An Open-Label Administration of Bioavailable-Form Curcumin in Patients With Pelizaeus-Merzbacher Disease.

BACKGROUND: Two preclinical studies using mouse models of Pelizeaus-Merzbacher disease (PMD) have revealed the potential therapeutic effects of curcumin. In this study, we examined the effects of curcumin in patients with PMD. METHODS: We conducted a study administering an open-label oral bioavailable form of curcumin in nine patients genetically confirmed to have PMD (five to 20 years; mean 11 years) for 12 months (low doses for two months followed by high doses for 10 months). We evaluated changes in clinical symptoms as the primary end point using two scales, Gross Motor Function Measure (GMFM) and the PMD Functional Disability Score (PMD-FDS). The level of myelination by brain magnetic resonance imaging (MRI) and the electrophysiological state by auditory brainstem response (ABR) were evaluated as secondary end points. The safety and tolerability of oral curcumin were also examined. RESULTS: Increase in GMFM and PMD-FDS were noted in five and three patients, respectively, but overall, no statistically significant improvement was demonstrated. We found no clear improvement in their brain MRI or ABR. No adverse events associated with oral administration of curcumin were observed. CONCLUSIONS: Although we failed to demonstrate any significant therapeutic effects of curcumin after 12 months, its tolerability and safety were confirmed. This study does not exclude the possibility of therapeutic effects of curcumin, and a trial of longer duration should be considered to compare the natural history of the disease with the effects of curcumin.

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.

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.

Allele-specific silencing as treatment for gene duplication disorders: proof-of-principle in autosomal dominant leukodystrophy.

Allele-specific silencing by RNA interference (ASP-siRNA) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. This approach has been effectively used to target autosomal dominant mutations and single nucleotide polymorphisms linked with aberrantly expanded trinucleotide repeats. Here, we propose ASP-siRNA as a preferable choice to target duplicated disease genes, avoiding potentially harmful excessive downregulation. As a proof-of-concept, we studied autosomal dominant adult-onset demyelinating leukodystrophy (ADLD) due to lamin B1 (LMNB1) duplication, a hereditary, progressive and fatal disorder affecting myelin in the CNS. Using a reporter system, we screened the most efficient ASP-siRNAs preferentially targeting one of the alleles at rs1051644 (average minor allele frequency: 0.45) located in the 3' untranslated region of the gene. We identified four siRNAs with a high efficacy and allele-specificity, which were tested in ADLD patient-derived fibroblasts. Three of the small interfering RNAs were highly selective for the target allele and restored both LMNB1 mRNA and protein levels close to control levels. Furthermore, small interfering RNA treatment abrogates the ADLD-specific phenotypes in fibroblasts and in two disease-relevant cellular models: murine oligodendrocytes overexpressing human LMNB1, and neurons directly reprogrammed from patients' fibroblasts. In conclusion, we demonstrated that ASP-silencing by RNA interference is a suitable and promising therapeutic option for ADLD. Moreover, our results have a broad translational value extending to several pathological conditions linked to gene-gain in copy number variations.

Pelizaeus-Merzbacher disease: cellular pathogenesis and pharmacologic therapy.

Pelizaeus-Merzbacher disease (PMD) is a rare leukodystrophy that causes severe dysmyelination in the central nervous system in infancy and early childhood. Many previous studies showed that various proteolipid protein 1 (plp1) mutations, including duplications, point mutations, and deletions, lead to oligodendrocyte dysfunction in patients with PMD. PMD onset and clinical severity range widely, depending on the type of plp1 mutation. Patients with PMD exhibit a delayed mental and physical development phenotype, but specific pharmacological therapy and clinical treatment for PMD are not yet well established. This review describes PMD pathology and establishment of new clinical treatment for PMD. These findings support the development of a new therapy for PMD and these treatments may improve the quality of life in patients with PMD.

Progesterone antagonist therapy in a Pelizaeus-Merzbacher mouse model.

Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disease, characterized by ataxia, intellectual disability, epilepsy, and premature death. In the majority of cases, PMD is caused by duplication of PLP1 that is expressed in myelinating oligodendrocytes. Despite detailed knowledge of PLP1, there is presently no curative therapy for PMD. We used a Plp1 transgenic PMD mouse model to test the therapeutic effect of Lonaprisan, an antagonist of the nuclear progesterone receptor, in lowering Plp1 mRNA overexpression. We applied placebo-controlled Lonaprisan therapy to PMD mice for 10 weeks and performed the grid slip analysis to assess the clinical phenotype. Additionally, mRNA expression and protein accumulation as well as histological analysis of the central nervous system were performed. Although Plp1 mRNA levels are increased 1.8-fold in PMD mice compared to wild-type controls, daily Lonaprisan treatment reduced overexpression at the RNA level to about 1.5-fold, which was sufficient to significantly improve the poor motor phenotype. Electron microscopy confirmed a 25% increase in the number of myelinated axons in the corticospinal tract when compared to untreated PMD mice. Microarray analysis revealed the upregulation of proapoptotic genes in PMD mice that could be partially rescued by Lonaprisan treatment, which also reduced microgliosis, astrogliosis, and lymphocyte infiltration.

Attenuation of endoplasmic reticulum stress in Pelizaeus-Merzbacher disease by an anti-malaria drug, chloroquine.

Pelizaeus-Merzbacher disease (PMD) is a hypomyelinating disorder caused by the duplication and missense mutations of the proteolipid protein 1 (PLP1) gene. PLP1 missense proteins accumulate in the endoplasmic reticulum (ER) of premature oligodendrocytes and induce severe ER stress followed by apoptosis of the cells. Here, we demonstrate that an anti-malaria drug, chloroquine, decreases the amount of an ER-resident mutant PLP1 containing an alanine-243 to valine (A243V) substitution, which induces severe PMD in human. By preventing mutant PLP1 translation through enhancing the phosphorylation of eukaryotic initiation factor 2 alpha, chloroquine ameliorated the ER stress induced by the mutant protein in HeLa cells. Chroloquine also attenuated ER stress in the primary oligodendrocytes obtained from myelin synthesis deficit (msd) mice, which carry the same PLP1 mutation. In the spinal cords of msd mice, chloroquine inhibited ER stress and upregulated the expression of marker genes of mature oligodendrocytes. Chloroquine-mediated attenuation of ER stress was observed in HeLa cells treated with tunicamycin, an N-glycosylation inhibitor, but not with thapsigargin, a sarco/ER Ca(2+)ATPase inhibitor, which confirms its efficacy against ER stress caused by nascent proteins. These findings indicate that chloroquine is an ER stress attenuator with potential use in treating PMD and possibly other ER stress-related diseases.

Effect of curcumin in a mouse model of Pelizaeus-Merzbacher disease.

PLP1 amino acid substitutions cause accumulation of misfolded protein and induce endoplasmic reticulum (ER) stress, causing Pelizaeus-Merzbacher disease (PMD), a hypomyelinating disorder of the central nerve system. Currently no effective therapy is available for PMD. Promoted by its curative effects in other genetic disease models caused by similar molecular mechanisms, we tested if curcumin, a dietary compound, can rescue the lethal phenotype of a PMD mouse model (myelin synthesis deficient, msd). Curcumin was administered orally to myelin synthesis deficit (msd) mice at 180 mg·kg(-1)·day(-1) from the postnatal day 3. We evaluated general and motor status, changes in myelination and apoptosis of oligodendrocytes by neuropathological and biochemical examination, and transcription levels for ER-related molecules. We also examined the pharmacological effect of curcumin in cell culture system. Oral curcumin treatment resulted in 25% longer survival (p<0.01). In addition, oligodendrocytes undergoing apoptosis were reduced in number (p<0.05). However, no apparent improvement in motor function, neurological phenotype, and myelin formation was observed. Curcumin treatment did not change the expression of ER stress markers and subcellular localization of the mutant protein in vitro and/or in vivo. Curcumin partially mitigated the clinical and pathological phenotype of msd mice, although molecular mechanisms underlying this curative effect are yet undetermined. Nonetheless, curcumin may serve as a potential therapeutic compound for PMD caused by PLP1 point mutations.