A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export reduces the neurotoxic effects of dipeptide repeat proteins.

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

DJ-1 promotes energy balance by regulating both mitochondrial and autophagic homeostasis.

The protein DJ-1 is mutated in rare familial forms of recessive Parkinson's disease and in parkinsonism accompanied by amyotrophic lateral sclerosis symptoms and dementia. DJ-1 is considered a multitasking protein able to confer protection under various conditions of stress. However, the precise cellular function still remains elusive. In the present work, we evaluated fruit flies lacking the expression of the DJ-1 homolog dj-1ß as compared to control aged-matched individuals. Behavioral evaluations included lifespan, locomotion in an open field arena, sensitivity to oxidative insults, and resistance to starvation. Molecular analyses were carried out by analyzing the mitochondrial morphology and functionality, and the autophagic response. We demonstrated that dj-1ß null mutant flies are hypoactive and display higher sensitivity to oxidative insults and food deprivation. Analysis of mitochondrial homeostasis revealed that loss of dj-1ß leads to larger and more circular mitochondria, characterized by impaired complex-I-linked respiration while preserving ATP production capacity. Additionally, dj-1ß null mutant flies present an impaired autophagic response, which is suppressed by treatment with the antioxidant molecule N-Acetyl-L-Cysteine. Overall, our data point to a mechanism whereby DJ-1 plays a critical role in the maintenance of energy homeostasis, by sustaining mitochondrial homeostasis and affecting the autophagic flux through the maintenance of the cellular redox state. In light of the involvement of DJ-1 in neurodegenerative diseases and considering that neurons are highly energy-demanding cells, particularly sensitive to redox stress, our study sheds light on a key role of DJ-1 in the maintenance of cellular homeostasis.

DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion.

Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.

An assessment of the rescue action of resveratrol in parkin loss of function-induced oxidative stress in Drosophila melanogaster.

Loss-of-function mutations in parkin is associated with onset of juvenile Parkinson's disease (PD). Resveratrol is a polyphenolic stilbene with neuroprotective activity. Here, we evaluated the rescue action of resveratrol in parkin mutant D. melanogaster. The control flies (w1118) received diet-containing 2% ethanol (vehicle), while the PD flies received diets-containing resveratrol (15, 30 and 60 mg/kg diet) for 21 days to assess survival rate. Consequently, similar treatments were carried out for 10 days to evaluate locomotor activity, oxidative stress and antioxidant markers. We also determined mRNA levels of Superoxide dismutase 1 (Sod1, an antioxidant gene) and ple, which encodes tyrosine hydroxylase, the rate-limiting step in dopamine synthesis. Our data showed that resveratrol improved survival rate and climbing activity of PD flies compared to untreated PD flies. Additionally, resveratrol protected against decreased activities of acetylcholinesterase and catalase and levels of non-protein thiols and total thiols displayed by PD flies. Moreover, resveratrol mitigated against parkin mutant-induced accumulations of hydrogen peroxide, nitric oxide and malondialdehyde. Resveratrol attenuated downregulation of ple and Sod1 and reduction in mitochondrial fluorescence intensity displayed by PD flies. Overall, resveratrol alleviated oxidative stress and locomotor deficit associated with parkin loss-of-function mutation and therefore might be useful for the management of PD.

Metallobiology and therapeutic chelation of biometals (copper, zinc and iron) in Alzheimer's disease: Limitations, and current and future perspectives.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent cause of cognitive impairment and dementia worldwide. The pathobiology of the disease has been studied in the form of several hypotheses, ranging from oxidative stress, amyloid-beta (Aß) aggregation, accumulation of tau forming neurofibrillary tangles (NFT) through metal dysregulation and homeostasis, dysfunction of the cholinergic system, and to inflammatory and autophagic mechanism. However, none of these hypotheses has led to confirmed diagnostics or approved cure for the disease. OBJECTIVE: This review is aimed as a basic and an encyclopedic short course into metals in AD and discusses the advances in chelation strategies and developments adopted in the treatment of the disease. Since there is accumulating evidence of the role of both biometal dyshomeostasis (iron (Fe), copper (Cu), and zinc (Zn)) and metal-amyloid interactions that lead to the pathogenesis of AD, this review focuses on unraveling therapeutic chelation strategies that have been considered in the treatment of the disease, aiming to sequester free and protein-bound metal ions and reducing cerebral metal burden. Promising compounds possessing chemically modified moieties evolving as multi-target ligands used as anti-AD drug candidates are also covered. RESULTS AND CONCLUSION: Several multidirectional and multifaceted studies on metal chelation therapeutics show the need for improved synthesis, screening, and analysis of compounds to be able to effectively present chelating anti-AD drugs. Most drug candidates studied have limitations in their physicochemical properties; some enhance redistribution of metal ions, while others indirectly activate signaling pathways in AD. The metal chelation process in vivo still needs to be established and the design of potential anti-AD compounds that bi-functionally sequester metal ions as well as inhibit the Aß aggregation by competing with the metal ions and reducing metal-induced oxidative damage and neurotoxicity may signal a bright end in chelation-based therapeutics of AD.

DJ-1: A promising therapeutic candidate for ischemia-reperfusion injury.

DJ-1 is a multifaceted protein with pleiotropic functions that has been implicated in multiple diseases, ranging from neurodegeneration to cancer and ischemia-reperfusion injury. Ischemia is a complex pathological state arising when tissues and organs do not receive adequate levels of oxygen and nutrients. When the blood flow is restored, significant damage occurs over and above that of ischemia alone and is termed ischemia-reperfusion injury. Despite great efforts in the scientific community to ameliorate this pathology, its complex nature has rendered it challenging to obtain satisfactory treatments that translate to the clinic. In this review, we will describe the recent findings on the participation of the protein DJ-1 in the pathophysiology of ischemia-reperfusion injury, firstly introducing the features and functions of DJ-1 and, successively highlighting the therapeutic potential of the protein.

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila.

Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research.

Characterization of Drosophila ATPsynC mutants as a new model of mitochondrial ATP synthase disorders.

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.

Axonal transport defects are a common phenotype in Drosophila models of ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. Here, we assessed the axonal transport of different cargos in multiple Drosophila models of ALS. We found that axonal transport defects are common across all models tested, although they often showed a differential effect between mitochondria and vesicle cargos. Motor deficits were also common across the models and generally worsened with age, though surprisingly there was not a clear correlation between the severity of axonal transport defects and motor ability. These results further support defects in axonal transport as a common factor in models of ALS that may contribute to the pathogenic process.

Superoxide Dismutase (SOD)-mimetic M40403 Is Protective in Cell and Fly Models of Paraquat Toxicity: IMPLICATIONS FOR PARKINSON DISEASE.

Parkinson disease is a debilitating and incurable neurodegenerative disorder affecting ∼1-2% of people over 65 years of age. Oxidative damage is considered to play a central role in the progression of Parkinson disease and strong evidence links chronic exposure to the pesticide paraquat with the incidence of the disease, most probably through the generation of oxidative damage. In this work, we demonstrated in human SH-SY5Y neuroblastoma cells the beneficial role of superoxide dismutase (SOD) enzymes against paraquat-induced toxicity, as well as the therapeutic potential of the SOD-mimetic compound M40403. Having verified the beneficial effects of superoxide dismutation in cells, we then evaluated the effects using Drosophila melanogaster as an in vivo model. Besides protecting against the oxidative damage induced by paraquat treatment, our data demonstrated that in Drosophila M40403 was able to compensate for the loss of endogenous SOD enzymes, acting both at a cytosolic and mitochondrial level. Because previous clinical trials have indicated that the M40403 molecule is well tolerated in humans, this study may have important implication for the treatment of Parkinson disease.

The Parkinson's disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy.

Compelling evidence indicates that two autosomal recessive Parkinson's disease genes, PINK1 (PARK6) and Parkin (PARK2), cooperate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain-containing protein Fbxo7 (encoded by PARK15) also cause early-onset autosomal recessive Parkinson's disease, by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and acts in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin, supporting a functional relationship between the two proteins. Parkinson's disease-causing mutations in Fbxo7 interfered with this process, emphasizing the importance of mitochondrial dysfunction in Parkinson's disease pathogenesis.

Temporally dynamic response to Wingless directs the sequential elaboration of the proximodistal axis of the Drosophila wing.

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. Here, we show that a temporally dynamic response to Wingless signalling sequentially elaborates the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, zfh-2, is initially activated by Wingless throughout the "wing primordium," but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. Our data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance.