Rapamycin Alleviates Protein Aggregates, Reduces Neuroinflammation, and Rescues Demyelination in Globoid Cell Leukodystrophy.

We have shown in vivo and in vitro previously that psychosine causes dysfunction of autophagy and the ubiquitin-proteasome system underlying the pathogenesis of globoid cell leukodystrophy (GLD), a devastating lysosomal storage disease complicated by global demyelination. Here, we investigated the therapeutic efficacy of the mTOR inhibitor rapamycin in twitcher mice, a murine model of infantile GLD, in biochemical, histochemical, and clinical aspects. Administration of rapamycin to twitcher mice inhibited mTOR signaling in the brains, and significantly reduced the accumulation of insoluble ubiquitinated protein and the formation of ubiquitin aggregates. The astrocytes and microglia reactivity were attenuated in that reactive astrocytes, ameboid microglia, and globoid cells were reduced in the brains of rapamycin-treated twitcher mice. Furthermore, rapamycin improved the cortical myelination, neurite density, and rescued the network complexity in the cortex of twitcher mice. The therapeutic action of rapamycin on the pathology of the twitcher mice's brains prolonged the longevity of treated twitcher mice. Overall, these findings validate the therapeutic efficacy of rapamycin and highlight enhancing degradation of aggregates as a therapeutic strategy to modulate neuroinflammation, demyelination, and disease progression of GLD and other leukodystrophies associated with intracellular aggregates.

Impact of Mitochondrial A3243G Heteroplasmy on Mitochondrial Bioenergetics and Dynamics of Directly Reprogrammed MELAS Neurons.

The MELAS syndrome primarily affecting the CNS is mainly caused by the m.A3243G mutation. The heteroplasmy in different tissues affects the phenotypic spectrum, yet the impact of various levels of m.A3243G heteroplasmy on CNS remains elusive due to the lack of a proper neuronal model harboring m.A3243G mutation. We generated induced neurons (iNs) through the direct reprogramming of MELAS patients, with derived fibroblasts harboring high (>95%), intermediate (68%), and low (20%) m.A3243G mutation. iNs demonstrated neuronal morphology with neurite outgrowth, branching, and dendritic spines. The heteroplasmy and deficiency of respiratory chain complexes were retained in MELAS iNs. High heteroplasmy elicited the elevation in ROS levels and the disruption of mitochondrial membrane potential. Furthermore, high and intermediate heteroplasmy led to the impairment of mitochondrial bioenergetics and a change in mitochondrial dynamics toward the fission and fragmentation of mitochondria, with a reduction in mitochondrial networks. Moreover, iNs derived from aged individuals manifested with mitochondrial fission. These results help us in understanding the impact of various heteroplasmic levels on mitochondrial bioenergetics and mitochondrial dynamics in neurons as the underlying pathomechanism of neurological manifestations of MELAS syndrome. Furthermore, these findings provide targets for further pharmacological approaches of mitochondrial diseases and validate iNs as a reliable platform for studies in neuronal aspects of aging, neurodegenerative disorders, and mitochondrial diseases.

Impairment of Proteasome and Autophagy Underlying the Pathogenesis of Leukodystrophy.

Impairment of the ubiquitin-proteasome-system (UPS) and autophagy causing cytoplasmic aggregation of ubiquitin andp62 have been implicated in the pathogenesis of most neurodegenerative disorders, yet, they have not been fully elucidated in leukodystrophies. The relationship among impairment of UPS, autophagy, and globoid cell leukodystrophy (GLD), one of the most common demyelinating leukodystrophies, is clarified in this study. We examined the ubiquitin and autophagy markers in the brains of twitcher mice, a murine model of infantile GLD, and in human oligodendrocytes incubated with psychosine. Immunohistochemical examinations showed spatiotemporal accumulation of ubiquitin- and p62-aggregates mainly in the white matter of brain and spinal cord at disease progression. Western blot analysis demonstrated a significant accumulation of ubiquitin, p62, and LC3-II in insoluble fraction in parallel with progressive demyelination and neuroinflammation in twitcher brains. In vitro study validated a dose- and time-dependent cytotoxicity of psychosine upon autophagy and UPS machinery. Inhibition of autophagy and UPS exacerbated the accumulation of insoluble ubiquitin, p62, and LC3-II proteins mediated by psychosine cytotoxicity as well as increased cytoplasmic deposition of ubiquitin- and p62-aggregates, and accumulation of autophagosomes and autolysosomes. Further, the subsequent accumulation of reactive oxygen species and reduction of mitochondrial respiration led to cell death. Our studies validate the impairment of proteasome and autophagy underlying the pathogenesis of GLD. These findings provide a novel insight into pathogenesis of GLD and suggest a specific pathomechanism as an ideal target for therapeutic approaches.

Oxidative Insults and Mitochondrial DNA Mutation Promote Enhanced Autophagy and Mitophagy Compromising Cell Viability in Pluripotent Cell Model of Mitochondrial Disease.

Dysfunction of mitochondria causes defects in oxidative phosphorylation system (OXPHOS) and increased production of reactive oxygen species (ROS) triggering the activation of the cell death pathway that underlies the pathogenesis of aging and various diseases. The process of autophagy to degrade damaged cytoplasmic components as well as dysfunctional mitochondria is essential for ensuring cell survival. We analyzed the role of autophagy inpatient-specific induced pluripotent stem (iPS) cells generated from fibroblasts of patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) with well-characterized mitochondrial DNA mutations and distinct OXPHOS defects. MELAS iPS cells recapitulated the pathogenesis of MELAS syndrome, and showed an increase of autophagy in comparison with its isogenic normal counterpart, whereas mitophagy is very scarce at the basal condition. Our results indicated that the existence of pathogenic mtDNA alone in mitochondrial disease was not sufficient to elicit the degradation of dysfunctional mitochondria. Nonetheless, oxidative insults induced bulk macroautophagy with the accumulation of autophagosomes and autolysosomes upon marked elevation of ROS, overload of intracellular calcium, and robust depolarization of mitochondrial membrane potential, while mitochondria respiratory function was impaired and widespread mitophagy compromised cell viability. Collectively, our studies provide insights into the dysfunction of autophagy and activation of mitophagy contributing to the pathological mechanism of mitochondrial disease.

Inflexibility of AMPK-mediated metabolic reprogramming in mitochondrial disease.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is most commonly caused by the A3243G mutation of mitochondrial DNA. The capacity to utilize fatty acid or glucose as a fuel source and how such dynamic switches of metabolic fuel preferences and transcriptional modulation of adaptive mechanism in response to energy deficiency in MELAS syndrome have not been fully elucidated. The fibroblasts from patients with MELAS syndrome demonstrated a remarkable deficiency of electron transport chain complexes I and IV, an impaired cellular biogenesis under glucose deprivation, and a decreased ATP synthesis. In situ analysis of the bioenergetic properties of MELAS cells demonstrated an attenuated fatty acid oxidation that concomitantly occurred with impaired mitochondrial respiration, while energy production was mostly dependent on glycolysis. Furthermore, the transcriptional modulation was mediated by the AMP-activated protein kinase (AMPK) signaling pathway, which activated its downstream modulators leading to a subsequent increase in glycolytic flux through activation of pyruvate dehydrogenase. In contrast, the activities of carnitine palmitoyltransferase for fatty acid oxidation and acetyl-CoA carboxylase-1 for fatty acid synthesis were reduced and transcriptional regulation factors for biogenesis were not altered. These results provide novel information that MELAS cells lack the adaptive mechanism to switch fuel source from glucose to fatty acid, as glycolysis rates increase in response to energy deficiency. The aberrant secondary cellular responses to disrupted metabolic homeostasis mediated by AMPK signaling pathway may contribute to the development of the clinical phenotype.

De novo MECP2 duplication derived from paternal germ line result in dysmorphism and developmental delay.

Xq28 duplications encompassing the methyl CpG binding protein 2 (MECP2) in males exhibit a distinct phenotype, including developmental delay, facial dysmorphism, muscular hypotonia, intellectual disability, poor or absent speech, recurrent infections and early death. The vast majority of affected males inherit the MECP2 duplication from their usually asymptomatic carrier mothers. Only a few cases with Xq28 duplication originating from de novo unbalanced X/Y translocation have been reported and the paternal origin of the aberration has only been validated in three males in the related literature. Here we present a karyotypically normal male with features characteristic of the MECP2 duplication syndrome. The genome-wide SNP genotyping shows a de novo 2.26-Mb duplication from Xq28 to the terminus. The genotypes of the SNPs within the duplicated region indicated a paternal origin. Furthermore, the results of fluorescence in situ hybridization (FISH) indicated a novel Xq:Yp translocation, characterized as der(Y)t(Y;X)(p11.32;q28), which suggests an aberrant that occurred during spermatogenesis. The phenotype is compared to the previously reported cases with Xq28 duplication originated from an unbalanced X/Y translocation, and there was no specific part of the phenotype that could be contributed to the origin of parental imbalances. This report further highlights the capacity of high-molecular cytogenetic methods, such as SNP array and FISH, in the identification of submicroscopic rearrangement, structural configuration and parental origin of aberrant while in the evaluation of children with idiopathic developmental delay and intellectual disability.

Compound heterozygous mutations in PYCR1 further expand the phenotypic spectrum of De Barsy syndrome.

De Barsy syndrome (DBS) is characterized by progeroid features, ophthalmological abnormalities, intrauterine growth retardation, and cutis laxa. Recently, PYCR1 mutations were identified in cutis laxa with progeroid features. Herein, we report on a DBS patient born to a nonconsanguineous Chinese family. The exceptional observation of congenital glaucoma, aortic root dilatation, and idiopathic hypertrophic pyloric stenosis in this patient widened the range of symptoms that have been noted in DBS. Mutation analysis of PYCR1 revealed compound heterozygous PYCR1 mutations, including a p.P115fsX7 null mutation allele and a second allele with two missense mutations in cis: p.G248E and p.G297R. The effect of mutation results in a reduction of PYCR1 mRNA expression and PYCR1 protein expression in skin fibroblasts from the patient. The findings presented here suggest a mutation screening of PYCR1 and cardiovascular survey in patients with DBS.

CNS-targeted AAV5 gene transfer results in global dispersal of vector and prevention of morphological and function deterioration in CNS of globoid cell leukodystrophy mouse model.

Globoid cell leukodystrophy (GLD) is a devastating lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). Currently, there is no definite cure for GLD. Several attempts with CNS-directed gene therapy in twitcher mice (a murine model of GLD) demonstrated restricted expression of GALC activity in CNS and failure of therapeutic efficacy in cerebellum and spinal cord, resulting in various degrees of correction of biochemical, pathological and clinical phenotype. More recently, twitcher mice receiving a combination of hematopoietic and viral vector gene transfer therapies were not protected from neurodegeneration and axonopathy in both cerebellum and spinal cord. This evidence indicates the requirement of sufficient and widespread GALC expression in CNS and rescue of cerebellum and spinal cord in the therapeutic intervention of murine model of GLD. In this study, we have optimized intracranial delivery of AAV2/5-GALC to the neocortex, hippocampus and cerebellum, instead of the thalamus as was previously conducted, of twitcher mice. The CNS-targeted AAV2/5 gene transfer effectively dispersed GALC transgene along the neuraxis of CNS as far as the lumbar spinal cord, and reduced the accumulation of psychosine in the CNS of twitcher mice. Most importantly, the treated twitcher mice were protected from loss of oligodendrocytes and Purkinje cells, axonopathy and marked gliosis, and had significantly improved neuromotor function and prolonged lifespan. These preclinical findings with our approach are encouraging, although a more robust response in the spinal cord would be desirable. Collectively, the information in this study validates the efficacy of this gene delivery approach to correct enzymatic deficiency, psychosine accumulation and neuropathy in CNS of GLD. Combining cell therapy such as bone marrow transplantation with treatment with the aim of reducing inflammation, replacing dead or dying oligodendrocytes and targeting PNS may provide a synergistic and more complete correction of this disease.

A novel mutation in PYCR1 causes an autosomal recessive cutis laxa with premature aging features in a family.

The autosomal recessive form of type II cutis laxa (ARCL II) is characterized by the appearance of redundant, inelastic skin with wrinkling, an aged look and additional variable systemic involvement including intrauterine growth retardation, failure to thrive, developmental delay, dysmorphism, osseous abnormality, and CNS manifestations. Several genetic defects have been found in patients and families with the clinical manifestations of ARCL II. Recently, mutations in PYCR1 have been linked to cutis laxa with progeroid features. We ascertained two siblings with of ARCL II born to non-consanguineous parents. Mutation analysis of PYCR1 revealed a novel single-base deletion (c.345delC) in exon 4 leading to frame-shift and premature stop of translation. The effect of this mutation results in a strong reduction of PYCR1 expression in skin fibroblasts from affected siblings. These two cases extend the genotypic spectrum of PYCR1-related ARCL II.

ND4 mutation in transitional cell carcinoma: Does mitochondrial mutation occur before tumorigenesis?

To investigate how mitochondrial mutation occurs in cancers, we analyzed ND4 mutation in 53 transitional cell carcinomas (TCCs) of the upper urinary tract and the normal counterpart (perirenal soft tissue). Three methods, i.e., DNA sequencing, restriction fragment length polymorphism (RFLP), and denaturing high-performance liquid chromatography (DHPLC), were employed because of their different sensitive of detecting mutation. The results of sequencing and RFLP showed that ND4 mutations were only found in 24.5% (13/53) of tumor. However, 11 of these mutations could also be identified in the normal tissue by DHPLC, indicating that most mitochondrial mutations identified in tumors preexist as minor components, which are too low in quantity to be detected by less sensitive methods such as DNA sequencing. The result suggests that mtDNA mutation occurs before tumorigenesis and become apparent in cancer cells.

Detection of mycobacteria in Crohn's disease by a broad spectrum polymerase chain reaction.

BACKGROUND: The role of mycobacterial infection, particularly related to Mycobacterium avium subsp paratuberculosis (Map), in Crohn's disease has long been debated. We developed primer pairs capable of detecting a broad spectrum of mycobacterium and employed them to investigate surgical specimens from patients with Crohn's disease. METHODS: Pan mycobacterium primers of the 65-kDa heat shock protein gene (Hsp65) were used in a polymerase chain reaction (PCR) to examine 12 surgically-resected, formalin-fixed, paraffin-embedded specimens from 11 patients with Crohn's disease. The DNA sequences of amplicons were aligned with those in GenBank. RESULTS: Mycobacterial DNA was found in specimens from three of 11 patients. M. mucogenicum was identified in a specimen from one patient and M. tuberculosis in two, but Map was not identified in any. CONCLUSION: Hsp65-based PCR can be employed to search for occult mycobacterial infection of the gastrointestinal tract in patients with a diagnosis or suspicion of Crohn's disease. This approach may have a therapeutic implication.

Oculopharyngeal somatic myopathy in a patient with a novel large-scale 3,399 bp deletion and a homoplasmic T5814C transition of the mitochondrial DNA.

We report a 65-year-old woman with a sporadic form of progressive oculopharyngeal somatic myopathy due to a novel large-scale 3,399 base pair (bp) deletion of the mitochondrial DNA (mtDNA) and co-occurrence of a homoplasmic T5814C transition. The onset of myopathy began from chronic progressive external ophthalmoplegia (CPEO) at age of 20 years. Bulbar weakness, neck and proximal limb paralysis, slowly progressed to eventual respiratory failure. The plasma levels of pyruvate (1.5 mg/dL) and lactate (20.2 mg/dL) were elevated. Muscle biopsy showed decreased enzymatic activity of cytochrome c oxidase, but no ragged-red fibers. Electron microscopy showed "parking-lot" paracrystalline inclusions in the enlarged mitochondria suggestive for mitochondrial myopathy. Sequencing of the whole mitochondrial genome of the patient's muscle and leukocytes showed 3,399 bp deletion of the mtDNA from nucleotide position 8,024 to 11,423 and a homoplasmic thymidine to cytosine transition at nucleotide position 5,814 of the tRNA(Cys) gene of mtDNA (T5814C). T5814C was absent in the white blood cells of the patient's daughter and in 205 normal controls. We conclude that a large-scale deletion may coexist with T5814C transition in patients with sporadic form of mitochondrial cytopathy manifested by slowly progressive oculopharyngeal somatic myopathy.

Evolutional analysis in determining pathogenic versus nonpathogenic mutations of ATPase 6 in human mitochondriopathy.

Because mitochondrial ATPase 6 plays an important role in ATP synthesis, mutations affecting ATPase 6 can undoubtedly cause human diseases. In contrast, the ATPase 6 gene is known to be a fast-evolving gene and has generated enough polymorphisms to allow identity investigation for forensic casework. To investigate these seemingly opposite views, we analyzed amino acid sequences of ATPase 6 in at least 1,266 humans, 102 mammals, and 213 vertebrates. The result showed that the amino acids of human ATPase 6 could be divided into the following four groups. Amino acid residue 192 (affected by alteration at nt 9101) and 79 other residues were variable, and therefore substitutions of these residues would not be pathogenic. Amino acid residue 156 (affected by alteration at nt 8993) and 93 other residues were conserved in Homo sapiens, but not in Mammalia. Therefore, they were potentially pathogenic if altered. Function studies would be necessary to confirm their role in pathogenesis. Amino acid residue 217 (affected by alteration at nt 9176) and 9 other residues were conserved across all species, including S. cerevisiae and E. coli. Mutations involving these residues would be pathogenic, some of which might even be life threatening. The remainder (42 residues) were conserved in Mammalia, but not in yeast and E. coli. They were probably pathogenic if mutated. The classification proposed in this study may, therefore, provide an algorithm for a diagnostic approach when a newly identified change of ATPase 6 is suspected for human mitochondriopathy.

Does surgical resection of hepatocellular carcinoma accelerate cancer dissemination?

AIM: This study was to investigate whether surgery could increase cancer dissemination and postoperative recurrence in patients with hepatocellular carcinoma (HCC) by detection of human alpha-fetoprotein messenger RNA (hAFP mRNA). hAFP mRNA in the peripheral blood of patients with HCC has been considered as a surrogate marker for circulating tumor cells. METHODS: Eighty-one consecutive patients who underwent curative resection for HCC entered this prospective cohort study. We examined hAFP mRNA from the peripheral blood obtained preoperatively, perioperatively, and postoperatively to correlate the prognosis after curative resections from HCC patients and from the control subjects. Detection of hAFP mRNA by reverse transcriptase and polymerase chain reaction amplification (RT-PCR) was performed with primers specifically. The relations between the clinical variables (age, sex, associated liver cirrhosis, hepatitis B virus infection, hepatitis C virus infection, serum ?-fetoprotein and Child-Pugh class), the histological variables (size, capsule, vascular permeation, grade of differentiation, and daughter nodules), hAFP mRNA in peripheral blood of 3 different sessions, and postoperative course (recurrence, and recurrence related death) were analysed. RESULTS: No hAFP mRNA was detected in control group subjects. Twenty-two (27%), 24 (30%) and 19 (23%) of 81 HCC patients had hAFP mRNA positivity in the preoperative, perioperative and postoperative peripheral blood. The preoperative presence did not influence the risk of HCC recurrence (55% vs 41%, P=0.280). In contrast, patients with postoperative presence had a significantly higher recurrence (90% vs 31%, P<0.001; odds ratio 19.2; 95% confidence interval: 4.0-91.7). In the multivariate analysis by COX proportional hazards model, postoperative positivity had a significant influence on recurrence (P=0.067) and recurrence related mortality (P=0.017). Whereas, the perioperative positivity of hAFP mRNA did not increase HCC recurrence (58% vs.39%, P=0.093). The correlation between perioperative hAFP mRNA positivity and recurrence related mortality had no statistical significance (P=0.836). CONCLUSION: From our study, perioperative detection of hAFP mRNA in peripheral blood of patients has no clinical relevance and significant role in the prediction of HCC recurrence. Surgical resection itself may not accelerate cancer dissemination and does not increase postoperative recurrence significantly either.

Melas with point mutations involving tRNALeu (A3243G) and tRNAGlu(A14693g).

The syndrome of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) is typically associated with a single point mutation in the mitochondrial genome (mtDNA). Because mtDNA is known to have a higher mutation rate than nuclear DNA, we speculate that some patients with MELAS syndrome may harbor more than one mutation in mtDNA. For this purpose, mtDNA extracted from muscle containing dysmorphic mitochondria from a 32-year-old man with MELAS was sequenced in its entirety to identify all possible mutations. The result showed a homoplasmic A14693G and a heteroplasmic A3243G. The A14693G transition was not present in 205 unrelated control individuals, was not seen in 76 species randomly selected from GenBank, and appears to disrupt the base pairing within the T-loop of mtDNA tRNA(Glu). His asymptomatic siblings' blood showed wild-type at these positions, whereas the blood of the patient's oligosymptomatic diabetic mother had a heteroplasmic A14693G and an apparent homoplasmic wild-type A3243, suggesting an association of A14693G with diabetes mellitus. This case demonstrates the importance of sequencing the mtDNA in its entirety to evaluate the molecular basis of mitochondriopathy.