A systems-biology approach connects aging mechanisms with Alzheimer's disease pathogenesis.

Age is the strongest risk factor for developing Alzheimer's disease, the most common neurodegenerative disorder. However, the mechanisms connecting advancing age to neurodegeneration in Alzheimer's disease are incompletely understood. We conducted an unbiased, genome-scale, forward genetic screen for age-associated neurodegeneration in Drosophila to identify the underlying biological processes required for maintenance of aging neurons. To connect genetic screen hits to Alzheimer's disease pathways, we measured proteomics, phosphoproteomics, and metabolomics in Drosophila models of Alzheimer's disease. We further identified Alzheimer's disease human genetic variants that modify expression in disease-vulnerable neurons. Through multi-omic, multi-species network integration of these data, we identified relationships between screen hits and tau-mediated neurotoxicity. Furthermore, we computationally and experimentally identified relationships between screen hits and DNA damage in Drosophila and human iPSC-derived neural progenitor cells. Our work identifies candidate pathways that could be targeted to attenuate the effects of age on neurodegeneration and Alzheimer's disease.

Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.

Missense mutations in the gene encoding the microtubule-associated protein TAU (current and approved symbol is MAPT) cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human TAU in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas-mediated gene editing to model frontotemporal dementia caused by the TAU P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for Tau P251L display age-dependent neurodegeneration, display metabolic defects, and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies, we performed single-cell RNA sequencing on approximately 130,000 cells from brains of Tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified. Gene expression was also altered in glial cells, suggestive of non-cell-autonomous regulation. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell type-specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy that faithfully recapitulates the genetic context and phenotypic features of the human disease, and use the results of comprehensive single-cell sequencing analysis to outline pathways of neurotoxicity and highlight the potential role of non-cell-autonomous changes in glia.

Deep sequencing of proteotoxicity modifier genes uncovers a Presenilin-2/beta-amyloid-actin genetic risk module shared among alpha-synucleinopathies.

Whether neurodegenerative diseases linked to misfolding of the same protein share genetic risk drivers or whether different protein-aggregation pathologies in neurodegeneration are mechanistically related remains uncertain. Conventional genetic analyses are underpowered to address these questions. Through careful selection of patients based on protein aggregation phenotype (rather than clinical diagnosis) we can increase statistical power to detect associated variants in a targeted set of genes that modify proteotoxicities. Genetic modifiers of alpha-synuclein (ɑS) and beta-amyloid (Aß) cytotoxicity in yeast are enriched in risk factors for Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. Here, along with known AD/PD risk genes, we deeply sequenced exomes of 430 ɑS/Aß modifier genes in patients across alpha-synucleinopathies (PD, Lewy body dementia and multiple system atrophy). Beyond known PD genes GBA1 and LRRK2, rare variants AD genes (CD33, CR1 and PSEN2) and Aß toxicity modifiers involved in RhoA/actin cytoskeleton regulation (ARGHEF1, ARHGEF28, MICAL3, PASK, PKN2, PSEN2) were shared risk factors across synucleinopathies. Actin pathology occurred in iPSC synucleinopathy models and RhoA downregulation exacerbated ɑS pathology. Even in sporadic PD, the expression of these genes was altered across CNS cell types. Genome-wide CRISPR screens revealed the essentiality of PSEN2 in both human cortical and dopaminergic neurons, and PSEN2 mutation carriers exhibited diffuse brainstem and cortical synucleinopathy independent of AD pathology. PSEN2 contributes to a common-risk signal in PD GWAS and regulates ɑS expression in neurons. Our results identify convergent mechanisms across synucleinopathies, some shared with AD.

Pathways controlling neurotoxicity and proteostasis in mitochondrial complex I deficiency.

Neuromuscular disorders caused by dysfunction of the mitochondrial respiratory chain are common, severe and untreatable. We recovered a number of mitochondrial genes, including electron transport chain components, in a large forward genetic screen for mutations causing age-related neurodegeneration in the context of proteostasis dysfunction. We created a model of complex I deficiency in the Drosophila retina to probe the role of protein degradation abnormalities in mitochondrial encephalomyopathies. Using our genetic model, we found that complex I deficiency regulates both the ubiquitin/proteasome and autophagy/lysosome arms of the proteostasis machinery. We further performed an in vivo kinome screen to uncover new and potentially druggable mechanisms contributing to complex I related neurodegeneration and proteostasis failure. Reduction of RIOK kinases and the innate immune signaling kinase pelle prevented neurodegeneration in complex I deficiency animals. Genetically targeting oxidative stress, but not RIOK1 or pelle knockdown, normalized proteostasis markers. Our findings outline distinct pathways controlling neurodegeneration and protein degradation in complex I deficiency and introduce an experimentally facile model in which to study these debilitating and currently treatment-refractory disorders.

Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.

Missense mutations in the gene encoding the microtubule-associated protein tau cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human tau in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease, but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas mediated gene editing to model frontotemporal dementia caused by the tau P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for tau P251L display age-dependent neurodegeneration, metabolic defects and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies we performed single-cell RNA sequencing on approximately 130,000 cells from brains of tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified and non-cell autonomously in glial cells. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell-type specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy, which faithfully recapitulates the genetic context and phenotypic features of the human disease and use the results of comprehensive single cell sequencing analysis to outline pathways of neurotoxicity and highlight the role of non-cell autonomous changes in glia.

A Drosophila model relevant to chemotherapy-related cognitive impairment.

Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, our goal was to validate a Drosophila model relevant to CRCI. We administered the chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin to adult Drosophila. Neurologic deficits were observed with all tested chemotherapies, with doxorubicin and in particular cisplatin also resulting in memory deficits. We then performed histologic and immunohistochemical analysis of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, our Drosophila model relevant to CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. Our new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI (and chemotherapy-induced neurotoxicity more generally) and pharmacologic screens to identify disease-modifying therapies.

Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging.

Aß peptides derived from the amyloid precursor protein (APP) have been strongly implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP and the importance of that role in neurodegenerative disease is less clear. We recover the Drosophila ortholog of APP, Appl, in an unbiased forward genetic screen for neurodegeneration mutants. We perform comprehensive single cell transcriptional and proteomic studies of Appl mutant flies to investigate Appl function in the aging brain. We find an unexpected role for Appl in control of multiple cellular pathways, including translation, mitochondrial function, nucleic acid and lipid metabolism, cellular signaling and proteostasis. We mechanistically define a role for Appl in regulating autophagy through TGFß signaling and document the broader relevance of our findings using mouse genetic, human iPSC and in vivo tauopathy models. Our results demonstrate a conserved role for APP in controlling age-dependent proteostasis with plausible relevance to Alzheimer's disease.

Circular RNAs in the human brain are tailored to neuron identity and neuropsychiatric disease.

Little is known about circular RNAs (circRNAs) in specific brain cells and human neuropsychiatric disease. Here, we systematically identify over 11,039 circRNAs expressed in vulnerable dopamine and pyramidal neurons laser-captured from 190 human brains and non-neuronal cells using ultra-deep, total RNA sequencing. 1526 and 3308 circRNAs are custom-tailored to the cell identity of dopamine and pyramidal neurons and enriched in synapse pathways. 29% of Parkinson's and 12% of Alzheimer's disease-associated genes produced validated circRNAs. circDNAJC6, which is transcribed from a juvenile-onset Parkinson's gene, is already dysregulated during prodromal, onset stages of common Parkinson's disease neuropathology. Globally, addiction-associated genes preferentially produce circRNAs in dopamine neurons, autism-associated genes in pyramidal neurons, and cancers in non-neuronal cells. This study shows that circular RNAs in the human brain are tailored to neuron identity and implicate circRNA-regulated synaptic specialization in neuropsychiatric diseases.

α-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to ß-spectrin.

α-synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. We show that α-synuclein binds directly to ß-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders we demonstrate that ß-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of ß-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na+/K+ ATPase, is mislocalized when human α-synuclein is expressed in Drosophila. Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. We examine the same pathway in human neurons and find that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na+/K+ ATPase, and membrane potential depolarization. Our findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies leads to neuronal dysfunction and death.

A Drosophila model of chemotherapy-related cognitive impairment.

Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, our goal was to validate a Drosophila model of CRCI. We administered the chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin to adult Drosophila. Neurocognitive deficits were observed with all tested chemotherapies, especially cisplatin. We then performed histologic and immunohistochemical analysis of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, our Drosophila model of CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. Our new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI and pharmacologic screens to identify novel therapies to ameliorate CRCI.

STAT3 Drives GFAP Accumulation and Astrocyte Pathology in a Mouse Model of Alexander Disease.

Alexander disease (AxD) is caused by mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament expressed by astrocytes in the central nervous system. AxD-associated mutations cause GFAP aggregation and astrogliosis, and GFAP is elevated with the astrocyte stress response, exacerbating mutant protein toxicity. Studies in mouse models suggest disease severity is tied to Gfap expression levels, and signal transducer and activator of transcription (STAT)-3 regulates Gfap during astrocyte development and in response to injury and is activated in astrocytes in rodent models of AxD. In this report, we show that STAT3 is also activated in the human disease. To determine whether STAT3 contributes to GFAP elevation, we used a combination of genetic approaches to knockout or reduce STAT3 activation in AxD mouse models. Conditional knockout of Stat3 in cells expressing Gfap reduced Gfap transactivation and prevented protein accumulation. Astrocyte-specific Stat3 knockout in adult mice with existing pathology reversed GFAP accumulation and aggregation. Preventing STAT3 activation reduced markers of reactive astrocytes, stress-related transcripts, and microglial activation, regardless of disease stage or genetic knockout approach. These results suggest that pharmacological inhibition of STAT3 could potentially reduce GFAP toxicity and provide a therapeutic benefit in patients with AxD.

Circular RNAs in the human brain are tailored to neuron identity and neuropsychiatric disease.

Little is known about circular RNAs (circRNAs) in specific brain cells and human neuropsychiatric disease. Here, we systematically identified over 11,039 circRNAs expressed in vulnerable dopamine and pyramidal neurons laser-captured from 190 human brains and non-neuronal cells using ultra-deep, total RNA sequencing. 1,526 and 3,308 circRNAs were custom-tailored to the cell identity of dopamine and pyramidal neurons and enriched in synapse pathways. 88% of Parkinson's and 80% of Alzheimer's disease-associated genes produced circRNAs. circDNAJC6, produced from a juvenile-onset Parkinson's gene, was already dysregulated during prodromal, onset stages of common Parkinson's disease neuropathology. Globally, addiction-associated genes preferentially produced circRNAs in dopamine neurons, autism-associated genes in pyramidal neurons, and cancers in non-neuronal cells. This study shows that circular RNAs in the human brain are tailored to neuron identity and implicate circRNA- regulated synaptic specialization in neuropsychiatric diseases.

α-Synuclein Promotes Neuronal Dysfunction and Death by Disrupting the Binding of Ankyrin to ß-Spectrin.

α-Synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. We show that α-synuclein binds directly to ß-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders, we demonstrate that ß-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of ß-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na+/K+ ATPase, is mislocalized when human α-synuclein is expressed in Drosophila Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. We examine the same pathway in human neurons and find that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na+/K+ ATPase, and membrane potential depolarization. Our findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies lead to neuronal dysfunction and death.SIGNIFICANCE STATEMENT The small synaptic vesicle associate protein α-synuclein plays a critical role in the pathogenesis of Parkinson's disease and related disorders, but the disease-relevant binding partners of α-synuclein and proximate pathways critical for neurotoxicity require further definition. We show that α-synuclein binds directly to ß-spectrin, a key cytoskeletal protein required for localization of plasma membrane proteins and maintenance of neuronal viability. Binding of α-synuclein to ß-spectrin alters the organization of the spectrin-ankyrin complex, which is critical for localization and function of integral membrane proteins, including Na+/K+ ATPase. These finding outline a previously undescribed mechanism of α-synuclein neurotoxicity and thus suggest potential new therapeutic approaches in Parkinson's disease and related disorders.

Nicotine-Mediated Rescue of α-Synuclein Toxicity Requires Synaptic Vesicle Glycoprotein 2 in Drosophila.

BACKGROUND: Parkinson's disease (PD) is characterized by α-synuclein aggregation and loss of dopamine neurons. Risk of PD arises due to a combination of genetic and environmental factors, which may interact, termed gene-environment (G×E) interactions. An inverse association between smoking and the risk of PD is well established, and a previous genome-wide G×E interaction study identified genetic variation in the synaptic-vesicle glycoprotein 2C (SV2C) locus as an important mediator of the degree to which smoking is inversely associated with PD. OBJECTIVE: We sought to determine the mechanism of the smoking-SV2C interaction in a Drosophila model of PD. METHODS: Flies expressing human α-synuclein in all neurons develop the hallmarks of PD, including motor dysfunction, loss of dopaminergic (DA) neurons, and formation of α-synuclein inclusions. We assessed the effects of increasing doses of nicotine on these parameters of neurodegeneration, in the presence or absence of knockdown of two Drosophila orthologues of SV2, hereafter referred to as SV2L1 and SV2L2. RESULTS: The α-synuclein-expressing flies treated with nicotine had improved locomotion, DA neuron counts, and α-synuclein aggregation. However, in α-synuclein-expressing flies in which SV2L1 and SV2L2 were knocked down, nicotine failed to rescue neurodegeneration. CONCLUSIONS: This work confirms a G×E interaction between nicotine and SV2, defines a role for this interaction in α-synuclein proteostasis, and suggests that future clinical trials on nicotine should consider genetic variation in SV2C. Furthermore, this provides proof of concept that our model can be used for the mechanistic study of G×E, paving the way for the investigation of additional G×E interactions or the identification of novel G×E. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Toward More Accessible Fully Automated 3D Volumetric MRI Decision Trees for the Differential Diagnosis of Multiple System Atrophy, Related Disorders, and Age-Matched Healthy Subjects.

Differentiating multiple system atrophy (MSA) from related neurodegenerative movement disorders (NMD) is challenging. MRI is widely available and automated decision-tree analysis is simple, transparent, and resistant to overfitting. Using a retrospective cohort of heterogeneous clinical MRIs broadly sourced from a tertiary hospital system, we aimed to develop readily translatable and fully automated volumetric diagnostic decision-trees to facilitate early and accurate differential diagnosis of NMDs. 3DT1 MRI from 171 NMD patients (72 MSA, 49 PSP, 50 PD) and 171 matched healthy subjects were automatically segmented using Freesurfer6.0 with brainstem module. Decision trees employing substructure volumes and a novel volumetric pons-to-midbrain ratio (3D-PMR) were produced and tenfold cross-validation performed. The optimal tree separating NMD from healthy subjects selected cerebellar white matter, thalamus, putamen, striatum, and midbrain volumes as nodes. Its sensitivity was 84%, specificity 94%, accuracy 84%, and kappa 0.69 in cross-validation. The optimal tree restricted to NMD patients selected 3D-PMR, thalamus, superior cerebellar peduncle (SCP), midbrain, pons, and putamen as nodes. It yielded sensitivities/specificities of 94/84% for MSA, 72/96% for PSP, and 73/92% PD, with 79% accuracy and 0.62 kappa. There was correct classification of 16/17 MSA, 5/8 PSP, 6/8 PD autopsy-confirmed patients, and 6/8 MRIs that preceded motor symptom onset. Fully automated decision trees utilizing volumetric MRI data distinguished NMD patients from healthy subjects and MSA from other NMDs with promising accuracy, including autopsy-confirmed and pre-symptomatic subsets. Our open-source methodology is well-suited for widespread clinical translation. Assessment in even more heterogeneous retrospective and prospective cohorts is indicated.

Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown etiology characterized by widespread aggregation of the protein alpha-synuclein in neurons and glia. Its orphan status, biological relationship to Parkinson's disease (PD), and rapid progression have sparked interest in drug development. One significant obstacle to therapeutics is disease heterogeneity. Here, we share our process of developing a clinical trial-ready cohort of MSA patients (69 patients in 2 years) within an outpatient clinical setting, and recruiting 20 of these patients into a longitudinal "n-of-few" clinical trial paradigm. First, we deeply phenotype our patients with clinical scales (UMSARS, BARS, MoCA, NMSS, and UPSIT) and tests designed to establish early differential diagnosis (including volumetric MRI, FDG-PET, MIBG scan, polysomnography, genetic testing, autonomic function tests, skin biopsy) or disease activity (PBR06-TSPO). Second, we longitudinally collect biospecimens (blood, CSF, stool) and clinical, biometric, and imaging data to generate antecedent disease-progression scores. Third, in our Mass General Brigham SCiN study (stem cells in neurodegeneration), we generate induced pluripotent stem cell (iPSC) models from our patients, matched to biospecimens, including postmortem brain. We present 38 iPSC lines derived from MSA patients and relevant disease controls (spinocerebellar ataxia and PD, including alpha-synuclein triplication cases), 22 matched to whole-genome sequenced postmortem brain. iPSC models may facilitate matching patients to appropriate therapies, particularly in heterogeneous diseases for which patient-specific biology may elude animal models. We anticipate that deeply phenotyped and genotyped patient cohorts matched to cellular models will increase the likelihood of success in clinical trials for MSA.