Remyelination in PNS and CNS: current and upcoming cellular and molecular strategies to treat disabling neuropathies.

Myelin is a lipid-rich nerve cover that consists of glial cell's plasmalemma layers and accelerates signal conduction. Axon-myelin contact is a source for many developmental and regenerative signals of myelination. Intra- or extracellular factors including both enhancers and inhibitors are other factors affecting the myelination process. Myelin damages are observed in several congenital and hereditary diseases, physicochemical conditions, infections, or traumatic insults, and remyelination is known as an intrinsic response to injuries. Here we discuss some molecular events and conditions involved in de- and remyelination and compare the phenomena of remyelination in CNS and PNS. We have explained applying some of these molecular events in myelin restoration. Finally, the current and upcoming treatment strategies for myelin restoration are explained in three groups of immunotherapy, endogenous regeneration enhancement, and cell therapy to give a better insight for finding the more effective rehabilitation strategies considering the underlying molecular events of a lesion formation and its current condition.

Defective myelination in an RNA polymerase III mutant leukodystrophic mouse.

RNA polymerase (Pol) III synthesizes abundant short noncoding RNAs that have essential functions in protein synthesis, secretion, and other processes. Despite the ubiquitous functions of these RNAs, mutations in Pol III subunits cause Pol III-related leukodystrophy, an early-onset neurodegenerative disease. The basis of this neural sensitivity and the mechanisms of disease pathogenesis are unknown. Here we show that mice expressing pathogenic mutations in the largest Pol III subunit, Polr3a, specifically in Olig2-expressing cells, have impaired growth and developmental delay, deficits in cognitive, sensory, and fine sensorimotor function, and hypomyelination in multiple regions of the cerebrum and spinal cord. These phenotypes reflect a subset of clinical features seen in patients. In contrast, the gross motor defects and cerebellar hypomyelination that are common features of severely affected patients are absent in the mice, suggesting a relatively mild form of the disease in this conditional model. Our results show that disease pathogenesis in the mice involves defects that reduce both the number of mature myelinating oligodendrocytes and the ability of these cells to produce a myelin sheath of normal thickness. The findings suggest unique sensitivities of oligodendrogenesis and myelination to perturbations of Pol III transcription.

Lipin1 Alleviates Autophagy Disorder in Sciatic Nerve and Improves Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is a chronic complication of diabetes, and its neural mechanisms underlying the pathogenesis remain unclear. Autophagy plays an important role in neurodegenerative diseases and nerve tissue injury. Lipin1 is a phosphatidic acid phosphatase enzyme that converts phosphatidic acid (PA) into diacylglycerol (DAG), a precursor of triacylglycerol and phospholipids which plays an important role in maintaining normal peripheral nerve conduction function. However, whether Lipin1 involved in the pathogenesis of DPN via regulation of autophagy is not elucidated. Here, we show that the Lipin1 expression was downregulated in streptozotocin (STZ)-induced DPN rat model. Interestingly, STZ prevented DAG synthesis, and resulted in autophagic hyperactivity, effects which may increase the apoptosis of Schwann cells and lead to demyelination in sciatic nerve in DPN rats. More importantly, upregulation of lipin1 in the DPN rats ameliorated autophagy disorders and pathological changes of the sciatic nerve, which associated with the increase of the motor nerve conductive velocity (MNCV) in DPN rats. In contrast, knockdown of lipin1 exacerbates neuronal abnormalities and facilitates the genesis of DPN phenotypes in rats. In addition, overexpression of lipin1 in RSC96 cells also significantly decreased the autophagic hyperactivity and apoptosis induced by hyperglycemia. These results suggest that lipin1 may exert neuroprotection within the sciatic nerve anomalies and may serve as a potential therapeutic target for the treatment of DPN.

Trigeminal neuropathy causes hypomyelination in the anterior cingulate cortex, disrupts the synchrony of neural circuitry, and impairs decision-making in male rats.

Infraorbital nerve-chronic constriction injury (ION-CCI) has become the most popular chronic trigeminal neuropathic pain (TNP) injury animal model which causes prolonged mechanical allodynia. Accumulative evidence suggests that TNP interferes with cognitive functions, however the underlying mechanisms are not known. The aim of this study was to investigate decision-making performance as well as synaptic and large-scale neural synchronized alterations in the spinal trigeminal nucleus (SpV) circuitry and anterior cingulate cortex (ACC) neural circuitry in male rats with TNP. Rat gambling task showed that ION-CCI led to decrease the proportion of good decision makers and increase the proportion of poor decision makers. Electrophysiological recordings showed long-lasting synaptic potentiation of local field potential in the trigeminal ganglia-SpV caudalis (SpVc) synapses in TNP rats. In this study, TNP led to disruption of ACC spike timing and basolateral amygdala (BLA) theta oscillation associated with suppressed synchronization of theta oscillation between the BLA and ACC, indicating reduced neuronal communications. Myelination is critical for information flow between brain regions, and myelin plasticity is an important feature for learning. Neural activity in the cortical regions impacts myelination by regulating oligodendrocyte (OL) proliferation, differentiation, and myelin formation. We characterized newly formed oligodendrocyte progenitor cells, and mature OLs are reduced in TNP and are associated with reduced myelin strength in the ACC region. The functional disturbances in the BLA-ACC neural circuitry is pathologically associated with the myelin defects in the ACC region which may be relevant causes for the deficits in decision-making in chronic TNP state.

Brain gray matter astroglia-specific connexin 43 ablation attenuates spinal cord inflammatory demyelination.

BACKGROUND: Brain astroglia are activated preceding the onset of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We characterized the effects of brain astroglia on spinal cord inflammation, focusing on astroglial connexin (Cx)43, because we recently reported that Cx43 has a critical role in regulating neuroinflammation. METHODS: Because glutamate aspartate transporter (GLAST)+ astroglia are enriched in the brain gray matter, we generated Cx43fl/fl;GLAST-CreERT2/+ mice that were brain gray matter astroglia-specific Cx43 conditional knockouts (Cx43 icKO). EAE was induced by immunization with myelin oligodendroglia glycoprotein (MOG) 35-55 peptide 10 days after tamoxifen injection. Cx43fl/fl mice were used as controls. RESULTS: Acute and chronic EAE signs were significantly milder in Cx43 icKO mice than in controls whereas splenocyte MOG-specific responses were unaltered. Histologically, Cx43 icKO mice showed significantly less demyelination and fewer CD45+ infiltrating immunocytes, including F4/80+ macrophages, and Iba1+ microglia in the spinal cord than controls. Microarray analysis of the whole cerebellum revealed marked upregulation of anti-inflammatory A2-specific astroglia gene sets in the pre-immunized phase and decreased proinflammatory A1-specific and pan-reactive astroglial gene expression in the onset phase in Cx43 icKO mice compared with controls. Astroglia expressing C3, a representative A1 marker, were significantly decreased in the cerebrum, cerebellum, and spinal cord of Cx43 icKO mice compared with controls in the peak phase. Isolated Cx43 icKO spinal microglia showed more anti-inflammatory and less proinflammatory gene expression than control microglia in the pre-immunized phase. In particular, microglial expression of Ccl2, Ccl5, Ccl7, and Ccl8 in the pre-immunized phase and of Cxcl9 at the peak phase was lower in Cx43 icKO than in controls. Spinal microglia circularity was significantly lower in Cx43 icKO than in controls in the peak phase. Significantly lower interleukin (IL)-6, interferon-γ, and IL-10 levels were present in cerebrospinal fluid from Cx43 icKO mice in the onset phase compared with controls. CONCLUSIONS: The ablation of Cx43 in brain gray matter astroglia attenuates EAE by promoting astroglia toward an anti-inflammatory phenotype and suppressing proinflammatory activation of spinal microglia partly through depressed cerebrospinal fluid proinflammatory cytokine/chemokine levels. Brain astroglial Cx43 might be a novel therapeutic target for MS.

Defective Oligodendroglial Lineage and Demyelination in Amyotrophic Lateral Sclerosis.

Motor neurons and their axons reaching the skeletal muscle have long been considered as the best characterized targets of the degenerative process observed in amyotrophic lateral sclerosis (ALS). However, the involvement of glial cells was also more recently reported. Although oligodendrocytes have been underestimated for a longer time than other cells, they are presently considered as critically involved in axonal injury and also conversely constitute a target for the toxic effects of the degenerative neurons. In the present review, we highlight the recent advances regarding oligodendroglial cell involvement in the pathogenesis of ALS. First, we present the oligodendroglial cells, the process of myelination, and the tight relationship between axons and myelin. The histological abnormalities observed in ALS and animal models of the disease are described, including myelin defects and oligodendroglial accumulation of pathological protein aggregates. Then, we present data that establish the existence of dysfunctional and degenerating oligodendroglial cells, the chain of events resulting in oligodendrocyte degeneration, and the most recent molecular mechanisms supporting oligodendrocyte death and dysfunction. Finally, we review the arguments in support of the primary versus secondary involvement of oligodendrocytes in the disease and discuss the therapeutic perspectives related to oligodendrocyte implication in ALS pathogenesis.

Mechanisms and repair strategies for white matter degeneration in CNS injury and diseases.

White matter degeneration is an important pathophysiological event of the central nervous system that is collectively characterized by demyelination, oligodendrocyte loss, axonal degeneration and parenchymal changes that can result in sensory, motor, autonomic and cognitive impairments. White matter degeneration can occur due to a variety of causes including trauma, neurotoxic exposure, insufficient blood flow, neuroinflammation, and developmental and inherited neuropathies. Regardless of the etiology, the degeneration processes share similar pathologic features. In recent years, a plethora of cellular and molecular mechanisms have been identified for axon and oligodendrocyte degeneration including oxidative damage, calcium overload, neuroinflammatory events, activation of proteases, depletion of adenosine triphosphate and energy supply. Extensive efforts have been also made to develop neuroprotective and neuroregenerative approaches for white matter repair. However, less progress has been achieved in this area mainly due to the complexity and multifactorial nature of the degeneration processes. Here, we will provide a timely review on the current understanding of the cellular and molecular mechanisms of white matter degeneration and will also discuss recent pharmacological and cellular therapeutic approaches for white matter protection as well as axonal regeneration, oligodendrogenesis and remyelination.

Neuronal hibernation following hippocampal demyelination.

Cognitive dysfunction occurs in greater than 50% of individuals with multiple sclerosis (MS). Hippocampal demyelination is a prominent feature of postmortem MS brains and hippocampal atrophy correlates with cognitive decline in MS patients. Cellular and molecular mechanisms responsible for neuronal dysfunction in demyelinated hippocampi are not fully understood. Here we investigate a mouse model of hippocampal demyelination where twelve weeks of treatment with the oligodendrocyte toxin, cuprizone, demyelinates over 90% of the hippocampus and causes decreased memory/learning. Long-term potentiation (LTP) of hippocampal CA1 pyramidal neurons is considered to be a major cellular readout of learning and memory in the mammalian brain. In acute slices, we establish that hippocampal demyelination abolishes LTP and excitatory post-synaptic potentials of CA1 neurons, while pre-synaptic function of Schaeffer collateral fibers is preserved. Demyelination also reduced Ca2+-mediated firing of hippocampal neurons in vivo. Using three-dimensional electron microscopy, we investigated the number, shape (mushroom, stubby, thin), and post-synaptic densities (PSDs) of dendritic spines that facilitate LTP. Hippocampal demyelination did not alter the number of dendritic spines. Surprisingly, dendritic spines appeared to be more mature in demyelinated hippocampi, with a significant increase in mushroom-shaped spines, more perforated PSDs, and more astrocyte participation in the tripartite synapse. RNA sequencing experiments identified 400 altered transcripts in demyelinated hippocampi. Gene transcripts that regulate myelination, synaptic signaling, astrocyte function, and innate immunity were altered in demyelinated hippocampi. Hippocampal remyelination rescued synaptic transmission, LTP, and the majority of gene transcript changes. We establish that CA1 neurons projecting demyelinated axons silence their dendritic spines and hibernate in a state that may protect the demyelinated axon and facilitates functional recovery following remyelination.

Glial progenitor cell-based repair of the dysmyelinated brain: Progression to the clinic.

Demyelinating disorders of the central white matter are among the most prevalent and disabling conditions in neurology. Since myelin-producing oligodendrocytes comprise the principal cell type deficient or lost in these conditions, their replacement by new cells generated from transplanted bipotential oligodendrocyte-astrocyte progenitor cells has emerged as a therapeutic strategy for a variety of primary dysmyelinating diseases. In this review, we summarize the research and clinical considerations supporting current efforts to bring this treatment approach to patients.

Clinical features of homozygous FIG4-p.Ile41Thr Charcot-Marie-Tooth 4J patients.

We describe the clinical, electrodiagnostic, and genetic findings of three homozygous FIG4-c.122T>C patients suffering from Charcot-Marie-Tooth disease type 4J (AR-CMT-FIG4). This syndrome usually involves compound heterozygosity associating FIG4-c.122T>C, a hypomorphic allele coding an unstable FIG4-p.Ile41Thr protein, and a null allele. While the compound heterozygous patients presenting with early onset usually show rapid progression, the homozygous patients described here show the signs of relative clinical stability. As FIG4 activity is known to be dose dependent, these patients' observations could suggest that the therapeutic perspective of increasing levels of the protein to improve the phenotype of AR-CMT-FIG4-patients might be efficient.

Acute motor deficit and subsequent remyelination-associated recovery following internal capsule demyelination in mice.

Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system (CNS), characterized by accumulated motor disability. However, whether remyelination promotes motor recovery following demyelinating injury remains unclear. Damage to the internal capsule (IC) is known to result in motor impairment in multiple sclerosis and stroke. Here, we induced focal IC demyelination in mice by lysophosphatidylcholine (LPC) injection, and examined its effect on motor behavior. We also compared the effect of LPC-induced IC damage to that produced by endothelin-1 (ET1), a potent vasoconstrictor used in experimental stroke lesions. We found that LPC or ET1 injections induced asymmetric motor deficit at 7 days post-lesion (dpl), and that both lesion types displayed increased microglia/macrophage density, myelin loss, and axonal dystrophy. The motor deficit and lesion pathology remained in ET1-injected mice at 28 dpl. In contrast, LPC-injected mice regained motor function by 28 dpl, with corresponding reduction in activated microglia/macrophage density, and recovery of myelin staining and axonal integrity in lesions. These results suggest that LPC-induced IC demyelination results in acute motor deficit and subsequent recovery through remyelination, and may be used to complement future drug screens to identify drugs for promoting remyelination.

Human stem cell-derived oligodendrocytes: From humanized animal models to cell therapy in myelin diseases.

Oligodendrocytes are main targets in demyelinating and dysmyelinating diseases of the central nervous system (CNS), but are also involved in accidental, neurodegenerative and psychiatric disorders. The underlying pathology of these diseases is not fully understood and treatments are still lacking. The recent discovery of the induced pluripotent stem cell (iPSC) technology has open the possibility to address the biology of human oligodendroglial cells both in the dish and in vivo via engraftment in animal models, and paves the way for the development of treatment for myelin disorders. In this review, we make a short overview of the different sources human oligodendroglial cells, and animal models available for pre-clinical cell therapy. We discuss the anatomical and functional benefit of grafted iPSC-progenitors over their brain counterparts, their use in disease modeling and the missing gaps that still prevent to study their biology in the most integrated way, and to translate iPSC-stem cell based therapy to the clinic.

The Intellicage system provides a reproducible and standardized method to assess behavioral changes in cuprizone-induced demyelination mouse model.

BACKGROUND: Multiple sclerosis is a neurodegenerative disorder characterized by myelin loss in the brain parenchyma. To mimic the disease, mice are fed a cuprizone-supplemented diet for 5 weeks, which leads to demyelination of white and grey matter regions, with the corpus callosum being the most susceptible to cuprizone intoxication. Although this model is highly exploited, classical behavioural tests showed inconsistent results. OBJECTIVE: In our study, we aimed to use the automated system Intellicage to phenotype the behaviour of cuprizone-fed mice. METHODS: Mice were continuously monitored during the 5 weeks of intoxication in their home cages, with minimal interference from the experimenter. Mice were assessed for spontaneous activity, fine movements, and impulsivity. RESULTS: Consistently, cuprizone-fed mice showed reduced activity and impulsivity throughout the test period. These behavioral results were confirmed by repeating the battery of behavioral tests in a second cohort of cuprizone-fed mice. Our results suggest that the behavioural phenotyping of cuprizone-fed mice using Intellicage is reproducible and sensitive enough to detect changes normally missed in standard behavioral test batteries. CONCLUSION: Using a reproducible and standardized method to assess behavioral changes in mice intoxicated with cuprizone is crucial to better understand the disease as well as the functional outcome of treatments.

Axonal mitochondria adjust in size depending on g-ratio of surrounding myelin during homeostasis and advanced remyelination.

Demyelinating pathology is common in many neurological diseases such as multiple sclerosis, stroke, and Alzheimer's disease and results in axonal energy deficiency, dysfunctional axonal propagation, and neurodegeneration. During myelin repair and also during myelin homeostasis, mutual regulative processes between axons and myelin sheaths are known to be essential. However, proficient tools are lacking to characterize axon-myelin interdependence during (re)myelination. Thus, we herein investigated adaptions in myelin sheath g-ratio as a proxy for myelin thickness and axon metabolic status during homeostasis and myelin repair, by using axonal mitochondrial size as a proxy for axonal metabolic status. We found that axons with thinner myelin sheaths had larger axonal mitochondria; this was true for across different central nervous system tracts as well as across species, including humans. The link between myelin sheath thickness and mitochondrial size was temporarily absent during demyelination but reestablished during advanced remyelination, as shown in two commonly used animal models of toxic demyelination. By further exploring this association in mice with either genetically induced mitochondrial or myelin dysfunction, we show that axonal mitochondrial size adjusts in response to the thickness of the myelin sheath but not vice versa. This pinpoints the relevance of mitochondrial adaptation upon myelin repair and might open a new therapeutic window for remyelinating therapies.

Myelin plasticity: sculpting circuits in learning and memory.

Throughout our lifespan, new sensory experiences and learning continually shape our neuronal circuits to form new memories. Plasticity at the level of synapses has been recognized and studied for decades, but recent work has revealed an additional form of plasticity - affecting oligodendrocytes and the myelin sheaths they produce - that plays a crucial role in learning and memory. In this Review, we summarize recent work characterizing plasticity in the oligodendrocyte lineage following sensory experience and learning, the physiological and behavioural consequences of manipulating that plasticity, and the evidence for oligodendrocyte and myelin dysfunction in neurodevelopmental disorders with cognitive symptoms. We also discuss the limitations of existing approaches and the conceptual and technical advances that are needed to move forward this rapidly developing field.

Ferroptosis Mediates Cuprizone-Induced Loss of Oligodendrocytes and Demyelination.

Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS. Cuprizone (CZ), a copper chelator, is widely used to study demyelination and remyelination in the CNS, in the context of MS. However, the mechanisms underlying oligodendrocyte (OL) cell loss and demyelination are not known. As copper-containing enzymes play important roles in iron homeostasis and controlling oxidative stress, we examined whether chelating copper leads to disruption of molecules involved in iron homeostasis that can trigger iron-mediated OL loss. We show that giving mice (male) CZ in the diet induces rapid loss of OL in the corpus callosum by 2 d, accompanied by expression of several markers for ferroptosis, a relatively newly described form of iron-mediated cell death. In ferroptosis, iron-mediated free radicals trigger lipid peroxidation under conditions of glutathione insufficiency, and a reduced capacity to repair lipid damage. This was further confirmed using a small-molecule inhibitor of ferroptosis that prevents CZ-induced loss of OL and demyelination, providing clear evidence of a copper-iron connection in CZ-induced neurotoxicity. This work has wider implications for disorders, such as multiple sclerosis and CNS injury.SIGNIFICANCE STATEMENT Cuprizone (CZ) is a copper chelator that induces demyelination. Although it is a widely used model to study demyelination and remyelination in the context of multiple sclerosis, the mechanisms mediating demyelination is not fully understood. This study shows, for the first time, that CZ induces demyelination via ferroptosis-mediated rapid loss of oligodendrocytes. This work shows that chelating copper with CZ leads to the expression of molecules that rapidly mobilize iron from ferritin (an iron storage protein), that triggers iron-mediated lipid peroxidation and oligodendrocyte loss (via ferroptosis). Such rapid mobilization of iron from cellular stores may also play a role in cell death in other neurologic conditions.

Ursolic acid treatment suppresses cuprizone-induced demyelination and motor dysfunction via upregulation of IGF-1.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system, characterized by apoptotic death of mature oligodendrocytes, neuroinflammation, and motor dysfunction. A pentacyclic triterpenoid compound, ursolic acid (UA), has various pharmacological activities, such as anti-inflammatory, anti-oxidative, and anti-apoptotic effects. In the present study, we investigated the effects of UA on cuprizone-induced demyelination, which is a model of MS. Oral administration of UA effectively suppressed cuprizone-induced demyelination and motor dysfunction via the enhancement of IGF-1 levels in the demyelinating lesions. Our results suggest that UA might be therapeutically useful for demyelination in MS.

Nano-hesperetin enhances the functional recovery and endogenous remyelination of the optic pathway in focal demyelination model.

Our recent report demonstrated that hesperetin (Hst) as a citrus flavonoid, significantly reduces the levels of demyelination in optic chiasm of rats. Previous evidence also indicated that nano-hesperetin (nano-Hst) possesses beneficial impacts in experimental models of Alzheimer's disease and autism. In this study, the effects of nano-Hst on latency of visual signals, demyelination levels, glial activation, and expression of Olig2 and MBP were evaluated in lysolecithin (LPC)-induced demyelination model. Focal demyelination was induced by injection of LPC (1%, 2 µL) into the rat optic chiasm. Animals received oral administration of nano-Hst at dose of 20 mg/kg for 14 or 21 days post LPC injection. Visual evoked potential (VEP) recording showed that nano-Hst reduces the latency of visual signals and ameliorates the extent of demyelination areas and glial activation. Expression levels of the Olig2 and MBP were also significantly increased in nano-Hst treated rats. Overall, our data suggest that nano-Hst reduces the latency of visual signals through its protective effects on myelin sheath, amelioration of glial activation, and enhancement of endogenous remyelination.

PPAR-γ Is Critical for HDAC3-Mediated Control of Oligodendrocyte Progenitor Cell Proliferation and Differentiation after Focal Demyelination.

Disruption of remyelination contributes to neurodegeneration and cognitive impairment in chronically disabled patients. Valproic acid (VPA) inhibits histone deacetylase (HDAC) function and probably promotes oligodendrocyte progenitor cell (OPC) proliferation and differentiation; however, the relevant molecular mechanisms remain unknown. Here, focal demyelinating lesions (FDLs) were generated in mice by two-point stereotactic injection of lysophosphatidylcholine (LPC) into the corpus callosum. Cognitive functions, sensorimotor abilities and histopathological changes were assessed for up to 28 days post-injury with or without VPA treatment. Primary OPCs were harvested and used to study the effect of VPA on OPC differentiation under inflammatory conditions. VPA dose-dependently attenuated learning and memory deficits and robustly protected white matter after FDL induction, as demonstrated by reductions in SMI-32 and increases in myelin basic protein staining. VPA also promoted OPC proliferation and differentiation and increased subsequent remyelination efficiency by day 28 post-FDL induction. VPA treatment did not affect HDAC1, HDAC2 or HDAC8 expression but reduced HDAC3 protein levels. In vitro, VPA improved the survival of mouse OPCs and promoted their differentiation into oligodendrocytes following lipopolysaccharide (LPS) stimulation. LPS caused OPCs to overexpress HDAC3, which translocated from the cytoplasm into the nucleus, where it directly interacted with the nuclear transcription factor PPAR-γ and negatively regulated PPAR-γ expression. VPA decreased the expression of HDAC3 and promoted remyelination and functional neurological recovery after FDL. These findings may support the use of strategies modulating HDAC3-mediated regulation of protein acetylation for the treatment of demyelination-related cognitive dysfunction.