Fragile X‐associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations

Fragile X‐associated tremor/ataxia syndrome (FXTAS) is a late‐onset neurodegenerative disorder that appears in at least one‐third of adult carriers of a premutation (55‐200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.     

HPDL deficiency causes a neuromuscular disease by impairing the mitochondrial respiration

Mitochondrial diseases are caused by variants in both mitochondrial and nuclear genomes. A nuclear gene HPDL(4-hydroxyphenylpyruvate dioxygenase-like), which encodes an intermembrane mitochondrial protein, has been recently implicated in causing a neurodegenerative disease characterized by pediatric-onset spastic movement phenotypes. Here, we report six Chinese patients with bi-allelic HPDL pathogenic variants from four unrelated families showing neuropathic symptoms of variable severity, including developmental delay/intellectual disability, spasm, and hypertonia. Seven different pathogenic variants are identified, of which five are novel. Both fibroblasts and immortalized lymphocytes derived from patients show impaired mitochondrial respiratory function, which is also observed in HPDL-knockdown(KD) He La cells. In these He La cells, overexpression of a wild-type HPDL gene can rescue the respiratory phenotype of oxygen consumption rate. In addition, a decreased activity of the oxidative phosphorylation(OXPHOS)complex II is observed in patient-derived lymphocytes and HPDL-KD He La cells, further supporting an essential role of HPDL in the mitochondrial respiratory chain. Collectively, our data expand the clinical and mutational spectra of this mitochondrial neuropathy and further delineate the possible disease mechanism involving the impairment of the OXPHOS complex II activity due to the bi-allelic inactivations of HPDL.     

The emerging molecular mechanisms for mitochondrial dysfunctions in FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by an expansion of 55-200 CGG repeats at 5UTR of FMR1 gene, known as premutation. The main clinical and neuropathological features of FXTAS include progressive intention tremor, gait ataxia, neuronal cell loss and presence of ubiquitin-positive intranuclear inclusions in neurons and astrocytes. Various mitochondrial dysfunctions are reported in in vitro/vivo models of FXTAS; however, the molecular mechanisms underlying such mitochondrial dysfunctions are unclear. CGG expansions are pathogenic through distinct mechanisms involving RNA gain of function, impaired DNA damage repair and FMRpolyG toxicity. Here, we have systematically reviewed the reports of mitochondrial dysfunctions under premutation condition. We have also focused on potential emerging mechanisms to understand mitochondrial associated pathology in FXTAS. This review highlights the important role of mitochondria in FXTAS and other related disorders; and suggests focus of future studies on mitochondrial dysfunction along with other prevailing mechanisms to alleviate neurodegeneration.     

Molecular imaging for mitochondrial metabolism and oxidative stress in mitochondrial diseases and neurodegenerative disorders

BackgroundIncreasing evidence from pathological and biochemical investigations suggests that mitochondrial metabolic impairment and oxidative stress play a crucial role in the pathogenesis of mitochondrial diseases, such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, and various neurodegenerative disorders. Recent advances in molecular imaging technology with positron emission tomography (PET) and functional magnetic resonance imaging (MRI) have accomplished a direct and non-invasive evaluation of the pathophysiological changes in living patients.Scope of reviewIn this review, we focus on the latest achievements of molecular imaging for mitochondrial metabolism and oxidative stress in mitochondrial diseases and neurodegenerative disorders.Major conclusionsMolecular imaging with PET and MRI exhibited mitochondrial metabolic changes, such as enhanced glucose utilization with lactic acid fermentation, suppressed fatty acid metabolism, decreased TCA-cycle metabolism, impaired respiratory chain activity, and increased oxidative stress, in patients with MELAS syndrome. In addition, PET imaging clearly demonstrated enhanced cerebral oxidative stress in patients with Parkinson's disease or amyotrophic lateral sclerosis. The magnitude of oxidative stress correlated well with clinical severity in patients, indicating that oxidative stress based on mitochondrial dysfunction is associated with the neurodegenerative changes in these diseases.General significanceMolecular imaging is a promising tool to improve our knowledge regarding the pathogenesis of diseases associated with mitochondrial dysfunction and oxidative stress, and this would facilitate the development of potential antioxidants and mitochondrial therapies.     

Ursodeoxycholic Acid Improves Mitochondrial Function and Redistributes Drp1 in Fibroblasts from Patients with Either Sporadic or Familial Alzheimer's Disease

Alzheimer's disease (AD) is the leading cause of dementia worldwide. Mitochondrial abnormalities have been identified in many cell types in AD, with deficits preceding the development of the classical pathological aggregations. Ursodeoxycholic acid (UDCA), a treatment for primary biliary cirrhosis, improves mitochondrial function in fibroblasts derived from Parkinson's disease patients as well as several animal models of AD and Parkinson's disease. In this paper, we investigated both mitochondrial function and morphology in fibroblasts from patients with both sporadic and familial AD. We show that both sporadic AD (sAD) and PSEN1 fibroblasts share the same impairment of mitochondrial membrane potential and alterations in mitochondrial morphology. Mitochondrial respiration, however, was decreased in sAD fibroblasts and increased in PSEN1 fibroblasts. Morphological changes seen in AD fibroblasts include reduced mitochondrial number and increased mitochondrial clustering around the cell nucleus as well as an increased number of long mitochondria. We show here for the first time in AD patient tissue that treatment with UDCA increases mitochondrial membrane potential and respiration as well as reducing the amount of long mitochondria in AD fibroblasts. In addition, we show reductions in dynamin-related protein 1 (Drp1) level, particularly the amount localized to mitochondria in both sAD and familial patient fibroblasts. Drp1 protein amount and localization were increased after UDCA treatment. The restorative effects of UDCA are abolished when Drp1 is knocked down. This paper highlights the potential use of UDCA as a treatment for neurodegenerative disease.     

Dominant optic atrophy,OPA1, and mitochondrial quality control: understanding mitochondrial network dynamics

Mitochondrial quality control is fundamental to all neurodegenerative diseases, including the most prominent ones, Alzheimer’s Disease and Parkinsonism. It is accomplished by mitochondrial network dynamics – continuous fission and fusion of mitochondria. Mitochondrial fission is facilitated by DRP1, while MFN1 and MFN2 on the mitochondrial outer membrane and OPA1 on the mitochondrial inner membrane are essential for mitochondrial fusion. Mitochondrial network dynamics are regulated in highly sophisticated ways by various different posttranslational modifications, such as phosphorylation, ubiquitination, and proteolytic processing of their key-proteins. By this, mitochondria process a wide range of different intracellular and extracellular parameters in order to adapt mitochondrial function to actual energetic and metabolic demands of the host cell, attenuate mitochondrial damage, recycle dysfunctional mitochondria via the mitochondrial autophagy pathway, or arrange for the recycling of the complete host cell by apoptosis. Most of the genes coding for proteins involved in this process have been associated with neurodegenerative diseases. Mutations in one of these genes are associated with a neurodegenerative disease that originally was described to affect retinal ganglion cells only. Since more and more evidence shows that other cell types are affected as well, we would like to discuss the pathology of dominant optic atrophy, which is caused by heterozygous sequence variants in OPA1, in the light of the current view on OPA1 protein function in mitochondrial quality control, in particular on its function in mitochondrial fusion and cytochrome C release. We think OPA1 is a good example to understand the molecular basis for mitochondrial network dynamics.     

Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedreich's ataxia

Respiratory chain dysfunction has been identified in several neurodegenerative disorders. In Friedreich's ataxia (FA) and Huntington's disease (HD), where the respective mutations are in nuclear genes encoding non-respiratory chain mitochondrial proteins, the defects in oxidative phosphorylation are clearly secondary. In Parkinson's disease (PD) the situation is less clear, with some evidence for a primary role of mitochondrial DNA in at least a proportion of patients. The pattern of the respiratory chain defect may provide some clue to its cause; in PD there appears to be a selective complex I deficiency; in HD and FA the deficiencies are most severe in complex II/III with a less severe defect in complex IV. Aconitase activity in HD and FA is severely decreased in brain and muscle, respectively, but appears to be normal in PD brain. Free radical generation is thought to be of importance in both HD and FA, via excitotoxicity in HD and abnormal iron handling in FA. The oxidative damage observed in PD may be secondary to the mitochondrial defect. Whatever the cause(s) and sequence of events, respiratory chain deficiencies appear to play an important role in the pathogenesis of neurodegeneration. The mitochondrial abnormalities induced may converge on the function of the mitochondrion in apoptosis. This mode of cell death is thought to play an important role in neurodegenerative diseases and it is tempting to speculate that the observed mitochondrial defects in PD, HD and FA result directly in apoptotic cell death, or in the lowering of a cell's threshold to undergo apoptosis. Clarifying the role of mitochondria in pathogenesis may provide opportunities for the development of treatments designed to reverse or prevent neurodegeneration.     

MicroRNA expression profiling in blood from fragile X‐associated tremor/ataxia syndrome patients

Fragile X‐associated tremor/ataxia syndrome (FXTAS) is a late‐onset neurodegenerative disorder associated with FMR1 gene premutation alleles (55–200 CGG repeats). Fragile X‐associated tremor/ataxia syndrome clinical core features include action tremor, gait ataxia, cognitive deficits progressing to dementia, and frequently parkinsonism. Although the pathogenic molecular mechanism of FXTAS is not completely understood, the restriction of the phenotype to the FMR1 premutation range has given rise to a model based on a RNA toxic gain‐of‐function. Since the identification of the first microRNAs (miRNAs) and their role in normal development, several studies have associated them with neurodegenerative diseases such as Parkinson, Alzheimer and Huntington diseases, suggesting that they play a key role in brain development, as well as in its morphogenesis. Herein, we present the characterization of miRNA expression profiles in FXTAS male patients using deep sequencing‐based technologies and microarray technology. Deep sequencing analysis evidenced 83 miRNAs that were significantly deregulated whereas microarray analysis showed 31. When comparing these results, 14 miRNAs were found deregulated in FXTAS patients. MiR‐424 and miR‐574‐3p showed significant fold change adjusted P‐values in both platforms in FXTAS patients. MiR‐424 has been founded substantially and specifically enriched in human cerebral cortical white matter of Alzheimer disease patients, which, together with cerebral atrophy, is a prominent imaging finding in individuals with FXTAS. The study provides the first systematic evidence of differential miRNA expression changes in FXTAS blood samples. Although further studies are necessary to better characterize the miRNA function in FXTAS disorder, our results suggest that they might contribute to its pathogenesis.     

Mitochondrial Complex Enzyme Activities and Cytochrome c Expression Changes in Multiple Sclerosis

Blood platelets have been widely proposed as biomarkers in studies of mitochondrial function and aging-related and neurodegenerative diseases. Defects in mitochondrial function were found not only in the substantia nigra of Parkinson’s disease patients but also in their blood platelets. Similarly, it has also been described in the blood platelet mitochondria of Alzheimer’s disease patients. To study mitochondrial aerobic metabolism function and protein expression in platelets of multiple sclerosis (MS) patients and control subjects, mitochondrial aconitase, mitochondrial superoxide dismutases 1 and 2 (SOD1 and SOD2), and respiratory complex enzyme activities in platelets of MS patients and control subjects were determined. Likewise, mitochondrial lipid peroxidation and mitochondrial SOD1 and cytochrome c expressions were investigated. Mitochondrial aconitase activity was higher in MS patients than in controls (P?<?0.05). A significant increase on all respiratory complex activities in MS patients was observed (P?<?0.05). Mitochondrial lipid peroxidation was significantly higher in MS patients than in controls (P?<?0.05). Significant changes of cytochrome c and mitochondrial SOD1 expressions were detected (P?<?0.05), with a decrease of 44?±?5 % and an increase of 46?±?6 %, respectively. Our study reveals that significant changes in mitochondrial aerobic metabolism function and mitochondrial SOD1 and cytochrome c expressions are produced in platelets of MS patients.     

Oxidative stress in human aging and mitochondrial disease-consequences of defective mitochondrial respiration and impaired antioxidant enzyme system

Respiratory function of mitochondria is compromised in aging human tissues and severely impaired in the patients with mitochondrial disease. A wide spectrum of mitochondrial DNA (mtDNA) mutations has been established to associate with mitochondrial diseases. Some of these mtDNA mutations also occur in various human tissues in an age-dependent manner. These mtDNA mutations cause defects in the respiratory chain due to impairment of the gene expression and structure of respiratory chain polypeptides that are encoded by the mitochondrial genome. Since defective mitochondria generate more reactive oxygen species (ROS) such as O2- and H2O2 via electron leak, we hypothesized that oxidative stress is a contributory factor for aging and mitochondrial disease. This hypothesis has been supported by the findings that oxidative stress and oxidative damage in tissues and culture cells are increased in elderly subjects and patients with mitochondrial diseases. Another line of supporting evidence is our recent finding that the enzyme activities of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) decrease with age in skin fibroblasts. By contrast, Mn-SOD activity increases up to 65 years of age and then slightly declines thereafter. On the other hand, we observed that the RNA, protein and activity levels of Mn-SOD are increased two- to three-fold in skin fibroblasts of the patients with CPEO syndrome but are dramatically decreased in patients with MELAS or MERRF syndrome. However, the other antioxidant enzymes did not change in the same manner. The imbalance in the expression of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may elicit an elevation of oxidative stress in the fibroblasts. Indeed, it was found that intracellular levels of H2O2 and oxidative damage to DNA and lipids in skin fibroblasts from elderly subjects or patients with mitochondrial diseases are significantly increased as compared to those of age-matched controls. Furthermore, Mn-SOD or GPx-1 gene knockout mice were found to display neurological disorders and enhanced oxidative damage similar to those observed in the patients with mitochondrial disease. These observations are reviewed in this article to support that oxidative stress elicited by defective respiratory function and impaired antioxidant enzyme system plays a key role in the pathophysiology of mitochondrial disease and human aging.     

线粒体功能缺陷和神经系统疾病

线粒体呼吸链缺陷一直被认为是诱发线粒体疾病的重要因素,这有助于研究人员阐释其遗传和临床多样性。然而,线粒体的其他功能也具有重要意义,包括蛋白质运输、细胞器动力学和细胞凋亡。调控这些功能的基因缺陷不仅导致神经和精神疾病,而且还导致年龄相关的神经变性疾病。因此,引起越来越多的关注。在讨论呼吸链缺陷引起相关神经系统疾病的一些致病难题后,就线粒体动力学改变引起的相关神经系统疾病病因和常见神经变性疾病的病理生理机制作一综述。     

The Role of Mitochondrial Quality Control in Cognitive Dysfunction in Diabetes

Type 2 diabetes (T2DM) is a well known risk factor for Alzheimer’s disease. Mitochondria are the center of intracellular energy metabolism and the main source of reactive oxygen species. Mitochondrial dysfunction has been identified as a key factor in diabetes-associated brain alterations contributing to neurodegenerative events. Defective insulin signaling may act in concert with neurodegenerative mechanisms leading to abnormalities in mitochondrial structure and function. Mitochondrial dysfunction triggers neuronal energy exhaustion and oxidative stress, leading to brain neuronal damage and cognitive impairment. The normality of mitochondrial function is basically maintained by mitochondrial quality control mechanisms. In T2DM, defects in the mitochondrial quality control pathway in the brain have been found to lead to mitochondrial dysfunction and cognitive impairment. Here, we discuss the association of mitochondrial dysfunction with T2DM and cognitive impairment. We also review the molecular mechanisms of mitochondrial quality control and impacts of mitochondrial quality control on the progression of cognitive impairment in T2DM.

     

Activation of mTOR Ameliorates Fragile X Premutation rCGG Repeat-Mediated Neurodegeneration

Fragile X associated tremor/ataxia syndrome (FXTAS) is a late onset neurodegenerative disorder caused by aberrant expansion of CGG repeats in 5′ UTR of FMR1 gene. The elevated mRNA confers a toxic gain-of-function thought to be the critical event of pathogenesis. Expressing rCGG₉₀ repeats of the human FMR1 5′UTR in Drosophila is sufficient to induce neurodegeneration. Rapamycin has been demonstrated to attenuate neurotoxicity by inducing autophagy in various animal models of neurodegenerative diseases. Surprisingly, we observed rapamycin exacerbated rCGG₉₀-induced neurodegenerative phenotypes through an autophagy-independent mechanism. CGG₉₀ expression levels of FXTAS flies exposed to rapamycin presented no significant differences. We further demonstrated that activation of the mammalian target of rapamycin (mTOR) signaling could suppress neurodegeneration of FXTAS. These findings indicate that rapamycin will exacerbate neurodegeneration, and that enhancing autophagy is insufficient to alleviate neurotoxicity in FXTAS. Moreover, these results suggest mTOR and its downstream molecules as new therapeutic targets for FXTAS by showing significant protection against neurodegeneration.     

Age related mitochondrial degenerative disorders in humans

Disorders caused by mitochondrial respiratory chain deficiency due to mutations in mitochondrial DNA have varied phenotypes but many involve neurological features often associated with cell loss within specific brain regions. These disorders, along with the increasing evidence of decline in mitochondrial function with ageing, have raised speculation that primary changes in mitochondria could have an important role in age-related neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Evidence supporting a role for mitochondria in common neurodegenerative diseases comes from studies with the toxin MPP+ and familial PD, which has been shown to involve proteins such as DJ-1 and Pink1 (both of which are predicted to have a role in mitochondrial function and oxidative stress). Mutations within the mitochondrial genome have been shown to accumulate with age and in common neurodegenerative diseases. Mitochondrial DNA haplogroups have also been shown to be associated with certain neurodegenerative conditions. This review covers the primary mitochondrial diseases but also discuss the potential role of mitochondria and mitochondrial DNA mutations in mitochondrial and neurodegenerative diseases, in particular in PD and in AD.     

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.     

Mitochondrial permeability transition pore is a potential drug target for neurodegeneration

Mitochondrial permeability transition pore (mPTP) plays a central role in alterations of mitochondrial structure and function leading to neuronal injury relevant to aging and neurodegenerative diseases including Alzheimer's disease (AD). mPTP putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT) and cyclophilin D (CypD). Reactive oxygen species (ROS) increase intra-cellular calcium and enhance the formation of mPTP that leads to neuronal cell death in AD. CypD-dependent mPTP can play a crucial role in ischemia/reperfusion injury. The interaction of amyloid beta peptide (Aβ) with CypD potentiates mitochondrial and neuronal perturbation. This interaction triggers the formation of mPTP, resulting in decreased mitochondrial membrane potential, impaired mitochondrial respiration function, increased oxidative stress, release of cytochrome c, and impaired axonal mitochondrial transport. Thus, the CypD-dependent mPTP is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of AD. Designing small molecules to block this interaction would lessen the effects of Aβ neurotoxicity. This review summarizes the recent progress on mPTP and its potential therapeutic target for neurodegenerative diseases including AD. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Mitochondria: A therapeutic target in neurodegeneration

Mitochondrial dysfunction has long been associated with neurodegenerative disease. Therefore, mitochondrial protective agents represent a unique direction for the development of drug candidates that can modify the pathogenesis of neurodegeneration. This review discusses evidence showing that mitochondrial dysfunction has a central role in the pathogenesis of Alzheimer's, Parkinson's and Huntington's diseases and amyotrophic lateral sclerosis. We also debate the potential therapeutic efficacy of metabolic antioxidants, mitochondria-directed antioxidants and Szeto–Schiller (SS) peptides. Since these compounds preferentially target mitochondria, a major source of oxidative damage, they are promising therapeutic candidates for neurodegenerative diseases. Furthermore, we will briefly discuss the novel action of the antihistamine drug Dimebon on mitochondria.     

Role of AMPK-mediated adaptive responses in human cells with mitochondrial dysfunction to oxidative stress

Background

Mitochondrial DNA (mtDNA) mutations are an important cause of mitochondrial diseases, for which there is no effective treatment due to complex pathophysiology. It has been suggested that mitochondrial dysfunction-elicited reactive oxygen species (ROS) plays a vital role in the pathogenesis of mitochondrial diseases, and the expression levels of several clusters of genes are altered in response to the elevated oxidative stress. Recently, we reported that glycolysis in affected cells with mitochondrial dysfunction is upregulated by AMP-activated protein kinase (AMPK), and such an adaptive response of metabolic reprogramming plays an important role in the pathophysiology of mitochondrial diseases.

Scope of review

We summarize recent findings regarding the role of AMPK-mediated signaling pathways that are involved in: (1) metabolic reprogramming, (2) alteration of cellular redox status and antioxidant enzyme expression, (3) mitochondrial biogenesis, and (4) autophagy, a master regulator of mitochondrial quality control in skin fibroblasts from patients with mitochondrial diseases.

Major conclusion

Induction of adaptive responses via AMPK–PFK2, AMPK–FOXO3a, AMPK–PGC-1α, and AMPK–mTOR signaling pathways, respectively is modulated for the survival of human cells under oxidative stress induced by mitochondrial dysfunction. We suggest that AMPK may be a potential target for the development of therapeutic agents for the treatment of mitochondrial diseases.

General significance

Elucidation of the adaptive mechanism involved in AMPK activation cascades would lead us to gain a deeper insight into the crosstalk between mitochondria and the nucleus in affected tissue cells from patients with mitochondrial diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.