Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response.

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.

Prion protein stabilizes amyloid-ß (Aß) oligomers and enhances Aß neurotoxicity in a Drosophila model of Alzheimer's disease.

The cellular prion protein (PrPC) can act as a cell-surface receptor for ß-amyloid (Aß) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aß isoforms and PrPC in the Drosophila brain. We found that co-expression of Aß and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aß deposition in the fly brain. In contrast, under the same conditions, expression of Aß or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aß as oligomeric assemblies and fragment-preformed Aß fibers. The ability of membrane-anchored PrPC to trap Aß as cytotoxic oligomers at the membrane surface and fragment inert Aß fibers suggests a mechanism by which PrPC exacerbates Aß deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aß toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aß and PrPC represents a potential therapeutic strategy.

The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aß.

Brain-derived neurotrophic factor (BDNF) has a crucial role in learning and memory by promoting neuronal survival and modulating synaptic connectivity. BDNF levels are lower in the brains of individuals with Alzheimer's disease (AD), suggesting a pathogenic involvement. The Drosophila orthologue of BDNF is the highly conserved Neurotrophin 1 (DNT1). BDNF and DNT1 have the same overall protein structure and can be cleaved, resulting in the conversion of a full-length polypeptide into separate pro- and mature-domains. While the BDNF mature-domain is neuroprotective, the role of the pro-domain is less clear. In flies and mammalian cells, we have identified a synergistic toxic interaction between the amyloid-ß peptide (Aß1-42) and the pro-domains of both DNT1 and BDNF. Specifically, we show that DNT1 pro-domain acquires a neurotoxic activity in the presence of Aß1-42. In contrast, DNT1 mature-domain is protective against Aß1-42 toxicity. Likewise, in SH-SY5Y cell culture, BDNF pro-domain is toxic only in the presence of Aß1-42. Western blots indicate that this synergistic interaction likely results from the Aß1-42-induced upregulation of the BDNF pro-domain receptor p75(NTR). The clinical relevance of these findings is underlined by a greater than thirty fold increase in the ratio of BDNF pro- to mature-domains in the brains of individuals with AD. This unbalanced BDNF pro:mature-domain ratio in patients represents a possible biomarker of AD and may offer a target for therapeutic intervention.

Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB.

Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.

Coordinate regulation of eIF2α phosphorylation by PPP1R15 and GCN2 is required during Drosophila development.

Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by the kinase GCN2 attenuates protein synthesis during amino acid starvation in yeast, whereas in mammals a family of related eIF2α kinases regulate translation in response to a variety of stresses. Unlike single-celled eukaryotes, mammals also possess two specific eIF2α phosphatases, PPP1R15a and PPP1R15b, whose combined deletion leads to a poorly understood early embryonic lethality. We report the characterisation of the first non-mammalian eIF2α phosphatase and the use of Drosophila to dissect its role during development. The Drosophila protein demonstrates features of both mammalian proteins, including limited sequence homology and association with the endoplasmic reticulum. Of note, although this protein is not transcriptionally regulated, its expression is controlled by the presence of upstream open reading frames in its 5'UTR, enabling induction in response to eIF2α phosphorylation. Moreover, we show that its expression is necessary for embryonic and larval development and that this is to oppose the inhibitory effects of GCN2 on anabolic growth.

Alzheimer's disease: insights from Drosophila melanogaster models.

The power of fruit fly genetics is being deployed against some of the most intractable and economically significant problems in modern medicine, the neurodegenerative diseases. Fly models of Alzheimer's disease can be exposed to the rich diversity of biological techniques that are available to the community and are providing new insights into disease mechanisms, and assisting in the identification of novel targets for therapy. Similar approaches might also help us to interpret the results of genome-wide association studies of human neurodegenerative diseases by allowing us to triage gene "hits" according to whether a candidate risk factor gene has a modifying effect on the disease phenotypes in fly model systems.

Retro-inversal of intracellular selected ß-amyloid-interacting peptides: implications for a novel Alzheimer's disease treatment.

The aggregation of ß-amyloid (Aß) into toxic oligomers is a hallmark of Alzheimer's disease pathology. Here we present a novel approach for the development of peptides capable of preventing amyloid aggregation based upon the previous selection of natural all-l peptides that bind Aß1-42. Using an intracellular selection system, successful library members were further screened via competition selection to identify the most effective peptides capable of reducing amyloid levels. To circumvent potential issues arising from stability and protease action for these structures, we have replaced all l residues with d residues and inverted the sequence. These retro-inverso (RI) peptide analogues therefore encompass reversed sequences that maintain the overall topological order of the native peptides. Our results demonstrate that efficacy in blocking and reversing amyloid formation is maintained while introducing desirable properties to the peptides. Thioflavin-T assays, circular dichroism, and oblique angle fluorescence microscopy collectively indicate that RI peptides can reduce amyloid load, while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrate modest reductions in cell toxicity. These conclusions are reinforced using Drosophila melanogaster studies to monitor pupal hatching rates and fly locomotor activity in the presence of RI peptides delivered via RI-trans-activating transcriptional activator peptide fusions. We demonstrate that the RI-protein fragment complementation assay approach can be used as a generalized method for deriving Aß-interacting peptides. This approach has subsequently led to several peptide candidates being further explored as potential treatments for Alzheimer's disease.

p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress.

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G(1) phase, although recent studies have identified a novel ER stress G(2) checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G(2) delay involving CHK1 and a later induction of G(1) arrest associated both with the induction of p53 target genes and loss of cyclin D(1). We show that substitution of p53/47 for p53 impairs the ER stress G(1) checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G(2) progression prior to ultimate G(1) arrest.

Suppression of Aß toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity.

The accumulation of ß-amyloid (Aß) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aß toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aß levels by clearing the Aß plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aß are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aß toxicity is indirect was supported by the finding that Aß is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aß toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aß toxicity.

ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism.

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-ß in Alzheimer's disease.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Expression in drosophila of tandem amyloid ß peptides provides insights into links between aggregation and neurotoxicity.

The generation and subsequent aggregation of amyloid ß (Aß) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aß(40)) or 42 residues (Aß(42)), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aß peptides to probe the manner in which changes in the aggregation kinetics of Aß affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aß(40) and Aß(42) compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aß(42) constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aß(42) peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aß(40) rather than simply to its higher rate of aggregation.

Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster.

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.

Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo.

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival.

Drosophila have emerged as a model system to study mammalian neurodegenerative diseases. In the present study we have generated Drosophila transgenic for ovine PrP (prion protein) to begin to establish an invertebrate model of ovine prion disease. We generated Drosophila transgenic for polymorphic variants of ovine PrP by PhiC31 site-specific germ-line transformation under expression control by the bi-partite GAL4/UAS (upstream activating sequence) system. Site-specific transgene insertion in the fly genome allowed us to test the hypothesis that single amino acid codon changes in ovine PrP modulate prion protein levels and the phenotype of the fly when expressed in the Drosophila nervous system. The Arg(154) ovine PrP variants showed higher levels of PrP expression in neuronal cell bodies and insoluble PrP conformer than did His(154) variants. High levels of ovine PrP expression in Drosophila were associated with phenotypic effects, including reduced locomotor activity and decreased survival. Significantly, the present study highlights a critical role for helix-1 in the formation of distinct conformers of ovine PrP, since expression of His(154) variants were associated with decreased survival in the absence of high levels of PrP accumulation. Collectively, the present study shows that variants of the ovine PrP are associated with different spontaneous detrimental effects in ovine PrP transgenic Drosophila.

Testing the therapeutic potential of doxycycline in a Drosophila melanogaster model of Alzheimer disease.

Therapies for Alzheimer disease that reduce the production of pathogenic amyloid ß (Aß) peptides have been associated with a range of unwanted effects. For this reason, alternative strategies that promote the clearance of the peptide by preventing its aggregation and deposition in the brain have been favored. In this context we have studied doxycycline, a member of the tetracycline family of antibiotics that has shown neuroprotective effects in a number of models of neurodegenerative disease. We investigated the neuroprotective potential of doxycycline in a Drosophila model of Aß toxicity and sought to correlate any effects with the aggregation state of the peptide. We found that administration of doxycycline to Aß42-expressing flies did not improve their lifespan but was able to slow the progression of their locomotor deficits. We also measured the rough eye phenotype of transgenic flies expressing the E22G variant of Aß42 and showed that doxycycline administration partially rescued the toxicity of Aß in the developing eye. We correlated these in vivo effects with in vitro observations using transmission electron microscopy, dynamic light scattering, and thioflavin T binding. We found that doxycycline prevents Aß fibrillization and favors the generation of smaller, non-amyloid structures that were non-toxic as determined by the lack of caspase 3 activation in a neuroblastoma cell line. Our confirmation that doxycycline can prevent amyloid ß toxicity both in vitro and in vivo supports its therapeutic potential in AD.

Iron promotes the toxicity of amyloid beta peptide by impeding its ordered aggregation.

We have previously shown that overexpressing subunits of the iron-binding protein ferritin can rescue the toxicity of the amyloid ß (Aß) peptide in our Drosophila model system. These data point to an important pathogenic role for iron in Alzheimer disease. In this study, we have used an iron-selective chelating compound and RNAi-mediated knockdown of endogenous ferritin to further manipulate iron in the brain. We confirm that chelation of iron protects the fly from the harmful effects of Aß. To understand the pathogenic mechanisms, we have used biophysical techniques to see how iron affects Aß aggregation. We find that iron slows the progression of the Aß peptide from an unstructured conformation to the ordered cross-ß fibrils that are characteristic of amyloid. Finally, using mammalian cell culture systems, we have shown that iron specifically enhances Aß toxicity but only if the metal is present throughout the aggregation process. These data support the hypothesis that iron delays the formation of well ordered aggregates of Aß and so promotes its toxicity in Alzheimer disease.

Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response.

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest.

Prion-induced toxicity in PrP transgenic Drosophila.

Prion diseases are fatal transmissible neurodegenerative diseases of humans and various vertebrate species. In their natural hosts these conditions are characterised by prolonged incubation times prior to the onset of clinical signs of terminal disease. Accordingly, tractable models of mammalian prion disease are required in order to better understand the mechanisms of prion replication and prion-induced neurotoxicity. Transmission of prion diseases can occur across a species barrier and this is facilitated in recipients transgenic for the same PrP gene as the individual from which the infectious prions are derived. Here we have tested the hypothesis that exogenous ovine prions can induce neurotoxicity in Drosophila melanogaster transgenic for ovine PrP. Drosophila that expressed ovine PrP pan neuronally and inoculated with ovine prions at the larval stage by oral exposure to scrapie-infected sheep brain homogenate showed markedly accelerated locomotor and survival defects. ARQ PrP transgenic Drosophila exposed to scrapie-infected brain homogenate showed a significant and progressive reduction in locomotor activity compared to similar flies exposed to normal sheep brain homogenate. The prion-induced locomotor defect was accompanied by the accumulation of potentially misfolded PrP in the brains of prion-inoculated flies. VRQ PrP transgenic Drosophila, which expressed less ovine PrP than ARQ flies, showed a reduced median survival compared to similar flies exposed to normal sheep brain homogenate. These prion-induced phenotypic effects were PrP-mediated since ovine prions were not toxic in non-PrP transgenic control flies. Our observations provide the basis of an invertebrate model of transmissible mammalian prion disease.

Characterisation of serpin polymers in vitro and in vivo.

Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation.