Generation of Pelizaeus-Merzbacher disease (PMD) mutant (PLP1-C33Y) in induced pluripotent stem cell (iPSC) by CRISPR/Cas9 genome editing.

Genetic alterations in the PLP1 gene, i.e. point mutations and duplications, are associated with demyelinating disease Pelizaeus-Merzbacher. Here, we describe the generation of a human iPSC line harboring a PLP1 variant in codon 33 which leads to an amino acid change from cysteine to tyrosine. The established PLP1C33Y iPSC line enables the study of PMD pathophysiology by investigating various cell types and -characteristics in our developed protocol for bioengineered neuronal organoids (BENOs)1.

Generation of functional human oligodendrocytes from dermal fibroblasts by direct lineage conversion.

Oligodendrocytes, the myelinating cells of the central nervous system, possess great potential for disease modeling and cell transplantation-based therapies for leukodystrophies. However, caveats to oligodendrocyte differentiation protocols ( Ehrlich et al., 2017; Wang et al., 2013; Douvaras and Fossati, 2015) from human embryonic stem and induced pluripotent stem cells (iPSCs), which include slow and inefficient differentiation, and tumorigenic potential of contaminating undifferentiated pluripotent cells, are major bottlenecks towards their translational utility. Here, we report the rapid generation of human oligodendrocytes by direct lineage conversion of human dermal fibroblasts (HDFs). We show that the combination of the four transcription factors OLIG2, SOX10, ASCL1 and NKX2.2 is sufficient to convert HDFs to induced oligodendrocyte precursor cells (iOPCs). iOPCs resemble human primary and iPSC-derived OPCs based on morphology and transcriptomic analysis. Importantly, iOPCs can differentiate into mature myelinating oligodendrocytes in vitro and in vivo. Finally, iOPCs derived from patients with Pelizaeus Merzbacher disease, a hypomyelinating leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene, showed increased cell death compared with iOPCs from healthy donors. Thus, human iOPCs generated by direct lineage conversion represent an attractive new source for human cell-based disease models and potentially myelinating cell grafts.

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.

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.

Gene suppressing therapy for Pelizaeus-Merzbacher disease using artificial microRNA.

Copy number increase or decrease of certain dosage-sensitive genes may cause genetic diseases with distinct phenotypes, conceptually termed genomic disorders. The most common cause of Pelizaeus-Merzbacher disease (PMD), an X-linked hypomyelinating leukodystrophy, is genomic duplication encompassing the entire proteolipid protein 1 (PLP1) gene. Although the exact molecular and cellular mechanisms underlying PLP1 duplication, which causes severe hypomyelination in the central nervous system, remain largely elusive, PLP1 overexpression is likely the fundamental cause of this devastating disease. Here, we investigated if adeno-associated virus-mediated (AAV-mediated) gene-specific suppression may serve as a potential cure for PMD by correcting quantitative aberrations in gene products. We developed an oligodendrocyte-specific Plp1 gene suppression therapy using artificial microRNA under the control of human CNP promoter in a self-complementary AAV (scAAV) platform. A single direct brain injection achieved widespread oligodendrocyte-specific Plp1 suppression in the white matter of WT mice. AAV treatment in Plp1-transgenic mice, a PLP1 duplication model, ameliorated cytoplasmic accumulation of Plp1, preserved mature oligodendrocytes from degradation, restored myelin structure and gene expression, and improved survival and neurological phenotypes. Together, our results provide evidence that AAV-mediated gene suppression therapy can serve as a potential cure for PMD resulting from PLP1 duplication and possibly for other genomic disorders.

Pathogenic variants in AIMP1 cause pontocerebellar hypoplasia.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) is a non-catalytic component of the multi-tRNA synthetase complex which catalyzes the ligation of amino acids to the correct tRNAs. Pathogenic variants in several aminoacyl-tRNA synthetases genes have been linked to various neurological disorders, including leukodystrophies and pontocerebellar hypoplasias (PCH). To date, loss-of-function variants in AIMP1 have been associated with hypomyelinating leukodystrophy-3 (MIM 260600). Here, we report a novel frameshift AIMP1 homozygous variant (c.160delA,p.Lys54Asnfs) in a child with pontocerebellar hypoplasia and simplified gyral pattern, a phenotype not been previously described with AIMP1 variants, thus expanding the phenotypic spectrum. AIMP1 should be included in diagnostic PCH gene panels.

Clinical and mutational spectrum of Colombian patients with Pelizaeus Merzbacher Disease / Heterogeneidad clínica y mutacional en pacientes colombianos con Pelizaeus Merzbacher

Abstract Case Presentation: Pelizaeus Merzbacher Disease (PMD) is an X-linked developmental defect of myelination that causes childhood chronic spastic encephalopathy. Its genetic etiology can be either a duplication (or other gene dosage alterations) or a punctual mutation at the PLP1 locus. Clinically, it presents with developmental delay, nystagmus and, spasticity, supported by neuroimaging in which the defect of myelination is evident. We present a series of seven Colombian patients diagnosed with this leucodystrophy, describing their genotypic and phenotypic characteristics and heterogeneity. Clinical Findings: All patients included were male, 6 months to 16 years of age. Mean age at onset of symptoms was 8 months. Mean age at diagnosis was 5 years 5 months, being classic PMD most frequently diagnosed, as compared to the connatal phenotype. All cases had a primary diagnosis of developmental delay on 100%, and in 28.7% of cases, early onset nystagmus was described. 85.7% of patients had spasticity, 71.4% cerebellar signs, 57.0% hypotonia, and 28.5% had an abnormal movement disorder. Only three patients were able to achieve gait, though altered. In the two patients who had a diagnosis of connatal PMD maturational ages in danger zones according to the WHO Abbreviated Scale of Psychosocial Development were documented. All cases had abnormalities in neuroimages. Molecular Analysis and Results: Molecular studies were used in the majority of the cases to confirm the diagnosis (85.7 %). For two cases molecular confirmation was not considered necessary given their affected male brothers had already been tested. PLP1 gene dosage alterations (duplications) were found in 28.5 % of the patients (two siblings), whereas three different single nucleotide variants were detected. Clinical Relevance: According to these findings, as authors we propose the diagnostic algorithm in Colombian population to begin on a high clinical suspicion, followed by paraclinical extension, moving on to the molecular confirmation by using approaches to simultaneously sequence the PLP1 gene in order to detect point mutations and in/dels and performing a copy number variation analysis for the detection of gene dosage alterations.

Resumen Descripción del caso: La enfermedad de Pelizaeus Merzbacher es una leucodistrofia ligada al X que causa encefalopatía espástica crónica en la infancia. Su etiología es genética, por duplicaciones u otros trastornos de la dosis génica o mutaciones puntuales del gen PLP1, lo que condiciona la formación anormal de las vainas de mielina principalmente en el sistema nervioso central. Clínicamente se caracteriza por un cuadro de retardo del neurodesarrollo, nistagmus y espasticidad, con neuroimágenes que evidencian la dismielinización. Presentamos una serie de siete casos colombianos con esta leucodistrofica en la que describimos fenotípica y genotípicamente la heterogeneidad de esta enfermedad en nuestra población. Hallazgos clínicos: Todos los pacientes analizados fueron de sexo masculino, con edad promedio de inicio de síntomas hacia los ocho meses de vida. La edad media al diagnóstico fue de 5 años 5 meses, siendo más frecuente el diagnóstico de PMD clásica que el tipo connatal. Se describe retardo del desarrollo motor en el 100% de los casos, acompañado de nistagmus en el 28.7%. 85.7% de los casos tenía algún grado de espasticidad, 71.4 % signos cerebelosos, 57.0% hipotonía, y hasta en 28.5% se evidenciaron movimientos anormales. Solo tres pacientes lograron marcha, aunque patológica. En los dos pacientes con la forma connatal se documentó una edad maduracional motora en el rango de Alerta, de acuerdo a la escala abreviada del desarrollo de la OMS. En todos los casos se detectó algún tipo de anormalidad en el estudio imagenológico cerebral. Estudios Moleculares y Resultados: El diagnóstico molecular se empleó en la mayoría de los casos (85.7%), encontrando alteraciones en la dosis génica en el 28.5% y tres diferentes mutaciones puntuales. Relevancia clínica: Dados los hallazgos en los resultados del estudio molecular, sugerimos que en el abordaje diagnóstico confirmatorio para la población colombiana se debería contemplar en un mismo tiempo tanto la secuenciación como el estudio de variantes del número de copias del gen afectado, contrario a lo sugerido en literatura mundial en la que se inicia con estudio para duplicación / deleción.

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.

Hypomyelinating leukodystrophy associated with a deleterious mutation in the ATRN gene.

Hypomyelinating leukodystrophies are a group of neurodevelopmental disorders that affect proper formation of the myelin sheath in the central nervous system. They are characterized by developmental delay, hypotonia, spasticity, and variable intellectual disability. We used whole exome analysis to study the molecular basis of hypomyelinating leukodystrophy in two sibs from a consanguineous family. A homozygous mutation, c.3068+5G>A, was identified in the ATRN gene, with the consequent insertion of an intronic sequence into the patients' cDNA and a predicted premature termination of the ATRN polypeptide. ATRN encodes Attractin, which was previously shown to play a critical role in central myelination. Several spontaneous ATRN rodent mutants exhibited impaired myelination which was attributed to oxidative stress and accelerated apoptosis. ATRN can now be added to the growing list of genes associated with hypomyelinating leukodystrophy. The disease seems to be confined to the CNS; however, given the young age of our patients, longer follow-up may be required.

A longitudinal study of a family with adult-onset autosomal dominant leukodystrophy: Clinical, autonomic and neuropsychological findings.

BACKGROUND AND PURPOSE: Adult-onset autosomal dominant leukodystrophy (ADLD) is a rare progressive neurological disorder caused by Lamin B1 duplication (LMNB1). Our aim was to investigate longitudinally the pattern of the autonomic dysfunction and the degree of neuropsychological involvement. METHODS: Three related ADLD patients and one asymptomatic carrier of LMNB1 duplication underwent a standardized evaluation of autonomic nervous system, including cardiovascular reflexes, pharmacological testing, microneurography, skin biopsy, Metaiodobenzylguanidine scintigraphy and a complete neuropsychological battery. RESULTS: An early neurogenic orthostatic hypotension was detected in all patients and confirmed by a low rise in noradrenaline levels on Tilt Test. However infusion of noradrenaline resulted in normal blood pressure rise as well as the infusion of clonidine. At the insulin tolerance test the increase in adrenaline resulted pathological in two out three patients. Microneurography failed to detect muscle sympathetic nerve activity bursts. Skin biopsy revealed a poor adrenergic innervation, while cardiac sympathetic nerves were normal. None of ADLD patients showed a global cognitive deficit but a selective impairment in the executive functions. CONCLUSION: Autonomic disorder in ADLD involves selectively the postganglionic sympathetic system including the sympatho-adrenal response. Cognitive involvement consisting in an early impairment of executive tasks that might precede brain MR abnormalities.

Myelin-associated glycoprotein gene mutation causes Pelizaeus-Merzbacher disease-like disorder.

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by mutations or rearrangements in PLP1. It presents in infancy with nystagmus, jerky head movements, hypotonia and developmental delay evolving into spastic tetraplegia with optic atrophy and variable movement disorders. A clinically similar phenotype caused by recessive mutations in GJC2 is known as Pelizaeus-Merzbacher-like disease. Both genes encode proteins associated with myelin. We describe three siblings of a consanguineous family manifesting the typical infantile-onset Pelizaeus-Merzbacher disease-like phenotype slowly evolving into a form of complicated hereditary spastic paraplegia with mental retardation, dysarthria, optic atrophy and peripheral neuropathy in adulthood. Magnetic resonance imaging and spectroscopy were consistent with a demyelinating leukodystrophy. Using genetic linkage and exome sequencing, we identified a homozygous missense c.399C>G; p.S133R mutation in MAG. This gene, previously associated with hereditary spastic paraplegia, encodes myelin-associated glycoprotein, which is involved in myelin maintenance and glia-axon interaction. This mutation is predicted to destabilize the protein and affect its tertiary structure. Examination of the sural nerve biopsy sample obtained in childhood in the oldest sibling revealed complete absence of myelin-associated glycoprotein accompanied by ill-formed onion-bulb structures and a relatively thin myelin sheath of the affected axons. Immunofluorescence, cell surface labelling, biochemical analysis and mass spectrometry-based proteomics studies in a variety of cell types demonstrated a devastating effect of the mutation on post-translational processing, steady state expression and subcellular localization of myelin-associated glycoprotein. In contrast to the wild-type protein, the p.S133R mutant was retained in the endoplasmic reticulum and was subjected to endoplasmic reticulum-associated protein degradation by the proteasome. Our findings identify involvement of myelin-associated glycoprotein in this family with a disorder affecting the central and peripheral nervous system, and suggest that loss of the protein function is responsible for the unique clinical phenotype.

Messenger RNA processing is altered in autosomal dominant leukodystrophy.

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.

Plp1 gene duplication inhibits airway responsiveness and induces lung inflammation.

Mice with Plp1 gene duplication model the most common form of Pelizaeus-Merzbacher disease (PMD), a CNS disease in which patients may suffer respiratory complications. We hypothesized that affected mice would lack airway responsiveness compared to wild-type and carrier mice during methacholine challenge. Wild-type (n = 10), carrier female (n = 6) and affected male (n = 8) mice were anesthetized-paralyzed, tracheostomized and ventilated. Respiratory mechanics were recorded at baseline and during escalating doses of nebulized methacholine followed by albuterol. Lung resistance (RL) was the primary endpoint. Lung tissues were assayed for inflammatory and histological differences. At baseline, phase angles were higher in carrier and affected mice than wild-type. Dose-response RL curves in affected and carrier mice indicated a lack of methacholine response. Albuterol reduced RL in wild-type and carrier, but not affected mice. Affected mice exhibited lower interleukin (IL)-6 tissue levels and alveolar inflammatory infiltrates. Affected and carrier mice, compared to wild-type, lacked airway reactivity during methacholine challenge, but only affected mice exhibited decreased lung tissue levels of IL-6 and inflammation.

Involvement of ER stress in dysmyelination of Pelizaeus-Merzbacher Disease with PLP1 missense mutations shown by iPSC-derived oligodendrocytes.

Pelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation.

Rearrangement structure-independent strategy of CNV breakpoint analysis.

Rare copy number variations (CNVs) generated by human genomic rearrangements have been shown to play an important role in pathogenesis of human diseases and cancers. CNV breakpoint analysis can help define genomic location, genetic content and sequence structure of pathogenic CNVs. This process is vital to elucidate CNV mutational mechanism and etiology of CNV-associated disorders. However, it is technically challenging to map CNV breakpoints at base-pair level, especially in the genomic regions with sequence complexity. In this study, we developed a new method of capture and breakpoint approaching sequencing (CBAS) to efficiently obtain CNV breakpoint sequences. This strategy is independent of CNV structures and applicable to various CNV types. As was demonstrated in CNV-associated patients with neurological disorders, CBAS achieved fine mapping of breakpoint sequences for compound deletion, complex duplication, and translocation. Intriguingly, CBAS also revealed unexpected CNV complexity involving long-range DNA rearrangement. Our observations showed that CBAS is an efficient method for obtaining CNV breakpoint sequence and mapping insertional events as well. This method can facilitate the researches on CNV-associated human diseases and cancers. CBAS is also applicable to mapping the integration sites of retrovirus (such as HIV) and transgenes in model organisms.

Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases.

Alternative splicing is an important regulator of the transcriptome. However, mutations may cause alteration of splicing patterns, which in turn leads to disease. During the past 10 years, exon skipping has been looked upon as a powerful tool for correction of missplicing in disease and progress has been made towards clinical trials. In this review, we discuss the use of antisense oligonucleotides to correct splicing defects through exon skipping, with a special focus on diseases affecting the nervous system, and the latest stage achieved in its progress.

Insertion of proteolipid protein into oligodendrocyte mitochondria regulates extracellular pH and adenosine triphosphate.

Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 PLP gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases adenosine triphosphate (ATP) in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and proinflammatory molecules are upregulated in Plp1 transgenic mice (Tatar et al. (2010) ASN Neuro 2:art:e00043). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients.

Lamin B1 fluctuations have differential effects on cellular proliferation and senescence.

The nuclear lamina consists of A- and B-type lamins. Mutations in LMNA cause many human diseases, including progeria, a premature aging syndrome, whereas LMNB1 duplication causes adult-onset autosomal dominant leukodystrophy (ADLD). LMNB1 is reduced in cells from progeria patients, but the significance of this reduction is unclear. In this paper, we show that LMNB1 protein levels decline in senescent human dermal fibroblasts and keratinocytes, mediated by reduced transcription and inhibition of LMNB1 messenger ribonucleic acid (RNA) translation by miRNA-23a. This reduction is also observed in chronologically aged human skin tissue. To determine whether altered LMNB1 levels cause senescence, we either increased or reduced LMNB1. Both LMNB1 depletion and overexpression inhibited proliferation, but only LMNB1 overexpression induced senescence, which was prevented by telomerase expression or inactivation of p53. This phenotype was exacerbated by a simultaneous reduction of LMNA/C. Our results demonstrate that altering LMNB1 levels inhibits proliferation and are relevant to understanding the molecular pathology of ADLD.

A case of cerebral hypomyelination with spondylo-epi-metaphyseal dysplasia.

We reported on a male patient with rare leukoencephalopathy and skeletal abnormalities. The condition was first noticed as a developmental delay, nystagmus and ataxia at 1 year of age. At 4 years of age, he was diagnosed as hypomyelination with skeletal abnormalities from clinical features, brain magnetic resonance imaging (MRI) and skeletal X-rays. His brain MRI revealed diffuse hypomyelination. These findings suggested the classical type of Pelizaeus-Merzbacher disease (PMD) caused by proteolipid protein (PLP)-1 gene or Pelizaeus-Merzbacher-like disease (PMLD). However, we found neither mutation nor duplication of PLP-1. The patient had severe growth retardation and general skeletal dysplasia compatible with spondylo-epi-metaphyseal dysplasia; however the mutation of discoidin domain receptor (DDR) 2 gene was absent. The co-morbidity of hypomyelination with skeletal abnormalities is rare. We performed array CGH and no causal copy number variation was recognized. Alternatively, this condition may have been caused by a mutation of the gene encoding a molecule that functions in both cerebral myelination and skeletal development.

Neural stem cell engraftment and myelination in the human brain.

Pelizaeus-Merzbacher disease (PMD) is a rare leukodystrophy caused by mutation of the proteolipid protein 1 gene. Defective oligodendrocytes in PMD fail to myelinate axons, causing global neurological dysfunction. Human central nervous system stem cells (HuCNS-SCs) can develop into oligodendrocytes and confer structurally normal myelin when transplanted into a hypomyelinating mouse model. A 1-year, open-label phase-1 study was undertaken to evaluate safety and to detect evidence of myelin formation after HuCNS-SC transplantation. Allogeneic HuCNS-SCs were surgically implanted into the frontal lobe white matter in four male subjects with an early-onset severe form of PMD. Immunosuppression was administered for 9 months. Serial neurological evaluations, developmental assessments, and cranial magnetic resonance imaging (MRI) and MR spectroscopy, including high-angular resolution diffusion tensor imaging (DTI), were performed at baseline and after transplantation. The neurosurgical procedure, immunosuppression regimen, and HuCNS-SC transplantation were well tolerated. Modest gains in neurological function were observed in three of the four subjects. No clinical or radiological adverse effects were directly attributed to the donor cells. Reduced T1 and T2 relaxation times were observed in the regions of transplantation 9 months after the procedure in the three subjects. Normalized DTI showed increasing fractional anisotropy and reduced radial diffusivity, consistent with myelination, in the region of transplantation compared to control white matter regions remote to the transplant sites. These phase 1 findings indicate a favorable safety profile for HuCNS-SCs in subjects with PMD. The MRI results suggest durable cell engraftment and donor-derived myelin in the transplanted host white matter.