A C Costa

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinsons disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.

Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson’s disease

Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson’s disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.

Mitofusin-mediated ER stress triggers neurodegeneration in pink1/parkin models of Parkinson's disease.

Mutations in PINK1 and PARKIN cause early-onset Parkinson’s disease (PD), thought to be due to mitochondrial toxicity. Here, we show that in Drosophila pink1 and parkin mutants, defective mitochondria also give rise to endoplasmic reticulum (ER) stress signalling, specifically to the activation of the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response (UPR). We show that enhanced ER stress signalling in pink1 and parkin mutants is mediated by mitofusin bridges, which occur between defective mitochondria and the ER. Reducing mitofusin contacts with the ER is neuroprotective, through suppression of PERK signalling, while mitochondrial dysfunction remains unchanged. Further, both genetic inhibition of dPerk-dependent ER stress signalling and pharmacological inhibition using the PERK inhibitor GSK2606414 were neuroprotective in both pink1 and parkin mutants. We conclude that activation of ER stress by defective mitochondria is neurotoxic in pink1 and parkin flies and that the reduction of this signalling is neuroprotective, independently of defective mitochondria. A video abstract for this article is available online in the supplementary information

Knockdown of Hsc70-5/mortalin Induces Loss of Synaptic Mitochondria in a Drosophila Parkinson’s Disease Model

Mortalin is an essential component of the molecular machinery that imports nuclear-encoded proteins into mitochondria, assists in their folding, and protects against damage upon accumulation of dysfunctional, unfolded proteins in aging mitochondria. Mortalin dysfunction associated with Parkinson’s disease (PD) increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, Drosophila melanogaster has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. We generated the first loss-of-Hsc70-5/mortalin-function Drosophila model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing Drosophila PD-models, which include reduced ATP levels, abnormal wing posture, shortened life span, and reduced spontaneous locomotor and climbing ability. Dopaminergic neurons seem to be more sensitive to the loss of mortalin than other neuronal sub-types and non-neuronal tissues. The loss of synaptic mitochondria is an early pathological change that might cause later degenerative events. It precedes both behavioral abnormalities and structural changes at the neuromuscular junction (NMJ) of mortalin-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. Ex vivo data from human fibroblasts identifies increased mitophagy as an early pathological change that precedes apoptosis. Given the specificity of the observed defects, we are confident that the loss-of-mortalin model presented in this study will be useful for further dissection of the complex network of pathways that underlie the development of mitochondrial parkinsonism.

Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson’s disease

The co-enzyme nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson’s disease (PD) associated with PARKIN mutations. Here, we uncovered alterations in NAD+ salvage metabolism in Drosophila parkin mutants. We show that a dietary supplementation with the NAD+ precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, we show that this increases levels of NAD+ and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. We conclude that strategies to enhance NAD+ levels by administration of dietary precursors or the inhibition of NAD+-dependent enzymes, such as PARP, that compete with mitochondria for NAD+ could be used to delay neuronal death associated with mitochondrial dysfunction.

Drosophila ref(2)P is required for the parkin -mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.

dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective

Neurons rely on mitochondria as their preferred source of energy. Mutations in PINK1 and PARKIN cause neuronal death in early-onset Parkinson’s disease (PD), thought to be due to mitochondrial dysfunction. In Drosophila pink1 and parkin mutants, mitochondrial defects lead to the compensatory upregulation of the mitochondrial one-carbon cycle metabolism genes by an unknown mechanism. Here we uncover that this branch is triggered by the activating transcription factor 4 (ATF4). We show that ATF4 regulates the expression of one-carbon metabolism genes SHMT2 and NMDMC as a protective response to mitochondrial toxicity. Suppressing Shmt2 or Nmdmc caused motor impairment and mitochondrial defects in flies. Epistatic analyses showed that suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. Conversely, the genetic enhancement of these one-carbon metabolism genes in pink1 or parkin mutants was neuroprotective. We conclude that mitochondrial dysfunction caused by mutations in the Pink1/Parkin pathway engages ATF4-dependent activation of one-carbon metabolism as a protective response. Our findings show a central contribution of ATF4 signalling to PD that may represent a new therapeutic strategy. A video abstract for this article is available at https://youtu.be/cFJJm2YZKKM.

Spatiotemporal diffusion of influenza A (H1N1): Starting point and risk factors

Influenza constitutes a major challenge to world health authorities due to high transmissibility and the capacity to generate large epidemics. This study aimed to characterize the diffusion process of influenza A (H1N1) by identifying the starting point of the epidemic as well as climatic and sociodemographic factors associated with the occurrence and intensity of transmission of the disease. The study was carried out in the Brazilian state of Paraná, where H1N1 caused the largest impact. The units of spatial and temporal analysis were the municipality of residence of the cases and the epidemiological weeks of the year 2009, respectively. Under the Bayesian paradigm, parametric inference was performed through a two-part spatiotemporal model and the integrated nested Laplace approximation (INLA) algorithm. We identified the most likely starting points through the effective distance measure based on mobility networks. The proposed estimation methodology allowed for rapid and efficient implementation of the spatiotemporal model, and provided evidence of different patterns for chance of occurrence and risk of influenza throughout the epidemiological weeks. The results indicate the capital city of Curitiba as the probable starting point, and showed that the interventions that focus on municipalities with greater migration and density of people, especially those with higher Human Development Indexes (HDIs) and the presence of municipal air and road transport, could play an important role in mitigation of effects of future influenza pandemics on public health. These results provide important information on the process of introduction and spread of influenza, and could contribute to the identification of priority areas for surveillance as well as establishment of strategic measures for disease prevention and control. The proposed model also allows identification of epidemiological weeks with high chance of influenza occurrence, which can be used as a reference criterion for creating an immunization campaign schedule.

Emerging Concepts Linking Mitochondrial Stress Signalling and Parkinson's Disease

Neurodegenerative diseases comprise of a large group of pathologies that affect a considerable number of individuals among the world’s population with a higher incidence in elderly people. The most common neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), are characterised by the presence of protein aggregates in different regions of the brain. The causes of these diseases remain undetermined. Nevertheless, new information on this topic is continuously being brought to light.