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Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show nonneural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood. We developed multiple models to investigate the impact of CNS stressors on motor function and found that Escherichia coli infections and SARS-CoV-2 protein expression caused reactive oxygen species (ROS) to accumulate in the brain. ROS induced expression of the cytokine Unpaired 3 (Upd3) in Drosophila and its ortholog, IL-6, in mice. CNS-derived Upd3/IL-6 activated the JAK-STAT pathway in skeletal muscle, which caused muscle mitochondrial dysfunction and impaired motor function. We observed similar phenotypes after expressing toxic amyloid-β (Aβ42) in the CNS. Infection and chronic disease therefore activate a systemic brain-muscle signaling axis in which CNS-derived cytokines bypass the connectome and directly regulate muscle physiology, highlighting IL-6 as a therapeutic target to treat disease-associated muscle dysfunction.

Background: The accumulation of amyloid beta (Aβ) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aβ fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts., Methods: To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of Aβ immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aβ42 flies, and Aβ42 seeded cultured neurons., Results: Amyloid fibrils are primarily composed by Aβ42 and unexpectedly harbor Aβ38 but generally lack Aβ40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, we confirmed 57 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic Aβ42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, we could functionally validate 20 genes as modifiers of Aβ42 toxicity in vivo., Conclusions: These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology., (© 2023. Editorial Group and BioMed Central Ltd., part of Springer Nature.)

Alzheimer's disease (AD) is the most prevalent neurodegenerative dementia in older adults worldwide. Sadly, there are no disease-modifying therapies available for treatment due to the multifactorial complexity of the disease. AD is pathologically characterized by extracellular deposition of amyloid beta (Aβ) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. Increasing evidence suggest that Aβ also accumulates intracellularly, which may contribute to the pathological mitochondrial dysfunction observed in AD. According with the mitochondrial cascade hypothesis, mitochondrial dysfunction precedes clinical decline and thus targeting mitochondria may result in new therapeutic strategies. Unfortunately, the precise mechanisms connecting mitochondrial dysfunction with AD are largely unknown. In this review, we will discuss how the fruit fly Drosophila melanogaster is contributing to answer mechanistic questions in the field, from mitochondrial oxidative stress and calcium dysregulation to mitophagy and mitochondrial fusion and fission. In particular, we will highlight specific mitochondrial insults caused by Aβ and tau in transgenic flies and will also discuss a variety of genetic tools and sensors available to study mitochondrial biology in this flexible organism. Areas of opportunity and future directions will be also considered., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Varte, Munkelwitz and Rincon-Limas.)

Objective: To identify potential diagnostic and prognostic biomarkers for clinical management and clinical trials in amyotrophic lateral sclerosis., Methods: We analysed proteomics data of ALS patient-induced pluripotent stem cell-derived motor neurons available through the AnswerALS consortium. After stratifying patients using clinical ALSFRS-R and ALS-CBS scales, we identified differentially expressed proteins indicative of ALS disease severity and progression rate as candidate ALS-related and prognostic biomarkers. Pathway analysis for identified proteins was performed using STITCH. Protein sets were correlated with the effects of drugs using the Connectivity Map tool to identify compounds likely to affect similar pathways. RNAi screening was performed in a Drosophila TDP-43 ALS model to validate pathological relevance. A statistical classification machine learning model was constructed using ridge regression that uses proteomics data to differentiate ALS patients from controls., Results: We identified 76, 21, 71 and 1 candidate ALS-related biomarkers and 22, 41, 27 and 64 candidate prognostic biomarkers from patients stratified by ALSFRS-R baseline, ALSFRS-R progression slope, ALS-CBS baseline and ALS-CBS progression slope, respectively. Nineteen proteins enhanced or suppressed pathogenic eye phenotypes in the ALS fly model. Nutraceuticals, dopamine pathway modulators, statins, anti-inflammatories and antimicrobials were predicted starting points for drug repurposing using the connectivity map tool. Ten diagnostic biomarker proteins were predicted by machine learning to identify ALS patients with high accuracy and sensitivity., Interpretation: This study showcases the powerful approach of iPSC-motor neuron proteomics combined with machine learning and biological confirmation in the prediction of novel mechanisms and diagnostic and predictive biomarkers in ALS., (© 2022 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Background: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology., Methods: We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo., Results: We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD., Conclusion: Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration., (© 2022. The Author(s).)

Background: Sodium present in NaCl is a fundamental nutrient required for many physiological processes but high salt consumption in western world is contributing health risk to all age individuals. Although high salt detection pathways have been studied in great detail, the mechanisms that regulate high salt consumption in animals are largely unknown. To understand how pre-exposure to high NaCl diet modulates subsequent feeding behavior, we looked into the neural mechanisms of high NaCl consumption in adult Drosophila., Method: We used Neuro-Genetics, imaging and behavioral assays to determine how flies respond to high NaCl exposure., Result: We studied the neural mechanism by which flies modify their acceptance of high salt as a function of diet, where a long-term high-salt exposure increases taste sensitivities of pharyngeal LSO (Labral sense organ) neurons and enhances high salt intake. We discovered that exposing flies to high NaCl diet(200mM NaCl in fly food) for three days modify their feeding responses to high levels of salt. High NaCl fed flies show decline in high salt aversion under starvation. Genetic suppression of LSO pharyngeal neurons in high NaCl fed flies inhibits excessive salt intake. We found that this modulation requires functional LSO neurons and starvation state, and that multiple independent taste receptor neurons and pathways are involved in this process. Silencing any one of multiple LSO neuronal types inhibits excessive salt intake., Conclusion: Our data support the idea that high dietary salt modulates and reshapes salt and other taste curves to promote over consumption of food in flies. Our study suggest flies can adapt to the amount of salt ingested over several days, indicating the presence of a critical mechanism to reset the salt appetite and related neural circuits. Identification of new molecular sensors for salt and related neural controls such as hormones, neuropeptides, and neurotransmitters may yield insights into the coordination of processes in the nervous system.

Nuclear depletion, abnormal modification, and cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43) are linked to a group of fatal neurodegenerative diseases called TDP-43 proteinopathies, which include amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Although our understanding of the physiological function of TDP-43 is rapidly advancing, the molecular mechanisms associated with its pathogenesis remain poorly understood. Accumulating evidence suggests that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are important players in TDP-43 pathology. However, while neurons derived from autopsied ALS and FTLD patients revealed TDP-43 deposits in the ER and displayed UPR activation, data originated from in vitro and in vivo TDP-43 models produced contradictory results. In this review, we will explore the complex interplay between TDP-43 pathology, ER stress, and the UPR by breaking down the evidence available in the literature and addressing the reasons behind these discrepancies. We also highlight underexplored areas and key unanswered questions in the field. A better synchronization and integration of methodologies, models, and mechanistic pathways will be crucial to discover the true nature of the TDP-43 and ER stress relationship and, ultimately, to uncover the full therapeutic potential of the UPR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Mena, Lopez-Scarim and Rincon-Limas.)

Transactive response DNA binding protein 43 (TDP-43) is a DNA/RNA binding protein involved in transcriptional regulation and RNA processing. It is linked to sporadic and familial amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 is predominantly nuclear, but it translocates to the cytoplasm under pathological conditions. Cytoplasmic accumulation, phosphorylation, ubiquitination and truncation of TDP-43 are the main hallmarks of TDP-43 proteinopathies. Among these processes, the pathways leading to TDP-43 fragmentation remain poorly understood. We review here the molecular and biochemical properties of several TDP-43 fragments, the mechanisms and factors mediating their production, and their potential role in disease progression. We also address the presence of TDP-43 C-terminal fragments in several neurological disorders, including Alzheimer's disease, and highlight their respective implications. Finally, we discuss features of animal models expressing TDP-43 fragments as well as recent therapeutic strategies to approach TDP-43 truncation., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Background: Self-assembly of the amyloid-β (Aβ) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer's disease (AD). However, it is clear that not all forms of Aβ are equally harmful and that linking a specific aggregate to toxicity also depends on the assays and model systems used (Haass et al., J Biol. Chem 269:17741-17748, 1994; Borchelt et al., Neuron 17:1005-1013, 1996). Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aβ deposition and neurodegeneration., Methods: In this study, we examined familial mutations of Aβ that increase aggregation and oligomerization, E22G and ΔE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aβ fusion technology and rAAV2/1-based somatic brain transgenesis in mice to selectively express individual mutant Aβ species in vivo. In parallel, we generated PhiC31-based transgenic Drosophila melanogaster expressing wild-type (WT) and Aβ40 and Aβ42 mutants, fused to the Argos signal peptide to assess the extent of Aβ42-induced toxicity as well as to interrogate the combined effect of different Aβ40 and Aβ42 species., Results: When expressed in the mouse brain for 6 months, Aβ42 E22G, Aβ42 E22Q/D23N, and Aβ42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aβ40WT showed no distinctive phenotype, Aβ40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aβ40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aβ42 E22G, E22Q, and S26C, but not Aβ40, were toxic to the eye of Drosophila. In contrast, flies expressing a copy of Aβ40 (WT or mutants), in addition to Aβ42WT, showed improved phenotypes, suggesting possible protective qualities for Aβ40., Conclusions: These studies suggest that while some Aβ40 mutants form unique amyloid aggregates in mouse brains, they do not exacerbate Aβ42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.

We present here PhotoGal4, a phytochrome B-based optogenetic switch for fine-tuned spatiotemporal control of gene expression in Drosophila explants. This switch integrates the light-dependent interaction between phytochrome B and PIF6 from plants with regulatory elements from the yeast Gal4/UAS system. We found that PhotoGal4 efficiently activates and deactivates gene expression upon red- or far-red-light irradiation, respectively. In addition, this optogenetic tool reacts to different illumination conditions, allowing for fine modulation of the light-dependent response. Importantly, by simply focusing a laser beam, PhotoGal4 induces intricate patterns of expression in a customized manner. For instance, we successfully sketched personalized patterns of GFP fluorescence such as emoji-like shapes or letterform logos in Drosophila explants, which illustrates the exquisite precision and versatility of this tool. Hence, we anticipate that PhotoGal4 will expand the powerful Drosophila toolbox and will provide a new avenue to investigate intricate and complex problems in biomedical research., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development., Competing Interests: The authors have declared that no competing interests exist.

The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.

The Drosophila ovary serves as a model for pioneering studies of stem cell niches, with defined cell types and signaling pathways supporting both germline and somatic stem cells. The establishment of the niche units begins during larval stages with the formation of terminal filament-cap structures; however, the genetics underlying their development remains largely unknown. Here, we show that the transcription factor Lmx1a is required for ovary morphogenesis. We found that Lmx1a is expressed in early ovarian somatic lineages and becomes progressively restricted to terminal filaments and cap cells. We show that Lmx1a is required for the formation of terminal filaments, during the larval-pupal transition. Finally, our data demonstrate that Lmx1a functions genetically downstream of Bric-à-Brac, and is crucial for the expression of key components of several conserved pathways essential to ovarian stem cell niche development. Importantly, expression of chicken Lmx1b is sufficient to rescue the null Lmx1a phenotype, indicating functional conservation across the animal kingdom. These results significantly expand our understanding of the mechanisms controlling stem cell niche development in the fly ovary., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder triggered by the accumulation of soluble assemblies of the amyloid-β42 (Aβ42) peptide. Despite remarkable advances in understanding the pathogenesis of AD, the development of palliative therapies is still lacking. Engineered anti-Aβ42 antibodies are a promising strategy to stall the progression of the disease. Single-chain variable fragment (scFv) antibodies increase brain penetration and offer flexible options for delivery while maintaining the epitope targeting of full antibodies. Here, we examined the ability of two anti-Aβ scFv antibodies targeting the N-terminal (scFv9) and C-terminal (scFv42.2) regions of Aβ42 to suppress the progressive memory decline induced by extracellular deposition of Aβ42 in Drosophila. Using olfactory classical conditioning, we observe that both scFv antibodies significantly improve memory performance in flies expressing Aβ42 in the mushroom body neurons, which are intimately involved in the coding and storage of olfactory memories. The scFvs effectively restore memory at all ages, from one-day post-eclosion to thirty-day-old flies, proving their ability to prevent the toxicity of different pathogenic assemblies. These data support the application of this paradigm of Aβ42-induced memory loss in Drosophila to investigate the protective activity of Aβ42-binding agents in an AD-relevant functional assay.

Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited. Our recent manuscript in PNAS revealed that an engineered form of secreted Hsp70 (secHsp70) is highly protective against toxicity induced by extracellular deposition of the amyloid-β42 (Aβ42) peptide. In this Extra View article, we extend our analysis to other members of the heat shock protein family. We created PhiC31-based transgenic lines for human Hsp27, Hsp40, Hsp60 and Hsp70 and compared their activities in parallel against extracellular Aβ42. Strikingly, only secreted Hsp70 exhibits robust protection against Aβ42-triggered toxicity in the extracellular milieu. These observations indicate that the ability of secHsp70 to suppress Aβ42 insults is quite unique and suggest that targeted secretion of Hsp70 may represent a new therapeutic approach against Aβ42 and other extracellular amyloids. The potential applications of this engineered chaperone are discussed.

Prion diseases (PrD) are unique neurodegenerative conditions with sporadic, genetic, and infectious etiologies. The agent responsible for these pathologies is a misfolded conformation of the prion protein (PrP). Although a process of autocatalytic "conversion" is known to mediate disease transmission, important gaps still remain regarding the physiological function of PrP and its relevance to pathogenesis, the molecular and cellular mechanisms mediating neurotoxicity and transmission, and the PrP conformations responsible for neurotoxicity. New Drosophila models expressing mammalian PrP have revealed physiological insight into PrP function and opened the door to significant progress in prion transmission and PrP neurotoxicity. Importantly, flies expressing human PrP showing a robust eye phenotype will allow performing genetic screens to uncover novel mechanisms mediating PrP neurotoxicity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. Disease heterogeneity is appreciated through varying comorbidities: dysarthria, dysphagia, oculomotor and/or retinal abnormalities, motor neuron pathology, epilepsy, cognitive impairment, autonomic dysfunction, and psychiatric manifestations. Our study focuses on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.3). We detail the clinical phenotype of four SCA13 kindreds that confirm causation of the KCNC3R423H allele. The heralding features demonstrate congenital onset with non-progressive, neurodevelopmental cerebellar hypoplasia and lifetime improvement in motor and cognitive function that implicate compensatory neural mechanisms. Targeted expression of human KCNC3R423H in Drosophila triggers aberrant wing veins, maldeveloped eyes, and fused ommatidia consistent with the neurodevelopmental presentation of patients. Furthermore, human KCNC3R423H expression in mammalian cells results in altered glycosylation and aberrant retention of the channel in anterograde and/or endosomal vesicles. Confirmation of the absence of plasma membrane targeting was based on the loss of current conductance in cells expressing the mutant channel. Mechanistically, genetic studies in Drosophila, along with cellular and biophysical studies in mammalian systems, demonstrate the dominant negative effect exerted by the mutant on the wild-type (WT) protein, which explains dominant inheritance. We demonstrate that ocular co-expression of KCNC3R423H with Drosophila epidermal growth factor receptor (dEgfr) results in striking rescue of the eye phenotype, whereas KCNC3R423H expression in mammalian cells results in aberrant intracellular retention of human epidermal growth factor receptor (EGFR). Together, these results indicate that the neurodevelopmental consequences of KCNC3R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum. Our results therefore confirm the KCNC3R423H allele as causative for SCA13, through a dominant negative effect on KCNC3WT and links with EGFR that account for dominant inheritance, congenital onset, and disease pathology.

Misfolding of the prion protein (PrP) is the key step in the transmission of spongiform pathologies in humans and several animals. Although PrP is highly conserved in mammals, a few changes in the sequence of endogenous PrP are proposed to confer protection to dogs, which were highly exposed to prion during the mad-cow epidemics. D159 is a unique amino acid found in PrP from dogs and other canines that was shown to alter surface charge, but its functional relevance has never been tested in vivo. Here, we show in transgenic Drosophila that introducing the N159D substitution on mouse PrP decreases its turnover. Additionally, mouse PrP-N159D demonstrates no toxicity and accumulates no pathogenic conformations, suggesting that a single D159 substitution is sufficient to prevent PrP conformational change and pathogenesis. Understanding the mechanisms mediating the protective activity of D159 is likely to lessen the burden of prion diseases in humans and domestic animals., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is the most prevalent of a large group of related proteinopathies for which there is currently no cure. Here, we used Drosophila to explore a strategy to block Aβ42 neurotoxicity through engineering of the Heat shock protein 70 (Hsp70), a chaperone that has demonstrated neuroprotective activity against several intracellular amyloids. To target its protective activity against extracellular Aβ42, we added a signal peptide to Hsp70. This secreted form of Hsp70 (secHsp70) suppresses Aβ42 neurotoxicity in adult eyes, reduces cell death, protects the structural integrity of adult neurons, alleviates locomotor dysfunction, and extends lifespan. SecHsp70 binding to Aβ42 through its holdase domain is neuroprotective, but its ATPase activity is not required in the extracellular space. Thus, the holdase activity of secHsp70 masks Aβ42 neurotoxicity by promoting the accumulation of nontoxic aggregates. Combined with other approaches, this strategy may contribute to reduce the burden of AD and other extracellular proteinopathies., Competing Interests: The authors declare no conflict of interest.

Alzheimer׳s disease is one of the main causes of dementia in the elderly and its frequency is on the rise worldwide. It is considered the result of complex interactions between genetic and environmental factors, being many of them unknown. Therefore, there is a dire necessity for the identification of novel molecular players for the understanding of this disease. In this data article we determined the protein expression profiles of whole protein extracts from cortex regions of brains from patients with Alzheimer׳s disease in comparison to a normal brain. We identified 721 iTRAQ-labeled polypeptides with more than 95% in confidence. We analyzed all proteins that changed in their expression level and located them in the KEGG metabolic pathways, as well as in the mitochondrial complexes of the electron transport chain and ATP synthase. In addition, we analyzed the over- and sub-expressed polypeptides through IPA software, specifically Core I and Biomarkers I modules. Data in this article is related to the research article "Identification of proteins that are differentially expressed in brains with Alzheimer's disease using iTRAQ labeling and tandem mass spectrometry" (Minjarez et al., 2016) [1].

Alzheimer's disease is one of the leading causes of dementia in the elderly. It is considered the result of complex events involving both genetic and environmental factors. To gain further insights into this complexity, we quantitatively analyzed the proteome of cortex region of brains from patients diagnosed with Alzheimer's disease, using a bottom-up proteomics approach. We identified 721 isobaric-tagged polypeptides. From this universe, 61 were found overexpressed and 69 subexpressed in three brains with Alzheimer's disease in comparison to a normal brain. We determined that the most affected processes involving the overexpressed polypeptides corresponded to ROS and stress responses. For the subexpressed polypeptides, the main processes affected were oxidative phosphorylation, organellar acidification and cytoskeleton. We used Drosophila to validate some of the hits, particularly those non-previously described as connected with the disease, such as Sideroflexin and Phosphoglucomutase-1. We manipulated their homolog genes in Drosophila models of Aβ- and Tau-induced pathology. We found proteins that can either modify Aβ toxicity, Tau toxicity or both, suggesting specific interactions with different pathways. This approach illustrates the potential of Drosophila to validate hits after MS studies and suggest that model organisms should be included in the pipeline to identify relevant targets for Alzheimer's disease., Biological Significance: We report a set of differentially expressed proteins in three Alzheimer's disease brains in comparison to a normal brain. Our analyses allowed us to identify that the main affected pathways were ROS and stress responses, oxidative phosphorylation, organellar acidification and cytoskeleton. We validated some identified proteins using genetic models of Amyloid-β and Tau-induced pathology in Drosophila melanogaster. With this approach, Sideroflexin and Phosphoglucomutase-1 were identified as novel proteins connected with Alzheimer's disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is the leading cause of dementia and the most common neurodegenerative disorder. AD is mostly a sporadic disorder and its main risk factor is age, but mutations in three genes that promote the accumulation of the amyloid-β (Aβ42) peptide revealed the critical role of amyloid precursor protein (APP) processing in AD. Neurofibrillary tangles enriched in tau are the other pathological hallmark of AD, but the lack of causative tau mutations still puzzles researchers. Here, we describe the contribution of a powerful invertebrate model, the fruit fly Drosophila melanogaster, to uncover the function and pathogenesis of human APP, Aβ42, and tau. APP and tau participate in many complex cellular processes, although their main function is microtubule stabilization and the to-and-fro transport of axonal vesicles. Additionally, expression of secreted Aβ42 induces prominent neuronal death in Drosophila, a critical feature of AD, making this model a popular choice for identifying intrinsic and extrinsic factors mediating Aβ42 neurotoxicity. Overall, Drosophila has made significant contributions to better understand the complex pathology of AD, although additional insight can be expected from combining multiple transgenes, performing genome-wide loss-of-function screens, and testing anti-tau therapies alone or in combination with Aβ42., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant disease resulting from mutations in KCNC3 (Kv3.3), a voltage-gated potassium channel. The KCNC3(R420H) mutation was first identified as causative for SCA13 in a four-generation Filipino kindred with over 20 affected individuals. Electrophysiological analyses in oocytes previously showed that this mutation did not lead to a functional channel and displayed a dominant negative phenotype. In an effort to identify the molecular basis of this allelic form of SCA13, we first determined that human KCNC3(WT) and KCNC3(R420H) display disparate post-translational modifications, and the mutant protein has reduced complex glycan adducts. Immunohistochemical analyses demonstrated that KCNC3(R420H) was not properly trafficking to the plasma membrane and surface biotinylation demonstrated that KCNC3(R420H) exhibited only 24% as much surface expression as KCNC3(WT). KCNC3(R420H) trafficked through the ER but was retained in the Golgi. KCNC3(R420H) expression results in altered Golgi and cellular morphology. Electron microscopy of KCNC3(R420H) localization further supports retention in the Golgi. These results are specific to the KCNC3(R420H) allele and provide new insight into the molecular basis of disease manifestation in SCA13., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Prion diseases are rare and aggressive neurodegenerative disorders caused by the accumulation of misfolded, toxic conformations of the prion protein (PrP). Therapeutic strategies directed at reducing the levels of PrP offer the best chance of delaying or halting disease progression. The challenge, though, is to define pharmacologic targets that result in reduced PrP levels. We previously reported that expression of wild type hamster PrP in flies induces progressive locomotor dysfunction and accumulation of pathogenic PrP conformations, while co-expression of human Hsp70 delayed these changes. To validate the therapeutic potential of Hsp70, we treated flies with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone. Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity. Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output. These findings can have important therapeutic applications for the devastating prion diseases and other related proteinopathies.

Prion diseases encompass a diverse group of neurodegenerative conditions characterized by the accumulation of misfolded prion protein (PrP) isoforms. Other conformational variants of PrP have also been proposed to contribute to neurotoxicity in prion diseases, including misfolded intermediates as well as cytosolic and transmembrane isoforms. To better understand PrP neurotoxicity, we analyzed the role of two highly conserved methionines in helix 3 on PrP biogenesis, folding and pathogenesis. Expression of the PrP-M205S and -M205,212S mutants in Drosophila led to hyperglycosylation, intracellular accumulation and widespread conformational changes due to failure of oxidative folding. Surprisingly, PrP-M205S and -M205,212S acquired a transmembrane topology (Ctm) previously linked to mutations in the signal peptide (SP) and the transmembrane domain (TMD). PrP-M205,212S also disrupted the accumulation of key neurodevelopmental proteins in lipid rafts, resulting in shortened axonal projections. These results uncover a new role for the hydrophobic domain in promoting oxidative folding and preventing the formation of neurotoxic Ctm PrP, mechanisms that may be relevant in the pathogenesis of both inherited and sporadic prion diseases.

Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. In this report we describe a new Drosophila expression vector (p∆TubHA4C) for ubiquitous expression of coding sequences under the control of a minimal 0.9 kb promoter of α1 tubulin (α1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the ∆Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9 kb ΔTub promoter induced similar expression levels to the intact 2.6 kb α1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible ∆TubHA4C vector.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Several neurodegenerative disorders are characterized by protein misfolding, a phenomenon that results in perturbation of cellular homeostasis. We recently identified the protective activity of the ER stress response factor XBP1 (X-box binding protein 1) against Amyloid-ß1-42 (Aß42) neurotoxicity in cellular and Drosophila models of Alzheimer's disease. Additionally, subtoxic concentrations of Aß42 soluble aggregates (oligomers) induced accumulation of spliced (active) XBP1 transcripts, supporting the involvement of the ER stress response in Aß42 neurotoxicity. Here, we tested the ability of three additional disease-related amyloidogenic proteins to induce ER stress by analyzing XBP1 activation at the RNA level. Treatment of human SY5Y neuroblastoma cells with homogeneous preparations of α-Synuclein (α-Syn), Prion protein (PrP106-126), and British dementia amyloid peptide (ABri1-34) confirmed the high toxicity of oligomers compared to monomers and fibers. Additionally, α-Syn oligomers, but not monomers or fibers, demonstrated potent induction of XBP1 splicing. On the other hand, PrP106-126 and ABri1-34 did not activate XBP1. These results illustrate the biological complexity of these structurally related assemblies and argue that oligomer toxicity depends on the activation of amyloid-specific cellular responses.

Alzheimer's, Parkinson's, and Huntington's disease are complex neurodegenerative conditions with high prevalence characterized by protein misfolding and deposition in the brain. Considerable progress has been made in the last two decades in identifying the genes and proteins responsible for several human 'proteinopathies'. A wide variety of wild type and mutant proteins associated with neurodegenerative conditions are structurally unstable, misfolded, and acquire conformations rich in ß-sheets (ß-state). These conformers form highly toxic self-assemblies that kill the neurons in stereotypical patterns. Unfortunately, the detailed understanding of the molecular and cellular perturbations caused by these proteins has not produced a single disease-modifying therapy. More than a decade ago, several groups demonstrated that human proteinopathies reproduce critical features of the disease in transgenic flies, including protein mis-folding, aggregation, and neurotoxicity. These initial reports led to an explosion of research that has contributed to a better understanding of the molecular mechanisms regulating conformational dynamics and neurotoxic cascades. To remain relevant in this competitive environment, Drosophila models will need to expand their flexible, innovative, and multidisciplinary approaches to find new discoveries and translational applications.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder clinically characterized by progressive cognitive impairment. A prominent pathologic hallmark in the AD brain is the abnormal accumulation of the amyloid-β 1-42 peptide (Aβ), but the exact pathways mediating Aβ neurotoxicity remain enigmatic. Endoplasmic reticulum (ER) stress is induced during AD, and has been indirectly implicated as a mediator of Aβ neurotoxicity. We report here that Aβ activates the ER stress response factor X-box binding protein 1 (XBP1) in transgenic flies and in mammalian cultured neurons, yielding its active form, the transcription factor XBP1s. XBP1s shows neuroprotective activity in two different AD models, flies expressing Aβ and mammalian cultured neurons treated with Aβ oligomers. Trying to identify the mechanisms mediating XBP1s neuroprotection, we found that in PC12 cells treated with Aβ oligomers, XBP1s prevents the accumulation of free calcium (Ca(2+)) in the cytosol. This protective activity can be mediated by the downregulation of a specific isoform of the ryanodine Ca(2+) channel, RyR3. In support of this observation, a mutation in the only ryanodine receptor (RyR) in flies also suppresses Aβ neurotoxicity, indicating the conserved mechanisms between the two AD models. These results underscore the functional relevance of XBP1s in Aβ toxicity, and uncover the potential of XBP1 and RyR as targets for AD therapeutics.

The Prion protein (PrP) is a membrane-tethered glycoprotein that plays a central role in a unique class of neurodegenerative diseases that affect humans and other mammals. Prion diseases have genetic and sporadic origins, but their infectious nature sets them apart from other neurodegenerative disorders. According to the "protein-only" hypothesis, misfolded PrP conformers (prions) are responsible for both spongiform degeneration of the brain and disease transmissibility. Thus, understanding PrP conformational dynamics is key to developing effective therapies. Classic studies showing the different susceptibility to prion disease in mammals have recently found support in structural and transgenic studies with PrP from susceptible (mouse, hamster) and resistant (rabbit, horse, dog) animals. These studies identify key residues in PrP that determine both PrP structure and its propensity to acquire a β-structure conformation proposed to be neurotoxic.

The prion protein (PrP) is best known for its association with prion diseases. However, a controversial new role for PrP in Alzheimer disease (AD) has recently emerged. In vitro studies and mouse models of AD suggest that PrP may be involved in AD pathogenesis through a highly specific interaction with amyloid-β (Aβ42) oligomers. Immobilized recombinant human PrP (huPrP) also exhibited high affinity and specificity for Aβ42 oligomers. Here we report the novel finding that aggregated forms of huPrP and Aβ42 are co-purified from AD brain extracts. Moreover, an anti-PrP antibody and an agent that specifically binds to insoluble PrP (iPrP) co-precipitate insoluble Aβ from human AD brain. Finally, using peptide membrane arrays of 99 13-mer peptides that span the entire sequence of mature huPrP, two distinct types of Aβ binding sites on huPrP are identified in vitro. One specifically binds to Aβ42 and the other binds to both Aβ42 and Aβ40. Notably, Aβ42-specific binding sites are localized predominantly in the octapeptide repeat region, whereas sites that bind both Aβ40 and Aβ42 are mainly in the extreme N-terminal or C-terminal domains of PrP. Our study suggests that iPrP is the major PrP species that interacts with insoluble Aβ42 in vivo. Although this work indicated the interaction of Aβ42 with huPrP in the AD brain, the pathophysiological relevance of the iPrP/Aβ42 interaction remains to be established.

Prion diseases are neurodegenerative disorders caused by misfolding of the normal prion protein (PrP) into a pathogenic "scrapie" conformation. To better understand the cellular and molecular mechanisms that govern the conformational changes (conversion) of PrP, we compared the dynamics of PrP from mammals susceptible (hamster and mouse) and resistant (rabbit) to prion diseases in transgenic flies. We recently showed that hamster PrP induces spongiform degeneration and accumulates into highly aggregated, scrapie-like conformers in transgenic flies. We show now that rabbit PrP does not induce spongiform degeneration and does not convert into scrapie-like conformers. Surprisingly, mouse PrP induces weak neurodegeneration and accumulates small amounts of scrapie-like conformers. Thus, the expression of three highly conserved mammalian prion proteins in transgenic flies uncovered prominent differences in their conformational dynamics. How these properties are encoded in the amino acid sequence remains to be elucidated.

The fruit fly Drosophila melanogaster has been a favored tool for genetic studies for over 100 years and has become an excellent model system to study development, signal transduction, cell biology, immunity and behavior. The relevance of Drosophila to humans is perhaps best illustrated by the fact that more than 75% of the genes identified in human diseases have counterparts in Drosophila. During the last decade, many fly models of neurodegenerative disorders have contributed to the identification of novel pathways mediating pathogenesis. However, the development of prion disease models in flies has been remarkably challenging. We recently reported a Drosophila model of sporadic prion pathology that shares relevant features with the typical disease in mammals. This new model provides the basis to explore relevant aspects of the biology of the prion protein, such as uncovering the genetic mechanisms regulating prion protein misfolding and prion-induced neurodegeneration, in a dynamic, genetically tractable in vivo system.

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C)) converts into a misfolded isoform (PrP(Sc)) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C); in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc), and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc)-specific conformational epitopes. In contrast to PrP(Sc) from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc). Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc)-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP(Sc) is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders., Competing Interests: The authors have declared that no competing interests exist.

apterous specifies dorsal cell fate and directs outgrowth of the wing during Drosophila wing development. Here we show that, in vertebrates, these functions appear to be performed by two separate proteins. Lmx-1 is necessary and sufficient to specify dorsal identity and Lhx2 regulates limb outgrowth. Our results suggest that Lhx2 is closer to apterous than Lmx-1, yet, in vertebrates, Lhx2 does not specify dorsal cell fate. This implies that in vertebrates, unlike Drosophila, limb outgrowth can be dissociated from the establishment of the dorsoventral axis.

There is growing evidence that some genetic predisposition is important in the etiology of schizophrenia. We have sought to implicate a major gene by performing a candidate gene association study comparing the allele frequencies of seven restriction fragment length polymorphisms (RFLPs) at six loci in both a psychiatrically normal control group (N = 51) and an affected (schizophrenia or schizoaffective disorder) group (N = 55). Each group comprised Caucasians of northern European origin. The candidate areas (D5S39, D5S78, dopamine receptor D2 (DRD2), D11S29, porphobilinogen deaminase (PBGD), and D11S84) were selected on the basis of prior cytogenetic findings in schizophrenics, linkage studies, and/or implicated gene products. The presence of a polymorphic ApaLI site within the PBGD gene showed a significant association with the presence of illness (P = 0.02). The relative risk of possessing the allele with the ApaLI site was 2.10. No significant association was found with any of the six other RFLPs. Our data suggests that either the PBGD gene itself or an unknown gene linked to and/or in linkage disequilibrium with the PBGD locus predisposes some individuals to schizophrenia. Independent replication of these findings will be required to determine their relevance to schizophrenia.