Myelin Vacuolation Research Articles

Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer's, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β-induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing ina demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.

Immediate versus delayed primary repair of the sciatic nerve in a nerve transaction model in rats.

The debate continues concerning surgical timing in a peripheral nerve injury. This study aims to evaluate the result of immediate versus delayed primary (after seven days) repair of peripheral nerve injury. In this study, Wistar rats were divided into four groups as follows: The nerve was sharply transected in Group 1, 2 and 4. It was immediately sutured in Group 1 and sutured seven days later in Group 2, and it was not sutured in Group 4. In Group 3, the left sciatic nerve was only explored. Eight weeks later, tissue samples were extracted from the injured nerve area. Both gastrocnemius muscles were weighed. The nerve samples were examined for axon degeneration. Myelin vacuolization, axon irregularity, and edema/inflammation parameters were evaluated. There were not any significant differences in the score of axon degeneration and the weight of the gastrocnemius muscle between the immediate and delayed primary repair groups. However, these parameters were significantly better in both repair groups than to be in the control group and significantly worse than to be in the sham-operated group. To delay the repair about one week did not affect the histological results and weight of the muscle that was innervated by the sectioned nerve comparing to be in the immediate repair in a sciatic nerve transaction model in rats.

Commentary: Closantel - A lesser-known evil.

Closantel is an antiparasitic agent routinely used in veterinary practice against some parasites of livestock. It is not used against agricultural and household pests. Recently, it has been discovered that closantel also acts against Onchocerca volvulus, a filarial nematode causing river blindness in humans.[1] Closantel belongs to the chemical class of the salicylanilides, which acts as an uncoupler of oxidative phosphorylation in the cell mitochondria and disturbs ATP production. This impairs the parasite's motility and probably other processes as well. Closantel also disturbs the liquid and ion transport mechanisms in the parasite's membranes. Closantel is a narrow-spectrum anti-helminthic effective against a few roundworms (mainly blood-sucking species), against flukes (e.g., Fasciola hepatica) and also against certain myiasis. It is ineffective against tapeworms or most external parasites. It is not used in swine, poultry, horses, or pets. It is used moderately in ruminants both as an injectable or a drench, often mixed with other anti-helminthics. After oral administration, closantel is readily absorbed into the bloodstream. Four days after treatment up to 60% of the injected and 30% of the drenched closantel is absorbed to blood. In the blood, >99% of the unchanged closantel binds strongly to plasma albumins. Peak plasma levels are reached between 10 and 48 h after administration, both after oral or intramuscular administration. Half-life in plasma is 3 to 4 weeks. Owing to the strong binding to plasma albumins, closantel residues in the tissues are rather low. Closantel is poorly metabolized. Approximately, 80% of the administered dose is excreted through the feces, of which >98% in the parent molecule form. Excretion 48 h after oral administration reaches ~45% of the administered dose, but only ~10% after intramuscular injection. Excretion half-life in the organism is 2 to 3 weeks. In dairy cows, about 1% of the administered dose is excreted unchanged through the milk. The routinely used safe-dose for the anti-helminthic action of closantel varies from 3 to 10 mg/kg depending upon the type of parasite and the animal affected. The established lethal dose limit for oral closantel in rats is 342 mg/kg, and for subcutaneous administration is 67 mg/kg of the active ingredient. Closantel intoxication has been reported in animals such as sheep, cattle, goats, and dogs.[2345] Closantel intoxication often presents with neurological and ocular features. The central nervous system, retina, and optic nerve are commonly affected. Histopathological changes noted are spongiosis in the brain white matter and spinal cord. These changes are symmetrical and affect periventricular white matter, optic radiation, brain stem, thalamic nuclei, and cerebellar peduncles. The induced histopathological changes in optic nerve following closantel overdosage include significant spongiform change, edema, and myelin vacuolization resulting in optic disc atrophy.[6] This results in edema in the intracanalicular portion of the optic nerve, which being in the osseous part gets compressed, resulting loss of myelinated axons.[6] In retina, there is destruction and apoptosis of the outer retinal layers, especially photoreceptors. The most frequent symptoms following closantel intoxication include loss of appetite, ataxia or uncoordinated movements, weakness, visual disturbances, and blindness. Other symptoms reported in sheep include depression, prostration, colic, reduced skin sensitivity, opisthotonos, nystagmus, mydriasis, loss of pupillary reflex, and blindness. Mortalities can also happen. The use of closantel in humans and milk-producing animals is strongly avoided. However, accidental ingestion of closantel leading to overdose and adverse effects in humans has been reported in literature.[78910] In humans, ocular symptoms following closantel overdosage range from sudden onset blurring of vision to total blindness. The onset of symptoms has been reported to occur from as early as 3 days to 22 years following closantel intake.[910] Depending upon the severity of damage, ocular features show optic nerve involvement ranging from blurring of disc margins and disc edema in acute stage to optic atrophy in the late stages. There is outer retinal layer thinning with spicule like hyper-pigmentation owing to the involvement of photoreceptor and retinal pigment epithelium layer. Destruction in outer retinal layers and visual pathway leads to changes in electroretinography and visual evoked potential (VEP) test. On electro-retinography, there is a reduction in both a- and b-wave amplitudes, whereas VEP shows a reduction in amplitude and delay in latency. The effects of closantel intoxication are often permanent and irreversible. There is no specific antidote for closantel. Treatment consists of supportive and symptomatic measures. Partial visual recovery with plasma exchange has been reported in a man who ingested closantel because of helminthiasis fearing.[8] To conclude, accidental closantel intake can lead to blinding visual symptoms due to outer retinal degeneration and optic atrophy.

Conventional and quantitative MRI in a novel feline model of demyelination and endogenous remyelination.

BackgroundThe feeding of irradiated food to healthy adult cats results in widespread, noninflammatory demyelination of the central nervous system (CNS); a return to a normal diet results in endogenous remyelination with functional recovery. This recently discovered, reversible disease might provide a compelling clinical neuroimaging model system for the development and testing of myelin‐directed MRI methods as well as future remyelination therapies.PurposeIdentify the noninvasive imaging characteristics of this new disease model and determine whether it features measurable changes on conventional and quantitative MRI.Study TypePilot study.Animal ModelTen adult cats at various stages of demyelinating disease induced by an irradiated diet (35–55 kGy), and during recovery following a return to a normal diet.Field Strength/SequenceConventional (T2‐weighted) and quantitative (diffusion tensor, magnetization transfer) at 3T.AssessmentMRI of the brain, optic nerves, and cervical spinal cord; a subset of diseased cats was euthanized for comparative histopathology.Statistical TestsDescriptive statistics.ResultsDisease produced T2 prolongation, progressing from patchy to diffuse throughout most of the cerebral white matter (eventually involving U‐fibers) and spinal cord (primarily dorsal columns, reminiscent of subacute combined degeneration but without evidence of B12 deficiency). Magnetization transfer parameters decreased by 50–53% in cerebral white matter and by 25–30% in optic nerves and spinal cord dorsal columns. Fractional diffusion anisotropy decreased by up to 20% in pyramidal tracts, primarily driven by increased radial diffusivity consistent with axon preservation. Histopathology showed scattered myelin vacuolation of major white matter tracts as well as many thin myelin sheaths consistent with remyelination in the recovery phase, which was detectable on magnetization transfer imaging.Data ConclusionFeline irradiated diet‐induced demyelination features noninvasively imageable and quantifiable demyelination and remyelination of the CNS. It is therefore a compelling clinical neuroimaging model system. Level of Evidence: 4 Technical Efficacy: Stage 2J. Magn. Reson. Imaging 2019;49:1304–1311.

Abnormal Morphology of Select Cortical and Subcortical Regions in Neurofibromatosis Type 1.

Purpose To evaluate whether patients with neurofibromatosis type 1 (NF1)-a multisystem neurodevelopmental disorder with myriad imaging manifestations, including focal transient myelin vacuolization within the deep gray nuclei, brainstem, and cerebellum-exhibit differences in cortical and subcortical structures, particularly in subcortical regions where these abnormalities manifest. Materials and Methods In this retrospective study, by using clinically obtained three-dimensional T1-weighted MR images and established image analysis methods, 10 intracranial volume-corrected subcortical and 34 cortical regions of interest (ROIs) were quantitatively assessed in 32 patients with NF1 and 245 age- and sex-matched healthy control subjects. By using linear models, ROI cortical thicknesses and volumes were compared between patients with NF1 and control subjects, as a function of age. With hierarchic cluster analysis and partial correlations, differences in the pattern of association between cortical and subcortical ROI volumes in patients with NF1 and control subjects were also evaluated. Results Patients with NF1 exhibited larger subcortical volumes and thicker cortices of select regions, particularly the hippocampi, amygdalae, cerebellar white matter, ventral diencephalon, thalami, and occipital cortices. For the thalami and pallida and 22 cortical ROIs in patients with NF1, a significant inverse association between volume and age was found, suggesting that volumes decrease with increasing age. Moreover, compared with those in control subjects, ROIs in patients with NF1 exhibited a distinct pattern of clustering and partial correlations. Discussion Neurofibromatosis type 1 is characterized by larger subcortical volumes and thicker cortices of select structures. Most apparent within the hippocampi, amygdalae, cerebellar white matter, ventral diencephalon, thalami and occipital cortices, these neurofibromatosis type 1-associated volumetric changes may, in part, be age dependent. © RSNA, 2018 Online supplemental material is available for this article.

Oligodendrocytes control potassium accumulation in white matter and seizure susceptibility.

The inwardly rectifying K+ channel Kir4.1 is broadly expressed by CNS glia and deficits in Kir4.1 lead to seizures and myelin vacuolization. However, the role of oligodendrocyte Kir4.1 channels in controlling myelination and K+ clearance in white matter has not been defined. Here, we show that selective deletion of Kir4.1 from oligodendrocyte progenitors (OPCs) or mature oligodendrocytes did not impair their development or disrupt the structure of myelin. However, mice lacking oligodendrocyte Kir4.1 channels exhibited profound functional impairments, including slower clearance of extracellular K+ and delayed recovery of axons from repetitive stimulation in white matter, as well as spontaneous seizures, a lower seizure threshold, and activity-dependent motor deficits. These results indicate that Kir4.1 channels in oligodendrocytes play an important role in extracellular K+ homeostasis in white matter, and that selective loss of this channel from oligodendrocytes is sufficient to impair K+ clearance and promote seizures.

Investigation of the histological changes in spinal nerve roots post intrathecal ketamine administration

Background: Ketamine is a general anesthetic agent, administered intramuscularly or intravenously. It is generally used for induction and less frequently for the continuation of surgical anesthesia. Although ketamine was initially thought to be used as an intrathecal agent, psychomimetic side effects were observed. Objectives: In this study, the differences in the spinal nerve roots postintrathecal ketamine injection have been examined in the light and electron microscope. Materials and Methods: 30 min, 1 h, and 24-h postintrathecal ketamine (0.5 mg/kg) injection; the rabbits were sacrificed. The tissue samples taken from the injection area fixed with 1st 2% glutaraldehyde and later 1% OsO4. Following dehydration tissue samples embedded in Araldite and sections were taken and examined in the light and electron microscope. Results: There were severe degenerations in the myelinated nerve fibers postintrathecal injection of ketamine. Fading on the myelin lamellae, shrinking and vacuolization of the axoplasm, endoneurial edema, organelle loss, and cystic parts were observed. The vacuolization and lack of organelles in the Schwann cell cytoplasm were clear. The degeneration of the large myelinated nerve fibers was observed more than unmyelinated fibers. Conclusion: The general and local anesthetics used have significant side effects such as respiratory depression or permanent neurological disorders. Therefore, new options are searched, and the effects of the drugs used in different fields of anesthesia were investigated.

Effect of nerve growth factor on chronic peripheral neuropathy in rats induced by 1-bromopropane

Objective: To observe the effect of nerve growth factor (NGF) and Mecobalamin on chronic peripheral neuropathy in rats induced by 1-bromopropane. Methods: 36 male SD rats were exposed to 1-bromopropane vapor at concentrations of 4 000 mg/m(3), 6 hours per day, 5 days per week for 12 weeks. The rats were randomed divided into 4 groups, and treated by Mecobalamin for 300 μg/kg qd, NGF for 40 μg/kg qd, Mecobalamin+NGF with the dose as mentioned above, respecively. The control group were fed in normal condition. The changes of Sciatic nerve conduction velocity (NCV) , electromyography (EMG) and pathology were observed 30 days later. Results: The nerve conduction velocity were decreased in all the rats. Compared with the control group, the motor nerve conduction velocity (MCV) was improved in group Mecobalamin and group Mecobalamin+NGF, The difference was statistically significant, as the sensory nerve conduction velocity (SCV) was improved only in group Mecobalamin+NGF. Sciatic nerve biopsy observed by electron microscope showed that myelinated nerve fibers were obvious swelling, lamellar separation, partial myelin vacuolization, and axonal degeneration. After treatment with exogenous nerve growth factor, the number and severity of damaged nerve fibers were restored. Conclusion: Exogenous nerve growth factor contributes to the recovery of peripheral nerve damage induced by 1-bromopropane.

Microbial Neuraminidase Induces a Moderate and Transient Myelin Vacuolation Independent of Complement System Activation.

Some central nervous system pathogens express neuraminidase (NA) on their surfaces. In the rat brain, a single intracerebroventricular (ICV) injection of NA induces myelin vacuolation in axonal tracts. Here, we explore the nature, the time course, and the role of the complement system in this damage. The spatiotemporal analysis of myelin vacuolation was performed by optical and electron microscopy. Myelin basic protein-positive area and oligodendrocyte transcription factor (Olig2)-positive cells were quantified in the damaged bundles. Neuronal death in the affected axonal tracts was assessed by Fluoro-Jade B and anti-caspase-3 staining. To evaluate the role of the complement, membrane attack complex (MAC) deposition on damaged bundles was analyzed using anti-C5b9. Rats ICV injected with the anaphylatoxin C5a were studied for myelin damage. In addition, NA-induced vacuolation was studied in rats with different degrees of complement inhibition: normal rats treated with anti-C5-blocking antibody and C6-deficient rats. The stria medullaris, the optic chiasm, and the fimbria were the most consistently damaged axonal tracts. Vacuolation peaked 7 days after NA injection and reverted by day 15. Olig2+ cell number in the damaged tracts was unaltered, and neurodegeneration associated with myelin alterations was not detected. MAC was absent on damaged axonal tracts, as revealed by C5b9 immunostaining. Rats ICV injected with the anaphylatoxin C5a displayed no myelin injury. When the complement system was experimentally or constitutively inhibited, NA-induced myelin vacuolation was similar to that observed in normal rats. Microbial NA induces a moderate and transient myelin vacuolation that is not caused either by neuroinflammation or complement system activation.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 regulates epidermal growth factor receptor signaling in astrocytes.

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

Isolated extrapontine myelinolysis due to hemodialysis in a normonatremic individual

Osmotic demyelination syndrome primarily occurs due to an imbalance of serum electrolytes and caused by rapid correction of hyponatremia. Isolated extrapontine myelinolysis is extremely rare, etiopathogenesis of which may be related to osmotic stress resulting from electrolyte disequilibrium. Extrapontine sites involved in osmotic demyelination syndrome show abundant oligodendrocytes, which are highly susceptible to osmotic stresses. Myelin vacuolization followed by intramyelinic splitting and disruption of myelin sheaths occur due to rapid osmolar fluctuations. Symmetrical hyperintensities showing restriction, which resolve in subsequent magnetic resonance scans can help in clinching the diagnosis of this condition in a given clinical setting.

Chapter 6 - Immunopathology: autoimmune glial diseases and differentiation from multiple sclerosis

While multiple sclerosis (MS) is often referred to as an autoimmune inflammatory demyelinating disease, neuromyelitis optica (NMO) is currently the only proven and well-characterized autoimmune disease affecting the glial cells. The target antigen is the water channel aquaporin-4 (AQP4), expressed on astrocytes, and antibodies against AQP4 (AQP4-IgG) are present in the serum of NMO patients. Clinical, serologic, cerebrospinal fluid, and neuroimaging criteria help differentiate NMO from other central nervous system inflammatory demyelinating disorders. Pathologically, the presence of dystrophic astrocytes, myelin vacuolation, granulocytic inflammatory infiltrates, vascular hyalinization, macrophages containing glial fibrillary acidic protein-positive debris and/or the absence of Creutzfeldt-Peters cells is more characteristic, but not specific, for NMO. These findings should prompt the neuropathologist to perform AQP4 immunohistochemistry, and recommend serologic testing for AQP4-IgG to exclude a diagnosis of NMO/NMO spectrum disorder (NMOSD). Loss of AQP4 on biopsied active demyelinating lesions and/or seropositivity for AQP4-IgG may confirm the diagnosis of NMO/NMOSD, which is important because treatments that are suitable for MS can aggravate NMO. Few other putative glial antigens have been postulated, but their pathogenic role remains to be demonstrated.

Soybean β-Conglycinin Prevents Age-Related Hearing Impairment.

Obesity-related complications are associated with the development of age-related hearing impairment. β-Conglycinin (β-CG), one of the main storage proteins in soy, offers multiple health benefits, including anti-obesity and anti-atherosclerotic effects. Here, to elucidate the potential therapeutic application of β-CG, we investigated the effect of β-CG on age-related hearing impairment. Male wild-type mice (age 6 months) were randomly divided into β-CG-fed and control groups. Six months later, the body weight was significantly lower in β-CG-fed mice than in the controls. Consumption of β-CG rescued the hearing impairment observed in control mice. Cochlear blood flow also increased in β-CG-fed mice, as did the expression of eNOS in the stria vascularis (SV), which protects vasculature. β-CG consumption also ameliorated oxidative status as assessed by 4-HNE staining. In the SV, lipofuscin granules of marginal cells and vacuolar degeneration of microvascular pericytes were decreased in β-CG-fed mice, as shown by transmission electron microscopy. β-CG consumption prevented loss of spiral ganglion cells and reduced the frequencies of lipofuscin granules, nuclear invaginations, and myelin vacuolation. Our observations indicate that β-CG ameliorates age-related hearing impairment by preserving cochlear blood flow and suppressing oxidative stress.

Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells in the hippocampal neurogenesis involving myelin vacuolation of cholinergic and glutamatergic inputs in mice.

Hexachlorophene (HCP) has been shown to induce myelin vacuolation due to intramyelinic edema of the nerve fibers in animal neural tissue. We investigated the maternal exposure effect of HCP on hippocampal neurogenesis in the offspring of pregnant mice supplemented with 0 (control), 33 or 100 ppm HCP in diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, offspring as examined in males exhibited decreased granule cell lineage populations expressing paired box 6, sex-determining region Y-box 2 and eomesodermin in the hippocampal subgranular zone (SGZ) accompanied by myelin vacuolation involving white matter tracts of the hippocampal fimbria at ≥ 33 ppm. However, SGZ cellular populations expressing brain lipid binding protein and doublecortin were unchanged at any dose. Transcript expression of cholinergic receptor genes, Chrna4 and Chrnb2, and glutamate receptor genes, Grm1 and Grin2d, examined at 100 ppm, decreased in the dentate gyrus. HCP exposure did not alter the number of proliferating or apoptotic cells in the SGZ, or reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)ergic interneurons in the dentate hilus, on PND 21 and PND 77. All neurogenesis-related changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77, suggesting that maternal HCP exposure at ≥ 33 ppm reversibly decreased type 2 intermediate-stage progenitor cells in the hippocampal neurogenesis. Myelin vacuolation might be responsible for changes in neurogenesis possibly by reducing nerve conduction velocity of cholinergic inputs from the septal-hippocampal pathway to granule cell lineages and/or GABAergic interneurons, and of glutamatergic inputs to granule cell lineages.

MLC1 protein: a likely link between leukodystrophies and brain channelopathies.

Megalencephalic leukoencephalopathy with subcortical cysts (MLCs) disease is a rare inherited, autosomal recessive form of childhood-onset spongiform leukodystrophy characterized by macrocephaly, deterioration of motor functions, epileptic seizures and mental decline. Brain edema, subcortical fluid cysts, myelin and astrocyte vacuolation are the histopathological hallmarks of MLC. Mutations in either the MLC1 gene (>75% of patients) or the GlialCAM gene (<20% of patients) are responsible for the disease. Recently, the GlialCAM adhesion protein was found essential for the membrane expression and function of the chloride channel ClC-2 indicating MLC disease caused by mutation in GlialCAM as the first channelopathy among leukodystrophies. On the contrary, the function of MLC1 protein, which binds GlialCAM, its functional relationship with ClC-2 and the molecular mechanisms underlying MLC1 mutation-induced functional defects are not fully understood yet. The human MLC1 gene encodes a 377-amino acid membrane protein with eight predicted transmembrane domains which shows very low homology with voltage-dependent potassium (K+) channel subunits. The high expression of MLC1 in brain astrocytes contacting blood vessels and meninges and brain alterations observed in MLC patients have led to hypothesize a role for MLC1 in the regulation of ion and water homeostasis. Recent studies have shown that MLC1 establishes structural and/or functional interactions with several ion/water channels and transporters and ion channel accessory proteins, and that these interactions are affected by MLC1 mutations causing MLC. Here, we review data on MLC1 functional properties obtained in in vitro and in vivo models and discuss evidence linking the effects of MLC1 mutations to brain channelopathies.

Vigabatrin-induced CNS changes in juvenile rats: Induction, progression and recovery of myelin-related changes

The purpose of this study was to expand on the knowledge previously published on the central nervous system effects of Vigabatrin in juvenile animals. By employing extended sectioning of the brain and by using four different tissue staining techniques it is demonstrated that oral administration of Vigabatrin to juvenile rats (treatment periods of post-natal day (PND) 4–7, 7–14 or 14–30) will cause histological CNS changes at dose levels of 15 and 50mg/kg/day, but not at a dose level of 5mg/kg/day.No evidence of neuronal degeneration or gliosis was seen at any stage of treatment. Consistent with previous reports microvacuolation, as well as effects on myelination and on oligodendrocytes were recorded. The present study expands on these findings and demonstrates that the variation in the location of the vigabatrin-induced lesions in the juvenile rat brain (both neuropil vacuolation and reduction of myelin) appears to be consistent with the process of myelination: In the youngest animals (PND 4–7) myelination occurs mainly in the hind brain (medulla oblongata and pons) where neuropil vacuolations is recorded. In animals dosed during PNDs 7–14 or during PNDs 14–30, the first changes were found in the thalamus. It seems likely that the earlier stages of myelination are more vulnerable to treatment related effects and the swollen oligodendrocytes seen as the initial change in the thalamus in animals treated during PNDs 4–7 and 7–14 represents an early stage in the development of the myelin lesion which is seen later as neuropil vacuolation.

Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells of the hippocampal neurogenesis in rat offspring via dysfunction of cholinergic inputs by myelin vacuolation.

Hexachlorophene (HCP) is known to induce myelin vacuolation corresponding to intramyelinic edema of nerve fibers in the central and peripheral nervous system in animals. This study investigated the effect of maternal exposure to HCP on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 100, or 300 ppm HCP in the diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, the numbers of T box brain 2(+) progenitor cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling(+) apoptotic cells in the hippocampal subgranular zone (SGZ) decreased in female offspring at 300 ppm, which was accompanied by myelin vacuolation and punctate tubulin beta-3 chain staining of nerve fibers in the hippocampal fimbria. In addition, transcript levels of the cholinergic receptor, nicotinic beta 2 (Chrnb2) and B-cell CLL/lymphoma 2 (Bcl2) decreased in the dentate gyrus. HCP-exposure did not alter the numbers of SGZ proliferating cells and reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)-ergic interneuron subpopulations in the dentate hilus on PND 21 and PND 77. Although some myelin vacuolation remained, all other changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77. These results suggest that maternal HCP exposure reversibly decreases type-2b intermediate-stage progenitor cells via the mitochondrial apoptotic pathway in offspring hippocampal neurogenesis at 300 ppm HCP. Neurogenesis may be affected by dysfunction of cholinergic inputs into granule cell lineages and/or GABAergic interneurons as indicated by decreased transcript levels of Chrnb2 and numbers of Chrnb2(+) interneurons caused by myelin vacuolation in the septal-hippocampal pathway.

Diagnostic utility of aquaporin-4 in the analysis of active demyelinating lesions.

To assess, in a surgical biopsy cohort of active demyelinating lesions, the diagnostic utility of aquaporin-4 (AQP4) immunohistochemistry in identifying neuromyelitis optica (NMO) or NMO spectrum disorder (NMOSD) and describe pathologic features that should prompt AQP4 immunohistochemical analysis and AQP4-immunoglobulin G (IgG) serologic testing. This was a neuropathologic cohort study of 20 surgical biopsies (19 patients; 11 cord/9 brain), performed because of diagnostic uncertainty, interpreted as active demyelinating disease and containing 2 or more of the following additional features: tissue vacuolation, granulocytic infiltrates, or astrocyte injury. AQP4 immunoreactivity was lost in 18 biopsies and increased in 2. Immunopathologic features of the AQP4 loss cohort were myelin vacuolation (18), dystrophic astrocytes and granulocytes (17), vascular hyalinization (16), macrophages containing glial fibrillary acid protein (GFAP)-positive debris (14), and Creutzfeldt-Peters cells (0). All 14 cases with available serum tested positive for AQP4-IgG after biopsy. Diagnosis at last follow-up was NMO/NMOSD (15) and longitudinally extensive transverse myelitis (1 each relapsing and single). Immunopathologic features of the AQP4 increased cohort were macrophages containing GFAP-positive debris and granulocytes (2), myelin vacuolation (1), dystrophic astrocytes (1), Creutzfeldt-Peters cells (1), and vascular hyalinization (1). Diagnosis at last follow-up was multiple sclerosis (MS) and both tested AQP4-IgG seronegative after biopsy. AQP4 immunohistochemistry with subsequent AQP4-IgG testing has diagnostic utility in identifying cases of NMO/NMOSD. This study highlights the importance of considering NMOSD in the differential diagnosis of tumefactive brain or spinal cord lesions. AQP4-IgG testing may avert biopsy and avoid ineffective therapies if these patients are erroneously treated for MS.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 regulates glial surface localization of GLIALCAM from fish to humans.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction

Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.