In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire.

The Psychiatric Cell Map Initiative: A Convergent Systems Biological Approach to Illuminating Key Molecular Pathways in Neuropsychiatric Disorders.

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.

Three dimensional and four dimensional live imaging to study mechanisms of progressive neurodegeneration.

Neurodegenerative diseases are complex and progressive, posing challenges to their study and understanding. Recent advances in microscopy imaging technologies have enabled the exploration of neurons in three spatial dimensions (3D) over time (4D). When applied to 3D cultures, tissues, or animals, these technologies can provide valuable insights into the dynamic and spatial nature of neurodegenerative diseases. This review focuses on the use of imaging techniques and neurodegenerative disease models to study neurodegeneration in 4D. Imaging techniques such as confocal microscopy, two-photon microscopy, miniscope imaging, light sheet microscopy, and robotic microscopy offer powerful tools to visualize and analyze neuronal changes over time in 3D tissue. Application of these technologies to in vitro models of neurodegeneration such as mouse organotypic culture systems and human organoid models provide versatile platforms to study neurodegeneration in a physiologically relevant context. Additionally, use of 4D imaging in vivo, including in mouse and zebrafish models of neurodegenerative diseases, allows for the investigation of early dysfunction and behavioral changes associated with neurodegeneration. We propose that these studies have the power to overcome the limitations of two-dimensional monolayer neuronal cultures and pave the way for improved understanding of the dynamics of neurodegenerative diseases and the development of effective therapeutic strategies.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Genetic Regulation of Neuronal Progranulin Reveals a Critical Role for the Autophagy-Lysosome Pathway.

Deficient progranulin levels cause dose-dependent neurological syndromes: haploinsufficiency leads to frontotemporal lobar degeneration (FTLD) and nullizygosity produces adult-onset neuronal ceroid lipofuscinosis. Mechanisms controlling progranulin levels are largely unknown. To better understand progranulin regulation, we performed a genome-wide RNAi screen using an ELISA-based platform to discover genes that regulate progranulin levels in neurons. We identified 830 genes that raise or lower progranulin levels by at least 1.5-fold in Neuro2a cells. When inhibited by siRNA or some by submicromolar concentrations of small-molecule inhibitors, 33 genes of the druggable genome increased progranulin levels in mouse primary cortical neurons; several of these also raised progranulin levels in FTLD model mouse neurons. "Hit" genes regulated progranulin by transcriptional or posttranscriptional mechanisms. Pathway analysis revealed enrichment of hit genes from the autophagy-lysosome pathway (ALP), suggesting a key role for this pathway in regulating progranulin levels. Progranulin itself regulates lysosome function. We found progranulin deficiency in neurons increased autophagy and caused abnormally enlarged lysosomes and boosting progranulin levels restored autophagy and lysosome size to control levels. Our data link the ALP to neuronal progranulin: progranulin levels are regulated by autophagy and, in turn, progranulin regulates the ALP. Restoring progranulin levels by targeting genetic modifiers reversed FTLD functional deficits, opening up potential opportunities for future therapeutics development.SIGNIFICANCE STATEMENT Progranulin regulates neuron and immune functions and is implicated in aging. Loss of one functional allele causes haploinsufficiency and leads to frontotemporal lobar degeneration (FTLD), the second leading cause of dementia. Progranulin gene polymorphisms are linked to Alzheimer's disease (AD) and complete loss of function causes neuronal ceroid lipofuscinosis. Despite the critical role of progranulin levels in neurodegenerative disease risk, almost nothing is known about their regulation. We performed an unbiased screen and identified specific pathways controlling progranulin levels in neurons. Modulation of these pathways restored levels in progranulin-deficient neurons and reversed FTLD phenotypes. We provide a new comprehensive understanding of the genetic regulation of progranulin levels and identify potential targets to treat FTLD and other neurodegenerative diseases, including AD.

The Arc of cognition: Signaling cascades regulating Arc and implications for cognitive function and disease.

The activity-regulated cytoskeletal (Arc) gene is implicated in numerous synaptic plasticity paradigms, including long-term potentiation and depression and homeostatic plasticity, and is critical for consolidating memory. How Arc facilitates these forms of plasticity is not fully understood. Unlike other neuronal immediate-early genes, Arc encodes a protein that shuttles between the somatodendritic and nuclear compartments to regulate synaptic plasticity. Little attention has been paid to Arc's role in the nucleus. Here, we highlight the regulatory elements and signaling cascades required to induce Arc transcription and discuss the significance of Arc nuclear localization for synaptic plasticity and scaling. We integrate these findings into the context of cognitive function and disease and propose a model in which Arc mediates an effect on memory as a "chaser" of synaptic activity through homeostatic scaling.

Functional genomics, genetic risk profiling and cell phenotypes in neurodegenerative disease.

Human genetics provides unbiased insights into the causes of human disease, which can be used to create a foundation for effective ways to more accurately diagnose patients, stratify patients for more successful clinical trials, discover and develop new therapies, and ultimately help patients choose the safest and most promising therapeutic option based on their risk profile. But the process for translating basic observations from human genetics studies into pathogenic disease mechanisms and treatments is laborious and complex, and this challenge has particularly slowed the development of interventions for neurodegenerative disease. In this review, we discuss the many steps in the process, the important considerations at each stage, and some of the latest tools and technologies that are available to help investigators translate insights from human genetics into diagnostic and therapeutic strategies that will lead to the sort of advances in clinical care that make a difference for patients.

Nrf2 mitigates LRRK2- and α-synuclein-induced neurodegeneration by modulating proteostasis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) and α-synuclein lead to Parkinson's disease (PD). Disruption of protein homeostasis is an emerging theme in PD pathogenesis, making mechanisms to reduce the accumulation of misfolded proteins an attractive therapeutic strategy. We determined if activating nuclear factor erythroid 2-related factor (Nrf2), a potential therapeutic target for neurodegeneration, could reduce PD-associated neuron toxicity by modulating the protein homeostasis network. Using a longitudinal imaging platform, we visualized the metabolism and location of mutant LRRK2 and α-synuclein in living neurons at the single-cell level. Nrf2 reduced PD-associated protein toxicity by a cell-autonomous mechanism that was time-dependent. Furthermore, Nrf2 activated distinct mechanisms to handle different misfolded proteins. Nrf2 decreased steady-state levels of α-synuclein in part by increasing α-synuclein degradation. In contrast, Nrf2 sequestered misfolded diffuse LRRK2 into more insoluble and homogeneous inclusion bodies. By identifying the stress response strategies activated by Nrf2, we also highlight endogenous coping responses that might be therapeutically bolstered to treat PD.

Clinical Trials in a Dish: The Potential of Pluripotent Stem Cells to Develop Therapies for Neurodegenerative Diseases.

Neurodegenerative diseases are a leading cause of death. No disease-modifying therapies are available, and preclinical animal model data have routinely failed to translate into success for therapeutics. Induced pluripotent stem cell (iPSC) biology holds great promise for human in vitro disease modeling because these cells can give rise to any cell in the human brain and display phenotypes specific to neurodegenerative diseases previously identified in postmortem and clinical samples. Here, we explore the potential and caveats of iPSC technology as a platform for drug development and screening, and the future potential to use large cohorts of disease-bearing iPSCs to perform clinical trials in a dish.

Assessing microscope image focus quality with deep learning.

BACKGROUND: Large image datasets acquired on automated microscopes typically have some fraction of low quality, out-of-focus images, despite the use of hardware autofocus systems. Identification of these images using automated image analysis with high accuracy is important for obtaining a clean, unbiased image dataset. Complicating this task is the fact that image focus quality is only well-defined in foreground regions of images, and as a result, most previous approaches only enable a computation of the relative difference in quality between two or more images, rather than an absolute measure of quality. RESULTS: We present a deep neural network model capable of predicting an absolute measure of image focus on a single image in isolation, without any user-specified parameters. The model operates at the image-patch level, and also outputs a measure of prediction certainty, enabling interpretable predictions. The model was trained on only 384 in-focus Hoechst (nuclei) stain images of U2OS cells, which were synthetically defocused to one of 11 absolute defocus levels during training. The trained model can generalize on previously unseen real Hoechst stain images, identifying the absolute image focus to within one defocus level (approximately 3 pixel blur diameter difference) with 95% accuracy. On a simpler binary in/out-of-focus classification task, the trained model outperforms previous approaches on both Hoechst and Phalloidin (actin) stain images (F-scores of 0.89 and 0.86, respectively over 0.84 and 0.83), despite only having been presented Hoechst stain images during training. Lastly, we observe qualitatively that the model generalizes to two additional stains, Hoechst and Tubulin, of an unseen cell type (Human MCF-7) acquired on a different instrument. CONCLUSIONS: Our deep neural network enables classification of out-of-focus microscope images with both higher accuracy and greater precision than previous approaches via interpretable patch-level focus and certainty predictions. The use of synthetically defocused images precludes the need for a manually annotated training dataset. The model also generalizes to different image and cell types. The framework for model training and image prediction is available as a free software library and the pre-trained model is available for immediate use in Fiji (ImageJ) and CellProfiler.

The Receptor-interacting Serine/Threonine Protein Kinase 1 (RIPK1) Regulates Progranulin Levels.

Progranulin (PGRN), a secreted growth factor, is a key regulator of inflammation and is genetically linked to two common and devastating neurodegenerative diseases. Haploinsufficiency mutations in GRN, the gene encoding PGRN, cause frontotemporal dementia (FTD), and a GRN SNP confers significantly increased risk for Alzheimer's disease (AD). Because cellular and animal data indicate that increasing PGRN can reverse phenotypes of both FTD and AD, modulating PGRN level has been proposed as a therapeutic strategy for both diseases. However, little is known about the regulation of PGRN levels. In this study, we performed an siRNA-based screen of the kinome to identify genetic regulators of PGRN levels in a rodent cell-based model system. We found that knocking down receptor-interacting serine/threonine protein kinase 1 (Ripk1) increased both intracellular and extracellular PGRN protein levels by increasing the translation rate of PGRN without affecting mRNA levels. We observed this effect in Neuro2a cells, wild-type primary mouse neurons, and Grn-haploinsufficient primary neurons from an FTD mouse model. We found that the effect of RIPK1 on PGRN is independent of the kinase activity of RIPK1 and occurs through a novel signaling pathway. These data suggest that targeting RIPK1 may be a therapeutic strategy in both AD and FTD.

αB-Crystallin overexpression in astrocytes modulates the phenotype of the BACHD mouse model of Huntington's disease.

Huntington's disease (HD) is caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. The polyQ expansion increases the propensity of htt to aggregate and accumulate, and manipulations that mitigate protein misfolding or facilitate the clearance of misfolded proteins are predicted to slow disease progression in HD models. αB-crystallin (αBc) or HspB5 is a well-characterized member of the small heat shock protein (sHsp) family that reduces mutant htt (mhtt) aggregation and toxicity in vitro and in Drosophila models of HD. Here, we determined if overexpressing αBc in vivo modulates aggregation and delays the onset and progression of disease in a full-length model of HD, BACHD mice. Expression of sHsps in neurodegenerative disease predominantly occurs in non-neuronal cells, and in the brain, αBc is mainly found in astrocytes and oligodendrocytes. Here, we show that directed αBc overexpression in astrocytes improves motor performance in rotarod and balance beam tests and improves cognitive function in the BACHD mice. Improvement in behavioral deficits correlated with mitigation of neuropathological features commonly observed in HD. Interestingly, astrocytic αBc overexpression was neuroprotective against neuronal cell loss in BACHD brains, suggesting αBc might be acting in a non-cell-autonomous manner. At the protein level, αBc decreased the level of soluble mhtt and decreased the size of mhtt inclusions in BACHD brain. Our results support a model in which elevating astrocytic αBc confers neuroprotection through a potential non-cell-autonomous pathway that modulates mhtt aggregation and protein levels.

Targeting TEAD/YAP-transcription-dependent necrosis, TRIAD, ameliorates Huntington's disease pathology.

Neuronal cell death in neurodegenerative diseases is not fully understood. Here we report that mutant huntingtin (Htt), a causative gene product of Huntington's diseases (HD) selectively induces a new form of necrotic cell death, in which endoplasmic reticulum (ER) enlarges and cell body asymmetrically balloons and finally ruptures. Pharmacological and genetic analyses revealed that the necrotic cell death is distinct from the RIP1/3 pathway-dependent necroptosis, but mediated by a functional deficiency of TEAD/YAP-dependent transcription. In addition, we revealed that a cell cycle regulator, Plk1, switches the balance between TEAD/YAP-dependent necrosis and p73/YAP-dependent apoptosis by shifting the interaction partner of YAP from TEAD to p73 through YAP phosphorylation at Thr77. In vivo ER imaging with two-photon microscopy detects similar ER enlargement, and viral vector-mediated delivery of YAP as well as chemical inhibitors of the Hippo pathway such as S1P recover the ER instability and necrosis in HD model mice. Intriguingly S1P completely stops the decline of motor function of HD model mice even after the onset of symptom. Collectively, we suggest approaches targeting the signalling pathway of TEAD/YAP-transcription-dependent necrosis (TRIAD) could lead to a therapeutic development against HD.

Sequence-Level Analysis of the Major European Huntington Disease Haplotype.

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.

iPS cells in the study of PD molecular pathogenesis.

Parkinson's disease (PD) is the second most common neurodegenerative disease and its pathogenic mechanisms are poorly understood. The majority of PD cases are sporadic but a number of genes are associated with familial PD. Sporadic and familial PD have many molecular and cellular features in common, suggesting some shared pathogenic mechanisms. Induced pluripotent stem cells (iPSCs) have been derived from patients harboring a range of different mutations of PD-associated genes. PD patient-derived iPSCs have been differentiated into relevant cell types, in particular dopaminergic neurons and used as a model to study PD. In this review, we describe how iPSCs have been used to improve our understanding of the pathogenesis of PD. We describe what cellular and molecular phenotypes have been observed in neurons derived from iPSCs harboring known PD-associated mutations and what common pathways may be involved.

Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1.

Over 30% of patients with amyotrophic lateral sclerosis (ALS) exhibit cognitive deficits indicative of frontotemporal dementia (FTD), suggesting a common pathogenesis for both diseases. Consistent with this hypothesis, neuronal and glial inclusions rich in TDP43, an essential RNA-binding protein, are found in the majority of those with ALS and FTD, and mutations in TDP43 and a related RNA-binding protein, FUS, cause familial ALS and FTD. TDP43 and FUS affect the splicing of thousands of transcripts, in some cases triggering nonsense-mediated mRNA decay (NMD), a highly conserved RNA degradation pathway. Here, we take advantage of a faithful primary neuronal model of ALS and FTD to investigate and characterize the role of human up-frameshift protein 1 (hUPF1), an RNA helicase and master regulator of NMD, in these disorders. We show that hUPF1 significantly protects mammalian neurons from both TDP43- and FUS-related toxicity. Expression of hUPF2, another essential component of NMD, also improves survival, whereas inhibiting NMD prevents rescue by hUPF1, suggesting that hUPF1 acts through NMD to enhance survival. These studies emphasize the importance of RNA metabolism in ALS and FTD, and identify a uniquely effective therapeutic strategy for these disorders.

Potential function for the Huntingtin protein as a scaffold for selective autophagy.

Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51-like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.

Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology--cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we show that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity and discovered that pathogenic mutations shorten TDP43 half-life. New compounds that stimulate autophagy improved TDP43 clearance and localization and enhanced survival in primary murine neurons and in human stem cell-derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance.

A three-groups model for high-throughput survival screens.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition characterized by the progressive deterioration of motor neurons in the cortex and spinal cord. Using an automated robotic microscope platform that enables the longitudinal tracking of thousands of single neurons, we examine the effects a large library of compounds on modulating the survival of primary neurons expressing a mutation known to cause ALS. The goal of our analysis is to identify the few potentially beneficial compounds among the many assayed, the vast majority of which do not extend neuronal survival. This resembles the large-scale simultaneous inference scenario familiar from microarray analysis, but transferred to the survival analysis setting due to the novel experimental setup. We apply a three-component mixture model to censored survival times of thousands of individual neurons subjected to hundreds of different compounds. The shrinkage induced by our model significantly improves performance in simulations relative to performing treatment-wise survival analysis and subsequent multiple testing adjustment. Our analysis identified compounds that provide insight into potential novel therapeutic strategies for ALS.

Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy.

Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.