Cross-seeding by prion protein inactivates TDP-43.

A common pathological denominator of various neurodegenerative diseases is the accumulation of protein aggregates. Neurotoxic effects are caused by a loss of the physiological activity of the aggregating protein and/or a gain of toxic function of the misfolded protein conformers. In transmissible spongiform encephalopathies or prion diseases, neurodegeneration is caused by aberrantly folded isoforms of the prion protein (PrP). However, it is poorly understood how pathogenic PrP conformers interfere with neuronal viability. Employing in vitro approaches, cell culture, animal models and patients' brain samples, we show that misfolded PrP can induce aggregation and inactivation of TAR DNA-binding protein-43 (TDP-43). Purified PrP aggregates interact with TDP-43 in vitro and in cells and induce the conversion of soluble TDP-43 into non-dynamic protein assemblies. Similarly, mislocalized PrP conformers in the cytosol bind to and sequester TDP-43 in cytosolic aggregates. As a consequence, TDP-43-dependent splicing activity in the nucleus is significantly decreased, leading to altered protein expression in cells with cytosolic PrP aggregates. Finally, we present evidence for cytosolic TDP-43 aggregates in neurons of transgenic flies expressing mammalian PrP and Creutzfeldt-Jakob disease patients. Our study identified a novel mechanism of how aberrant PrP conformers impair physiological pathways by cross-seeding.

Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration.

BACKGROUND: Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. RESULTS: Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. CONCLUSIONS: The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.

Human TRMT2A methylates tRNA and contributes to translation fidelity.

5-Methyluridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme for m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

Drosophila melanogaster as a versatile model organism to study genetic epilepsies: An overview.

Epilepsy is one of the most prevalent neurological disorders, affecting more than 45 million people worldwide. Recent advances in genetic techniques, such as next-generation sequencing, have driven genetic discovery and increased our understanding of the molecular and cellular mechanisms behind many epilepsy syndromes. These insights prompt the development of personalized therapies tailored to the genetic characteristics of an individual patient. However, the surging number of novel genetic variants renders the interpretation of pathogenetic consequences and of potential therapeutic implications ever more challenging. Model organisms can help explore these aspects in vivo. In the last decades, rodent models have significantly contributed to our understanding of genetic epilepsies but their establishment is laborious, expensive, and time-consuming. Additional model organisms to investigate disease variants on a large scale would be desirable. The fruit fly Drosophila melanogaster has been used as a model organism in epilepsy research since the discovery of "bang-sensitive" mutants more than half a century ago. These flies respond to mechanical stimulation, such as a brief vortex, with stereotypic seizures and paralysis. Furthermore, the identification of seizure-suppressor mutations allows to pinpoint novel therapeutic targets. Gene editing techniques, such as CRISPR/Cas9, are a convenient way to generate flies carrying disease-associated variants. These flies can be screened for phenotypic and behavioral abnormalities, shifting of seizure thresholds, and response to anti-seizure medications and other substances. Moreover, modification of neuronal activity and seizure induction can be achieved using optogenetic tools. In combination with calcium and fluorescent imaging, functional alterations caused by mutations in epilepsy genes can be traced. Here, we review Drosophila as a versatile model organism to study genetic epilepsies, especially as 81% of human epilepsy genes have an orthologous gene in Drosophila. Furthermore, we discuss newly established analysis techniques that might be used to further unravel the pathophysiological aspects of genetic epilepsies.

Regulation of the collagen IV network by the basement membrane protein perlecan is crucial for squamous epithelial cell morphogenesis and organ architecture.

All epithelia have their basal side in contact with a specialized extracellular matrix, the basement membrane (BM). During development, the BM contributes to the shaping of epithelial organs via its mechanical properties. These properties rely on two core components of the BM, collagen type IV and perlecan/HSPG2, which both interact with another core component, laminin, the initiator of BM assembly. While collagen type IV supplies the BM with rigidity to constrain the tissue, perlecan antagonizes this effect. Nevertheless, the number of organs that has been studied is still scarce, and given that epithelial tissues exhibit a wide array of shapes, their forms are bound to be regulated by distinct mechanisms. This is underscored by mounting evidence that BM composition and assembly/biogenesis is tissue-specific. Moreover, previous reports have essentially focused on the mechanical role of the BM in morphogenesis at the tissue scale, but not the cell scale. Here, we took advantage of the robust conservation of core BM proteins and the limited genetic redundancy of the Drosophila model system to address how this matrix shapes the wing imaginal disc, a complex organ comprising a squamous, a cuboidal and a columnar epithelium. With the use of a hypomorphic allele, we show that the depletion of Trol (Drosophila perlecan) affects the morphogenesis of the three epithelia, but particularly that of the squamous one. The planar surface of the squamous epithelium (SE) becomes extremely narrow, due to a function for Trol in the control of the squamous shape of its cells. Furthermore, we find that the lack of Trol impairs the biogenesis of the BM of the SE by modifying the structure of the collagen type IV lattice. Through atomic force microscopy and laser surgery, we demonstrate that Trol provides elasticity to the SE's BM, thereby regulating the mechanical properties of the SE. Moreover, we show that Trol acts via collagen type IV, since the global reduction in the trol mutant context of collagen type IV or the enzyme that cross-links its 7S -but not the enzyme that cross-links its NC1- domain substantially restores the morphogenesis of the SE. In addition, a stronger decrease in collagen type IV achieved by the overexpression of the matrix metalloprotease 2 exclusively in the BM of the SE, significantly rescues the organization of the two other epithelia. Our data thus sustain a model in which Trol counters the rigidity conveyed by collagen type IV to the BM of the SE, via the regulation of the NC1-dependant assembly of its scaffold, allowing the spreading of the squamous cells, spreading which is compulsory for the architecture of the whole organ.

Standardized hypoxia-reoxygenation protocol to assess posthypoxic neurobehavioral impairments and molecular mechanisms in Drosophila melanogaster.

Hypoxia plays a pivotal role in the pathogenesis of major causes of mortality such as cerebral ischemia. Here, we present a standardized protocol for the induction of global hypoxia and reoxygenation in Drosophila melanogaster, with details on subsequent analysis of mortality, neurobehavioral impairments, and molecular mechanisms. This protocol emphasizes the importance of controlling and monitoring specific environmental parameters to ensure reproducible results. It also highlights profound differences that can arise from variations in the age and genotype of the flies. For complete details on the use and execution of this protocol, please refer to Habib et al. (2021).

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death.

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

CK1BP Reduces α-Synuclein Oligomerization and Aggregation Independent of Serine 129 Phosphorylation.

The pathological accumulation of α-Synuclein (α-Syn) is the hallmark of neurodegenerative α-synucleinopathies, including Parkinsons's disease (PD). In contrast to the mostly non-phosphorylated soluble α-Syn, aggregated α-Syn is usually phosphorylated at serine 129 (S129). Therefore, S129-phosphorylation is suspected to interfere with α-Syn aggregation. Among other kinases, protein kinase CK1 (CK1) is known to phosphorylate α-Syn at S129. We overexpressed CK1 binding protein (CK1BP) to inhibit CK1 kinase activity. Using Bimolecular Fluorescence Complementation (BiFC) in combination with biochemical methods, we monitored the S129 phosphorylation and oligomerization of α-Syn in HEK293T cells. We found that CK1BP reduced the overall protein levels of α-Syn. Moreover, CK1BP concomitantly reduced S129 phosphorylation, oligomerization and the amount of insoluble α-Syn. Analyzing different α-Syn variants including S129 mutations, we show that the effects of CK1BP on α-Syn accumulation were independent of S129 phosphorylation. Further analysis of an aggregating polyglutamine (polyQ) protein confirmed a phosphorylation-independent decrease in aggregation. Our results imply that the inhibition of CK1 activity by CK1BP might exert beneficial effects on NDDs in general. Accordingly, CK1BP represents a promising target for the rational design of therapeutic approaches to cease or at least delay the progression of α-synucleinopathies.

Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin.

Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.

The Evolutionary Conserved Transmembrane BAX Inhibitor Motif (TMBIM) Containing Protein Family Members 5 and 6 Are Essential for the Development and Survival of Drosophila melanogaster.

The mammalian Transmembrane BAX Inhibitor Motif (TMBIM) protein family consists of six evolutionarily conserved hydrophobic proteins that affect programmed cell death and the regulation of intracellular calcium levels. The bacterial ortholog BsYetJ is a pH-dependent calcium channel. We here identified seven TMBIM family members in Drosophila melanogaster and describe their expression levels in diverse tissues and developmental stages. A phylogenetic analysis revealed that CG30379 represents the ortholog of human TMBIM4 although these two proteins are much less related than TMBIM5 (CG2076 and CG1287/Mics1) and TMBIM6 (CG7188/Bi-1) to their respective orthologs. For TMBIM1-3 the assignment is more dubious because the fly and the human proteins cluster together. We conducted a functional analysis based on expression levels and the availability of RNAi lines. This revealed that the ubiquitous knockdown of CG3798/Nmda1 and CG3814/Lfg had no effect on development while knockdown of CG2076/dTmbim5 resulted in death at the pupa stage and knockdown of CG7188/dTmbim6 in death at the embryonic stage. Ubiquitous knockdown of the second TMBIM5 paralog CG1287/Mics1 ensued in male sterility. Knockdown of dTmbim5 and 6 in muscle and neural tissue also greatly reduced lifespan through different mechanisms. Knockdown of the mitochondrial family member dTmbim5 resulted in reduced ATP production and a pro-apoptotic expression profile while knockdown of the ER protein dTmbim6 increased the ER calcium levels similar to findings in mammalian cells. Our data demonstrate that dTmbim5 and 6 are essential for fly development and survival but affect cell survival through different mechanisms.

Posthypoxic behavioral impairment and mortality of Drosophila melanogaster are associated with high temperatures, enhanced predeath activity and oxidative stress.

Hypoxia is an underlying pathophysiological condition of a variety of devastating diseases, including acute ischemic stroke (AIS). We are faced with limited therapeutic options for AIS patients, and even after successful restoration of cerebral blood flow, the poststroke mortality is still high. More basic research is needed to explain mortality after reperfusion and to develop adjunct neuroprotective therapies. Drosophila melanogaster (D.m.) is a suitable model to analyze hypoxia; however, little is known about the impacts of hypoxia and especially of the subsequent reperfusion injury on the behavior and survival of D.m. To address this knowledge gap, we subjected two wild-type D.m. strains (Canton-S and Oregon-R) to severe hypoxia (<0.3% O2) under standardized environmental conditions in a well-constructed hypoxia chamber. During posthypoxic reperfusion (21% O2), we assessed fly activity (evoked and spontaneous) and analyzed molecular characteristics (oxidative stress marker abundance, reactive oxygen species (ROS) production, and metabolic activity) at various timepoints during reperfusion. First, we established standard conditions to induce hypoxia in D.m. to guarantee stable and reproducible experiments. Exposure to severe hypoxia under defined conditions impaired the climbing ability and reduced the overall activity of both D.m. strains. Furthermore, a majority of the flies died during the early reperfusion phase (up to 24 h). Interestingly, the flies that died early exhibited elevated activity before death compared to that of the flies that survived the entire reperfusion period. Additionally, we detected increases in ROS and stress marker (Catalase, Superoxide Dismutase and Heat Shock Protein 70) levels as well as reductions in metabolic activity in the reperfusion phase. Finally, we found that changes in environmental conditions impacted the mortality rate. In particular, decreasing the temperature during hypoxia or the reperfusion phase displayed a protective effect. In conclusion, our data suggest that reperfusion-dependent death might be associated with elevated temperatures, predeath activity, and oxidative stress.

An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils.

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates. In flies, AS69 reduced [Formula: see text]-synuclein aggregates and the locomotor deficit resulting from [Formula: see text]-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-[Formula: see text]-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes.

Quantification of Protein Aggregates Using Bimolecular Fluorescence Complementation.

Neuronal accumulations of the presynaptic protein α-synuclein represent a hallmark of Parkinson's disease (PD). Multiple system atrophy and dementia with Lewy bodies are other neurodegenerative diseases in which α-synuclein aggregates are the main pathological characteristic. This is why these diseases are summarized as synucleinopathies. The abnormal α-Synuclein accumulation eventually results in the formation of Lewy bodies and Lewy neurites in surviving neurons of the brain. α-Synuclein aggregation has been widely researched in vitro but little is known about α-synuclein aggregation in vivo. This is why it is still unclear whether α-synuclein accumulation and/or which of the resulting aggregates (soluble oligomers or insoluble fibrils) actually trigger neurotoxic events. In our study, we employed the robust genetic makeup of Drosophila melanogaster in combination with bimolecular fluorescence complementation and developed an in vivo assay allowing to determine abundance of soluble α-synuclein aggregates (most likely oligomers). Insoluble α-synuclein aggregates (most likely fibrils) are detected by filter retardation assay. We provide an experimental outline to verify the reported assay system. The described method is easy, inexpensive, fast, and sensitive. Altogether, the described assay system is ideal for large-scale screening approaches aiming to test/identify compounds/conditions that affect α-synuclein aggregation in vivo.Moreover, the experimental outline reported here could serve as a template to analyze aggregation of proteins other than α-synuclein. As accumulation of specific proteins is a hallmark of almost every neurodegenerative disease, only minor changes to our protocol should be necessary for a similar analysis. We have put special focus on critical evaluation steps to consider when adapting our protocol to other aggregate-forming proteins.

Monitoring α-synuclein multimerization in vivo.

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α-synuclein (α-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α-Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α-Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α-Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α-Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α-Syn at serine 129 enhances the accumulation and toxicity of α-Syn. Altogether, our study establishes a novel model to study α-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.-Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α-synuclein multimerization in vivo.

Linking amyotrophic lateral sclerosis and spinal muscular atrophy through RNA-transcriptome homeostasis: a genomics perspective.

In this review, we present our most recent understanding of key biomolecular processes that underlie two motor neuron degenerative disorders, amyotrophic lateral sclerosis, and spinal muscular atrophy. We focus on the role of four multifunctional proteins involved in RNA metabolism (TDP-43, FUS, SMN, and Senataxin) that play a causal role in these diseases. Recent results have led to a novel scenario of intricate connections between these four proteins, bringing transcriptome homeostasis into the spotlight as a common theme in motor neuron degeneration. We review reported functional and physical interactions between these four proteins, highlighting their common association with nuclear bodies and small nuclear ribonucleoprotein particle biogenesis and function. We discuss how these interactions are turning out to be particularly relevant for the control of transcription and chromatin homeostasis, including the recent identification of an association between SMN and Senataxin required to ensure the resolution of DNA-RNA hybrid formation and proper termination by RNA polymerase II. These connections strongly support the existence of common pathways underlying the spinal muscular atrophy and amyotrophic lateral sclerosis phenotype. We also discuss the potential of genome-wide expression profiling, in particular RNA sequencing derived data, to contribute to unravelling the underlying mechanisms. We provide a review of publicly available datasets that have addressed both diseases using these approaches, and highlight the value of investing in cross-disease studies to promote our understanding of the pathways leading to neurodegeneration.

α-Synuclein enhances histone H3 lysine-9 dimethylation and H3K9me2-dependent transcriptional responses.

α-Synuclein (αS) is a protein linked to Parkinson's disease (PD) and related neurodegenerative disorders. It is mostly localized within synapses, but αS has also been suggested to play a role in the nucleus. We used transgenic Drosophila and inducible SH-SY5Y neuroblastoma cells to investigate the effects of αS on chromatin with a particular focus on histone modifications. Overexpression of αS in male flies as well as in retinoic acid pre-treated neuroblastoma cells led to an elevation of histone H3K9 methylations, mostly mono- (H3K9me1) and di- (H3K9me2). The transient increase of H3K9 methylation in αS-induced SH-SY5Y cells was preceded by mRNA induction of the euchromatic histone lysine N-methyltransferase 2 (EHMT2). EHMT2 and H3K9me2 can function within the REST complex. Chromatin immunoprecipitation (ChIP) analyses of selected candidate, REST regulated genes showed significantly increased H3K9me2 promoter occupancy of genes encoding the L1CAM cell adhesion molecule and the synaptosomal-associated protein SNAP25, whose reduced expression levels were confirmed by RT-qPCR in αS induced cells. Treatment with EHMT inhibitor UNC0638 restored the mRNA levels of L1CAM and SNAP25. Thus, αS overexpression enhances H3K9 methylations via ΕΗΜΤ2 resulting in elevated H3K9me2 at the SNAP25 promoter, possibly affecting SNARE complex assembly and hence synaptic vesicle fusion events regulated by αS.

The mitochondrial kinase PINK1: functions beyond mitophagy.

Mutations in the genes encoding the mitochondrial kinase PINK1 and the E3 ubiquitin ligase Parkin cause autosomal recessive Parkinson's disease (PD). Pioneering work in Drosophila melanogaster revealed that the loss of PINK1 or Parkin function causes similar phenotypes including dysfunctional mitochondria. Further research showed that PINK1 can act upstream of Parkin in a mitochondrial quality control pathway to induce removal of damaged mitochondria in a process called mitophagy. Albeit the PINK1/Parkin-induced mitophagy pathway is well established and has recently been elucidated in great detail, its pathophysiological relevance is being debated. Mounting evidence indicates that PINK1 has additional functions, for example, in regulating complex I activity and maintaining neuronal viability in response to stress. Here, we discuss mitophagy-dependent and -independent functions of PINK1 and their possible role in PD pathogenesis. Mutations in the PINK1 gene, encoding a mitochondrial kinase, are associated with autosomal recessive Parkinson's disease. In this review, we summarize and discuss the functional roles of PINK1 in maintaining mitochondrial integrity, eliminating damaged mitochondria, and promoting cell survival. This article is part of a special issue on Parkinson disease.

Rab7 induces clearance of α-synuclein aggregates.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over-expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP-Rab7-decorated vesicles contain α-synuclein. Rab7 over-expression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Time-lapse microscopy confirmed that particles frequently disappeared with Rab7 over-expression. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 over-expression, presumably representing autolysosomes. Rab7 over-expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease. Cells over-expressing aggregation-prone A53T alpha-synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time-lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over-expression, indicating that availability of Rab7 is rate-limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.

Cellular and molecular modifier pathways in tauopathies: the big picture from screening invertebrate models.

Abnormal tau accumulations were observed and documented in post-mortem brains of patients affected by Alzheimer's disease (AD) long before the identification of mutations in the Microtubule-associated protein tau (MAPT) gene, encoding the tau protein, in a different neurodegenerative disease called Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). The discovery of mutations in the MAPT gene associated with FTDP-17 highlighted that dysfunctions in tau alone are sufficient to cause neurodegeneration. Invertebrate models have been diligently utilized in investigating tauopathies, contributing to the understanding of cellular and molecular pathways involved in disease etiology. An important discovery came with the demonstration that over-expression of human tau in Drosophila leads to premature mortality and neuronal dysfunction including neurodegeneration, recapitulating some key neuropathological features of the human disease. The simplicity of handling invertebrate models combined with the availability of a diverse range of experimental resources make these models, in particular Drosophila a powerful invertebrate screening tool. Consequently, several large-scale screens have been performed using Drosophila, to identify modifiers of tau toxicity. The screens have revealed not only common cellular and molecular pathways, but in some instances the same modifier has been independently identified in two or more screens suggesting a possible role for these modifiers in regulating tau toxicity. The purpose of this review is to discuss the genetic modifier screens on tauopathies performed in Drosophila and C. elegans models, and to highlight the common cellular and molecular pathways that have emerged from these studies. Here, we summarize results of tau toxicity screens providing mechanistic insights into pathological alterations in tauopathies. Key pathways or modifiers that have been identified are associated with a broad range of processes including, but not limited to, phosphorylation, cytoskeleton organization, axonal transport, regulation of cellular proteostasis, transcription, RNA metabolism, cell cycle regulation, and apoptosis. We discuss the utility and application of invertebrate models in elucidating the cellular and molecular functions of novel and uncharacterized disease modifiers identified in large-scale screens as well as for investigating the function of genes identified as risk factors in genome-wide association studies from human patients in the post-genomic era. In this review, we combined and summarized several large-scale modifier screens performed in invertebrate models to identify modifiers of tau toxicity. A summary of the screens show that diverse cellular processes are implicated in the modification of tau toxicity. Kinases and phosphatases are the most predominant class of modifiers followed by components required for cellular proteostasis and axonal transport and cytoskeleton elements.

Quantitative interaction proteomics of neurodegenerative disease proteins.

Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.