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.     

RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA-mediated neurodegenerative disease caused by the titration of RNA-binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses a premutation-length repeat (90 CGG repeats) from the 5' UTR of the human FMR1 gene and displays neuronal degeneration. Here, we show that overexpression of RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppresses the phenotype of the CGG transgenic fly. Furthermore, we show that hnRNP A2/B1 directly interacts with riboCGG repeats and that the CUGBP1 protein interacts with the riboCGG repeats via hnRNP A2/B1.     

Methylation mosaicism of 5'-(CGG)(n)-3' repeats in fragile X, premutation and normal individuals

Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5′-(CGG)_n-3′ repeat in the promoter and 5′-untranslated region (5′-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M-SssI-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5′-(CGG)_n-3′ repeats and in the levels of methylation in the repeat and the 5′-UTR. In one patient (OEl) with high repeat length heterogeneity (n = 15 to >200), shorter repeats (n = 20–80) were methylated or unmethylated, longer repeats (n = 100–150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5′-CG-3′ sequences were found in some repeats and 5′-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M-SssI-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.     

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A₁ increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A₁) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell''s compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.     

Pur alpha binds to rCGG repeats and modulates repeat-mediated neurodegeneration in a Drosophila model of fragile X tremor/ataxia syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently recognized neurodegenerative disorder in fragile X premutation carriers with FMR1 alleles containing 55-200 CGG repeats. Previously, we developed a Drosophila model of FXTAS and demonstrated that transcribed premutation repeats alone are sufficient to cause neurodegeneration, suggesting that rCGG-repeat-binding proteins (RBPs) may be sequestered from their normal function by rCGG binding. Here, we identify Pur alpha and hnRNP A2/B1 as RBPs. We show that Pur alpha and rCGG repeats interact in a sequence-specific fashion that is conserved between mammals and Drosophila. Overexpression of Pur alpha in Drosophila could suppress rCGG-mediated neurodegeneration in a dose-dependent manner. Furthermore, Pur alpha is also present in the inclusions of FXTAS patient brains. These findings support the disease mechanism of FXTAS of rCGG repeat sequestration of specific RBPs, leading to neuronal cell death, and implicate that Pur alpha plays an important role in the pathogenesis of FXTAS.     

Structural characterization of a dimer of RNA duplexes composed of 8-bromoguanosine modified CGG trinucleotide repeats: a novel architecture of RNA quadruplexes

Fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS) are neurodegenerative disorders caused by the pathogenic expansion of CGG triplet repeats in the FMR1 gene. FXTAS is likely to be caused by a ‘toxic’ gain-of-function of the FMR1 mRNA. We provide evidence for the existence of a novel quadruplex architecture comprising CGG repeats. The 8-bromoguanosine (^BrG)-modified molecule GC^BrGGCGGC forms a duplex in solution and self-associates via the major groove to form a four-stranded, antiparallel (GC^BrGGCGGC)₄ RNA quadruplex with ^BrG3:G6:^BrG3:G6 tetrads sandwiched between mixed G:C:G:C tetrads. Self-association of Watson–Crick duplexes to form a four-stranded structure has previously been predicted; however, no experimental evidence was provided. This novel four-stranded RNA structure was characterized using a variety of experimental methods, such as native gel electrophoresis, NMR spectroscopy, small-angle X-ray scattering and electrospray ionization mass spectrometry.     

The quadruplex r(CGG)n destabilizing cationic porphyrin TMPyP4 cooperates with hnRNPs to increase the translation efficiency of fragile X premutation mRNA

The 5′ untranslated region of the FMR1 gene which normally includes 4–55 d(CGG) repeats expands to > 55–200 repeats in carriers of fragile X syndrome premutation. Although the levels of premutation FMR1 mRNA in carrier cells are 5–10-fold higher than normal, the amount of the product FMR protein is unchanged or reduced. We demonstrated previously that premutation r(CGG)_n tracts formed quadruplex structures that impeded translation and lowered the efficiency of protein synthesis. Normal translation could be restored in vivo by the quadruplex r(CGG)_n destabilizing action of CBF-A and hnRNP A2 proteins. Here we report that the quadruplex-interacting cationic porphyrin TMPyP4 by itself and in cooperation with CBF-A or hnRNP A2 also unfolded quadruplex r(CGG)_n and increased the efficiency of translation of 5′-(CGG)₉₉ containing reporter firefly (FL) mRNA. TMPyP4 destabilized in vitro a (CGG)₃₃ intramolecular quadruplex structure and enhanced the translation of 5′-(CGG)₉₉-FL mRNA in a rabbit reticulocyte lysate and in HEK293 cells. The efficiency of translation of (CGG)₉₉-FL mRNA was additively increased in cells exposed to TMPyP4 together with CBF-A. Whereas low doses of TMPyP4, CBF-A or hnRNP A2 by themselves did not affect the in vivo utilization of (CGG)₉₉-FL mRNA, introduction of TMPyP4 together with either protein synergistically augmented its translation efficiency.     

YB-1 Synthesis Is Regulated by mTOR Signaling Pathway

YB-1 is a eukaryotic protein with numerous intra- and extracellular functions based on its ability to interact with RNA, DNA, and many proteins. In spite of achievements in studying its functions, regulation of YB-1 synthesis in the cell remains poorly understood. In the current study Western and Northern blotting were used to determine the amounts of YB-1 and YB-1 mRNA in rabbit organs and several cell lines. As found, in the majority of studied eukaryotic cells a considerable proportion of YB-1 mRNA was stored in free mRNPs, i.e., was poorly translated. Also, we demonstrated that YB-1 synthesis depended on conditions that determined the rate of cell division. Specific suppression of YB-1 synthesis resulted from inhibition of the mTOR signaling pathway with inhibitor PP242, but not rapamycin. Experiments on reporter constructs showed that dependence of YB-1 mRNA translation on activity of the mTOR signaling pathway was dictated by 5′ untranslated regions of this mRNA, irrelatively of the TOP-like sequences at the beginning of 5′ UTR.     

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Fragile X‐associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′‐UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA‐binding proteins by the expanded CGG repeats. Sam68 is an RNA‐binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA‐binding proteins sequentially, first Sam68, then hnRNP‐G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing‐regulatory function. Consequently, Sam68‐responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain‐of‐function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.     

Hsp90 Interacts Specifically with Viral RNA and Differentially Regulates Replication Initiation of Bamboo mosaic virus and Associated Satellite RNA

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication.     

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Interruption of the fragile X syndrome expanded sequence d(CGG)(n) by interspersed d(AGG) trinucleotides diminishes the formation and stability of d(CGG)(n) tetrahelical structures

Fragile X syndrome is caused by expansion of a d(CGG) trinucleotide repeat sequence in the 5′ untranslated region of the first exon of the FMR1 gene. Repeat expansion is thought to be instigated by formation of d(CGG)_n secondary structures. Stable FMR1 d(CGG)_n runs in normal individuals consist of 6–52 d(CGG) repeats that are interrupted every 9–11 triplets by a single d(AGG) trinucleotide. By contrast, individuals having fragile X syndrome premutation or full mutation present >54–200 or >200–2000 monotonous d(CGG) repeats, respectively. Here we show that the presence of interspersed d(AGG) triplets diminished in vitro formation of bimolecular tetrahelical structures of d(CGG)₁₈ oligomers. Tetraplex structures formed by d(CGG)_n oligomers containing d(AGG) interspersions had lower thermal stability. In addition, tetraplex structures of d(CGG)₁₈ oligomers interspersed by d(AGG) triplets were unwound by human Werner syndrome DNA helicase at rates and to an extent that exceeded the unwinding of tetraplex form consisting of monotonous d(CGG)₁₈. Diminished formation and stability of tetraplex structures of d(AGG)-containing FMR1 d(CGG)_2–50 tracts might restrict their expansion in normal individuals.     

Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.     

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR''s ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington''s disease and phospholipase C (norpA)–mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.