Pelizaeus-Merzbacher disease: on the cusp of myelin medicine.

Pelizaeus-Merzbacher disease (PMD) is caused by mutations in the proteolipid protein 1 (PLP1) gene encoding proteolipid protein (PLP). As a major component of myelin, mutated PLP causes progressive neurodegeneration and eventually death due to severe white matter deficits. Medical care has long been limited to symptomatic treatments, but first-in-class PMD therapies with novel mechanisms now stand poised to enter clinical trials. Here, we review PMD disease mechanisms and outline rationale for therapeutic interventions, including PLP1 suppression, cell transplantation, iron chelation, and intracellular stress modulation. We discuss available preclinical data and their implications on clinical development. With several novel treatments on the horizon, PMD is on the precipice of a new era in the diagnosis and treatment of patients suffering from this debilitating disease.

A novel non-human primate model of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disorder of the central nervous system (CNS) linked to mutations in the proteolipid protein-1 (PLP1) gene. Although there are multiple animal models of PMD, few of them fully mimic the human disease. Here, we report three spontaneous cases of male neonatal rhesus macaques with the clinical symptoms of hypomyelinating disease, including intention tremors, progressively worsening motor dysfunction, and nystagmus. These animals demonstrated a paucity of CNS myelination accompanied by reactive astrogliosis, and a lack of PLP1 expression throughout white matter. Genetic analysis revealed that these animals were related to one another and that their parents carried a rare, hemizygous missense variant in exon 5 of the PLP1 gene. These animals therefore represent the first reported non-human primate model of PMD, providing a novel and valuable opportunity for preclinical studies that aim to promote myelination in pediatric hypomyelinating diseases.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90ß (Hsp90ß) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90ß-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Pathology of the neurovascular unit in leukodystrophies.

The blood-brain barrier is a dynamic endothelial cell barrier in the brain microvasculature that separates the blood from the brain parenchyma. Specialized brain endothelial cells, astrocytes, neurons, microglia and pericytes together compose the neurovascular unit and interact to maintain blood-brain barrier function. A disturbed brain barrier function is reported in most common neurological disorders and may play a role in disease pathogenesis. However, a comprehensive overview of how the neurovascular unit is affected in a wide range of rare disorders is lacking. Our aim was to provide further insights into the neuropathology of the neurovascular unit in leukodystrophies to unravel its potential pathogenic role in these diseases. Leukodystrophies are monogenic disorders of the white matter due to defects in any of its structural components. Single leukodystrophies are exceedingly rare, and availability of human tissue is unique. Expression of selective neurovascular unit markers such as claudin-5, zona occludens 1, laminin, PDGFRß, aquaporin-4 and α-dystroglycan was investigated in eight different leukodystrophies using immunohistochemistry. We observed tight junction rearrangements, indicative of endothelial dysfunction, in five out of eight assessed leukodystrophies of different origin and an altered aquaporin-4 distribution in all. Aquaporin-4 redistribution indicates a general astrocytic dysfunction in leukodystrophies, even in those not directly related to astrocytic pathology or without prominent reactive astrogliosis. These findings provide further evidence for dysfunction in the orchestration of the neurovascular unit in leukodystrophies and contribute to a better understanding of the underlying disease mechanism.

Spinal cord involvement and paroxysmal events in "Infantile Onset Transient Hypomyelination" due to TMEM63A mutation.

Monoallelic mutations on TMEM63A have been recently reported as cause of a previously unrecognized disorder named "infantile-onset transient hypomyelination". Clinical and neuroradiological presentation is described as highly similar to Pelizaeus-Merzbacher Disease but evolution over time was surprisingly benign with a progressive spontaneous improving course. We report on a new TMEM63A-mutated girl. The clinical picture was similar to the one already described except for the presence of recurrent episodes of unilateral eyelid twitching, and for the evidence of spinal cord involvement on MRI. These are interesting findings helping in distinguishing this condition from classic PMD since early disease stages. However, additional observations are needed to confirm if these are common features of this condition.

One-step Reprogramming of Human Fibroblasts into Oligodendrocyte-like Cells by SOX10, OLIG2, and NKX6.2.

Limited access to human oligodendrocytes impairs better understanding of oligodendrocyte pathology in myelin diseases. Here, we describe a method to robustly convert human fibroblasts directly into oligodendrocyte-like cells (dc-hiOLs), which allows evaluation of remyelination-promoting compounds and disease modeling. Ectopic expression of SOX10, OLIG2, and NKX6.2 in human fibroblasts results in rapid generation of O4+ cells, which further differentiate into MBP+ mature oligodendrocyte-like cells within 16 days. dc-hiOLs undergo chromatin remodeling to express oligodendrocyte markers, ensheath axons, and nanofibers in vitro, respond to promyelination compound treatment, and recapitulate in vitro oligodendroglial pathologies associated with Pelizaeus-Merzbacher leukodystrophy related to PLP1 mutations. Furthermore, DNA methylome analysis provides evidence that the CpG methylation pattern significantly differs between dc-hiOLs derived from fibroblasts of young and old donors, indicating the maintenance of the source cells' "age." In summary, dc-hiOLs represent a reproducible technology that could contribute to personalized medicine in the field of myelin diseases.

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next-generation sequencing.

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A-related leukodystrophy, Peroxisomal biogenesis disorders, POLR3-related Leukodystrophy, Vanishing White Matter, and Pelizaeus-Merzbacher Disease. Despite the relative frequency of these conditions, carrier-screening laboratories regularly test only 20 of the 55 leukodystrophy-related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.

Neural stem cells restore myelin in a demyelinating model of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease is a fatal X-linked leukodystrophy caused by mutations in the PLP1 gene, which is expressed in the CNS by oligodendrocytes. Disease onset, symptoms and mortality span a broad spectrum depending on the nature of the mutation and thus the degree of CNS hypomyelination. In the absence of an effective treatment, direct cell transplantation into the CNS to restore myelin has been tested in animal models of severe forms of the disease with failure of developmental myelination, and more recently, in severely affected patients with early disease onset due to point mutations in the PLP1 gene, and absence of myelin by MRI. In patients with a PLP1 duplication mutation, the most common cause of Pelizaeus-Merzbacher disease, the pathology is poorly defined because of a paucity of autopsy material. To address this, we examined two elderly patients with duplication of PLP1 in whom the overall syndrome, including end-stage pathology, indicated a complex disease involving dysmyelination, demyelination and axonal degeneration. Using the corresponding Plp1 transgenic mouse model, we then tested the capacity of transplanted neural stem cells to restore myelin in the context of PLP overexpression. Although developmental myelination and axonal coverage by endogenous oligodendrocytes was extensive, as assessed using electron microscopy (n = 3 at each of four end points) and immunostaining (n = 3 at each of four end points), wild-type neural precursors, transplanted into the brains of the newborn mutants, were able to effectively compete and replace the defective myelin (n = 2 at each of four end points). These data demonstrate the potential of neural stem cell therapies to restore normal myelination and protect axons in patients with PLP1 gene duplication mutation and further, provide proof of principle for the benefits of stem cell transplantation for other fatal leukodystrophies with 'normal' developmental myelination.

Development and Optimization of a High-Content Analysis Platform to Identify Suppressors of Lamin B1 Overexpression as a Therapeutic Strategy for Autosomal Dominant Leukodystrophy.

Autosomal dominant leukodystrophy (ADLD) is a fatal, progressive adult-onset disease characterized by widespread central nervous system (CNS) demyelination and significant morbidity. The late age of onset together with the relatively slow disease progression provides a large therapeutic window for the disorder. However, no treatment exists for ADLD, representing an urgent and unmet clinical need. We have previously shown that ADLD is caused by duplications of the lamin B1 gene causing increased expression of the lamin B1 protein, a major constituent of the nuclear lamina, and demonstrated that transgenic mice with oligodendrocyte-specific overexpression of lamin B1 exhibit temporal and histopathological features reminiscent of the human disease. As increased levels of lamin B1 are the causative event triggering ADLD, approaches aimed at reducing lamin B1 levels and associated functional consequences represent a promising strategy for discovery of small-molecule ADLD therapeutics. To this end, we have created an inducible cell culture model of lamin B1 overexpression and developed high-content analysis in connection with multivariate analysis to define, analyze, and quantify lamin B1 expression and its associated abnormal nuclear phenotype in mouse embryonic fibroblasts (MEFs). The assay has been optimized to meet high-throughput screening (HTS) criteria in multiday variability studies. To control for batch-to-batch variation in the primary MEFs, we have implemented a screening strategy that employs sentinel cells to avoid costly losses during HTS. We posit the assay will identify bona fide suppressors of lamin B1 pathophysiology as candidates for development into potential therapies for ADLD.

CNP deficiency causes severe hypomyelinating leukodystrophy in humans.

Myelin pathologies are an important cause of multifactorial, e.g., multiple sclerosis, and Mendelian, e.g., leukodystrophy, neurological disorders. CNP encodes a major component of myelin and its CNS expression is exclusive to myelin-forming oligodendrocytes. Deficiency of CNP in mouse causes a lethal white matter neurodegenerative phenotype. However, a corresponding human phenotype has not been described to date. Here, we describe a multiplex consanguineous family from Oman in which multiple affected members display a remarkably consistent phenotype of neuroregression with profound brain white matter loss. A novel homozygous missense variant in CNP was identified by combined autozygome/exome analysis. Immunoblot analysis suggests that this is a null allele in patient fibroblasts, which display abnormal F-actin organization. Our results suggest the establishment of a novel CNP-related hypomyelinating leukodystrophy in humans.

Oligodendrocyte Death in Pelizaeus-Merzbacher Disease Is Rescued by Iron Chelation.

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

Long-Term Safety, Immunologic Response, and Imaging Outcomes following Neural Stem Cell Transplantation for Pelizaeus-Merzbacher Disease.

Four boys with Pelizaeus-Merzbacher disease, an X-linked leukodystrophy, underwent transplantation with human allogeneic central nervous system stem cells (HuCNS-SC). Subsequently, all subjects were followed for an additional 4 years in this separate follow-up study to evaluate safety, neurologic function, magnetic resonance imaging (MRI) data, and immunologic response. The neurosurgical procedure, immunosuppression, and HuCNS-SC transplantation were well tolerated and all four subjects were alive at the conclusion of the study period. At year 2, all subjects exhibited diffusion MRI changes at the implantation sites as well as in more distant brain regions. There were persistent, increased signal changes in the three patients who were studied up to year 5. Two of four subjects developed donor-specific HLA alloantibodies, demonstrating that neural stem cells can elicit an immune response when injected into the CNS, and suggesting the importance of monitoring immunologic parameters and identifying markers of engraftment in future studies.

Elucidation of the pathogenic mechanism and potential treatment strategy for a female patient with spastic paraplegia derived from a single-nucleotide deletion in PLP1.

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder caused by abnormalities in the gene PLP1. Most females harboring heterozygous PLP1 abnormalities are basically asymptomatic. However, as a result of abnormal patterns of X-chromosome inactivation, it is possible for some female carriers to be symptomatic. Whole-exome sequencing of a female patient with unknown spastic paraplegia was performed to obtain a molecular diagnosis. As a result, a de novo heterozygous single-nucleotide deletion in PLP1 [NM_000533.5(PLP1_v001):c.783del; p.Thr262Leufs*20] was identified. RNA sequencing was performed in a patient-derived lymphoblastoid cell line, confirming mono-allelic expression of the mutated allele and abnormal inactivation of the wild-type allele. The patient-derived lymphoblastoid cell line was then treated with VX680 or 5azadC, which resulted in restored expression of the wild-type allele. These two agents thus have the potential to reverse inappropriately-skewed inactivation of the X-chromosome.

Ketogenic diet ameliorates axonal defects and promotes myelination in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is an untreatable and fatal leukodystrophy. In a model of PMD with perturbed blood-brain barrier integrity, cholesterol supplementation promotes myelin membrane growth. Here, we show that in contrast to the mouse model, dietary cholesterol in two PMD patients did not lead to a major advancement of hypomyelination, potentially because the intact blood-brain barrier precludes its entry into the CNS. We therefore turned to a PMD mouse model with preserved blood-brain barrier integrity and show that a high-fat/low-carbohydrate ketogenic diet restored oligodendrocyte integrity and increased CNS myelination. This dietary intervention also ameliorated axonal degeneration and normalized motor functions. Moreover, in a paradigm of adult remyelination, ketogenic diet facilitated repair and attenuated axon damage. We suggest that a therapy with lipids such as ketone bodies, that readily enter the brain, can circumvent the requirement of a disrupted blood-brain barrier in the treatment of myelin disease.

Glucose metabolism in the brain in LMNB1-related autosomal dominant leukodystrophy.

OBJECTIVE: LMNB1-related autosomal dominant leukodystrophy is caused by an overexpression of the protein lamin B1, usually due to a duplication of the LMNB1 gene. Symptoms start in 5th to 6th decade. This slowly progressive disease terminates with death. We studied brain glucose metabolism in this disease using 18 F-fluorodeoxyglucose positron emission tomography (PET). METHODS: We examined 8 patients, aged 48-64 years, in varying stages of clinical symptomatology. Two patients were investigated with quantitative PET on clinical indications after which six more patients were recruited. Absolute glucose metabolism was analyzed with the PVElab software in 6 patients and 18 healthy controls. A semiquantitative analysis using the CortexID software was performed in seven investigations, relating local metabolism levels to global glucose metabolism. RESULTS: The clinical quantitative PET revealed low global glucose metabolism, with the most marked reduction in the cerebellum. In the PVElab analysis, patients presented low mean glucose metabolism in the cerebellum, brainstem and global grey matter. In the semiquantitative analysis, 2 patients showed a decreased metabolism in the cerebellum and 4 patients a relatively higher metabolism in parts of the temporal lobes. Since none of the patients showed an increased metabolism in the quantitative analysis, we interpret these increases as "pseudo-increases" related to a globally reduced metabolism. CONCLUSIONS: Global reduction of grey matter glucose metabolism in this white matter disease most likely depends on a combination of cortical afferent dysfunction and, in later stages, neuronal loss. The lowest metabolism in the cerebellum is consistent with histopathological findings and prominent cerebellar symptoms.

Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease.

Pelizaeus-Merzbacher disease (PMD) is a fatal X-linked disorder caused by loss of myelinating oligodendrocytes and consequent hypomyelination. The underlying cellular and molecular dysfunctions are not fully defined, but therapeutic enhancement of oligodendrocyte survival could restore functional myelination in patients. Here we generated pure, scalable quantities of induced pluripotent stem cell-derived oligodendrocyte progenitor cells (OPCs) from a severe mouse model of PMD, Plp1jimpy. Temporal phenotypic and transcriptomic studies defined an early pathological window characterized by endoplasmic reticulum (ER) stress and cell death as OPCs exit their progenitor state. High-throughput phenotypic screening identified a compound, Ro 25-6981, which modulates the ER stress response and rescues mutant oligodendrocyte survival in jimpy, in vitro and in vivo, and in human PMD oligocortical spheroids. Surprisingly, increasing oligodendrocyte survival did not restore subsequent myelination, revealing a second pathological phase. Collectively, our work shows that PMD oligodendrocyte loss can be rescued pharmacologically and defines a need for multifactorial intervention to restore myelination.

Neurogenetics of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is an X-linked disorder caused by mutations in the PLP1 gene, which encodes the proteolipid protein of myelinating oligodendroglia. PMD exhibits phenotypic variability that reflects its considerable genotypic heterogeneity, but all forms of the disease result in central hypomyelination associated with early neurologic dysfunction, progressive deterioration, and ultimately death. PMD has been classified into three major subtypes, according to the age of presentation: connatal PMD, classic PMD, and transitional PMD, combining features of both connatal and classic forms. Two other less severe phenotypes were subsequently described, including the spastic paraplegia syndrome and PLP1-null disease. These disorders may be associated with duplications, as well as with point, missense, and null mutations within the PLP1 gene. A number of clinically similar Pelizaeus-Merzbacher-like disorders (PMLD) are considered in the differential diagnosis of PMD, the most prominent of which is PMLD-1, caused by misexpression of the GJC2 gene encoding connexin-47. No effective therapy for PMD exists. Yet, as a relatively pure central nervous system hypomyelinating disorder, with limited involvement of the peripheral nervous system and little attendant neuronal pathology, PMD is an attractive therapeutic target for neural stem cell and glial progenitor cell transplantation, efforts at which are now underway in a number of centers internationally.

Oxidative stress and mitochondrial dynamics malfunction are linked in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a fatal hypomyelinating disorder characterized by early impairment of motor development, nystagmus, choreoathetotic movements, ataxia and progressive spasticity. PMD is caused by variations in the proteolipid protein gene PLP1, which encodes the two major myelin proteins of the central nervous system, PLP and its spliced isoform DM20, in oligodendrocytes. Large duplications including the entire PLP1 gene are the most frequent causative mutation leading to the classical form of PMD. The Plp1 overexpressing mouse model (PLP-tg66/66 ) develops a phenotype very similar to human PMD, with early and severe motor dysfunction and a dramatic decrease in lifespan. The sequence of cellular events that cause neurodegeneration and ultimately death is poorly understood. In this work, we analyzed patient-derived fibroblasts and spinal cords of the PLP-tg66/66 mouse model, and identified redox imbalance, with altered antioxidant defense and oxidative damage to several enzymes involved in ATP production, such as glycolytic enzymes, creatine kinase and mitochondrial proteins from the Krebs cycle and oxidative phosphorylation. We also evidenced malfunction of the mitochondria compartment with increased ROS production and depolarization in PMD patient's fibroblasts, which was prevented by the antioxidant N-acetyl-cysteine. Finally, we uncovered an impairment of mitochondrial dynamics in patient's fibroblasts which may help explain the ultrastructural abnormalities of mitochondria morphology detected in spinal cords from PLP-tg66/66 mice. Altogether, these results underscore the link between redox and metabolic homeostasis in myelin diseases, provide insight into the pathophysiology of PMD, and may bear implications for tailored pharmacological intervention.

Modeling the Mutational and Phenotypic Landscapes of Pelizaeus-Merzbacher Disease with Human iPSC-Derived Oligodendrocytes.

Pelizaeus-Merzbacher disease (PMD) is a pediatric disease of myelin in the central nervous system and manifests with a wide spectrum of clinical severities. Although PMD is a rare monogenic disease, hundreds of mutations in the X-linked myelin gene proteolipid protein 1 (PLP1) have been identified in humans. Attempts to identify a common pathogenic process underlying PMD have been complicated by an incomplete understanding of PLP1 dysfunction and limited access to primary human oligodendrocytes. To address this, we generated panels of human induced pluripotent stem cells (hiPSCs) and hiPSC-derived oligodendrocytes from 12 individuals with mutations spanning the genetic and clinical diversity of PMD-including point mutations and duplication, triplication, and deletion of PLP1-and developed an in vitro platform for molecular and cellular characterization of all 12 mutations simultaneously. We identified individual and shared defects in PLP1 mRNA expression and splicing, oligodendrocyte progenitor development, and oligodendrocyte morphology and capacity for myelination. These observations enabled classification of PMD subgroups by cell-intrinsic phenotypes and identified a subset of mutations for targeted testing of small-molecule modulators of the endoplasmic reticulum stress response, which improved both morphologic and myelination defects. Collectively, these data provide insights into the pathogeneses of a variety of PLP1 mutations and suggest that disparate etiologies of PMD could require specific treatment approaches for subsets of individuals. More broadly, this study demonstrates the versatility of a hiPSC-based panel spanning the mutational heterogeneity within a single disease and establishes a widely applicable platform for genotype-phenotype correlation and drug screening in any human myelin disorder.