimulate Schwann cells to adapt their behavior after a lesion to generate adequate conditions for PNS regeneration remain unknown. In this work, we report that adult rat dorsal root ganglion (DRG) neurons are able, after a lesion performed in vivo or when they are dissociated and cultured in vitro, to synthesize transforming growth factor β (TGFβ), a pleiotropic growth factor implicated in wound healing processes and in carcinogenesis. This TGFβ is tentatively identified as the β-1 isoform. Adult rat DRG neurons release a biologically active form of TGFβ which is able to elicit multiple Schwann cell responses including a stimulation to proliferate. Moreover, purified TGFβ-1 produces a Schwann cell morphology alteration and decreases the secretion of tissue-type plasminogen activator (tPA) and enhances the secretion of plasminogen activator inhibitor (PAI) by Schwann cells. This generates conditions which are thought to favor a successful neuritic regrowth. Furthermore, purified TGFβ-1 stimulates type IV collagen mRNA expression in Schwann cells. This subtype of collagen is associated with the process of myelinization. Finally, TGFβ-1 decreases nerve growth factor (NGF) mRNA expression by Schwann cells, an effect which could participate in the maintenance of a distoproximal NGF gradient during nerve regeneration. We propose that neuronal TGFβ plays an essential role as a neuronoglial signal that modulates the response of Schwann cells to injury and participates in the successful regeneration processes observed in the PNS. © 1993 Wiley-Liss, Inc.     

Toll‐like receptor expression in the peripheral nerve

Toll‐like receptors comprise a family of evolutionary conserved pattern recognition receptors that act as a first defense line in the innate immune system. Upon stimulation with microbial ligands, they orchestrate the induction of a host defense response by activating different signaling cascades. Interestingly, they appear to detect the presence of endogenous signals of danger as well and as such, neurodegeneration is thought to trigger an immune response through ligation of TLRs. Though recent data report the expression of various TLRs in the central nervous system, TLR expression patterns in the peripheral nervous system have not been determined yet. We observed that Schwann cells express relatively high levels of TLRs, with especially TLR3 and TLR4 being prominent. Sensory and motor neurons hardly express TLRs at all. Through the use of NF‐κB signaling as read‐out, we could show that all TLRs are functional in Schwann cells and that bacterial lipoprotein, a ligand for TLR1/TLR2 receptors yields the strongest response. In sciatic nerve, basal levels of TLRs closely reflect the expression patterns as determined in Schwann cells. TLR3, TLR4, and TLR7 are majorly expressed, pointing to their possible role in immune surveillance. Upon axotomy, TLR1 becomes strongly induced, while most other TLR expression levels remain unaffected. Altogether, our data suggest that similar to microglia in the brain, Schwann cells might act as sentinel cells in the PNS. Furthermore, acute neurodegeneration induces a shift in TLR expression pattern, most likely illustrating specialized functions of TLRs in basal versus activated conditions of the peripheral nerve. © 2010 Wiley‐Liss, Inc.     

Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo

Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM‐CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM‐CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine‐β‐hydroxylase‐expressing neurons in mice [DβH^Cre/0;VGLUT2^flox/flox mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM‐CA neurons in cKO vs. control mice (DβH^Cre/0), using channelrhodopsin‐2 (ChR2–mCherry) optogenetics. ChR2–mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (~400 neurons), with identical selectivity for catecholaminergic neurons (90–99% colocalisation with tyrosine hydroxylase). RVLM‐CA neurons had similar morphology and axonal projections in DβH^Cre/0 and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2‐positive RVLM‐CA neurons in DβH^Cre/0 and cKO mice. Photostimulation in conscious mice produced frequency‐dependent respiratory activation in DβH^Cre/0 mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβH^Cre/0 mice only. Vagal responses were unaffected by α₁‐adrenoreceptor blockade. In conclusion, two responses evoked by RVLM‐CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM‐CA neurons are mediated by glutamate.     

Acetyl‐CoA production from pyruvate is not necessary for preservation of myelin

Oligodendrocytes and Schwann cells not only form myelin in the central and peripheral nervous system, but also provide metabolic and trophic support to the axons they ensheathe. Acetyl‐CoA is potentially a key molecule in Schwann cells and oligodendrocytes because it is at the crossroads of cellular lipid biosynthesis and energy generation. The main route for acetyl‐CoA production is the oxidation of pyruvate by the pyruvate dehydrogenase complex (PDC). PDC deficiency in humans results in neurodegeneration and developmental impairments in both white and gray matter structures. To address the importance of PDC in myelinating glia, we deleted Pdha1 gene specifically in oligodendrocytes and Schwann cells. Surprisingly, sciatic and optic nerve morphology and the motor performance of Pdha1^f/Y; Cnp^Cre/+ mice are undistinguishable from those of controls at 1 month of age. In addition, myelin is stably maintained for at least 10 months. However, Pdha1^f/Y; Cnp^Cre/+ mice showed reduced fiber density and signs of axonal degeneration in both sciatic and optic nerves from 6 months of age. In contrast, 10 month‐old mice bearing a floxed Pdha1 gene with either P0‐Cre (expressed only by Schwann cells) or NG2‐Cre^ER (expressed in oligodendrocyte precursor cells) do not show any sign of axonal pathology or alterations in myelin structure or thickness. This indicates that the axonopathy is specific to the Pdha1^f/Y; Cnp^Cre/+ mice. Taken together, these results suggest that acetyl‐CoA derived from pyruvate is not necessary for myelin maintenance and, thus, myelin‐forming cells are not likely to contribute to the pathophysiology of PDC deficiency.