Understanding the molecular mechanisms of skeletal muscle regeneration is crucial to exploiting this pathway for use in tissue repair. Our data demonstrate that the MEF2A transcription factor plays an essential role in skeletal muscle regeneration in adult mice. Injured Mef2a knockout mice display widespread necrosis and impaired myofiber formation. MEF2A controls this process through its direct regulation of the largest known mammalian microRNA (miRNA) cluster, the Gtl2-Dio3 locus. A subset of the Gtl2-Dio3 miRNAs represses secreted Frizzled-related proteins (sFRPs), inhibitors of WNT signaling. Consistent with these data, Gtl2-Dio3-encoded miRNAs are downregulated in regenerating Mef2a knockout muscle, resulting in upregulated sFRP expression and attenuated WNT activity. Furthermore, myogenic differentiation in Mef2a-deficient myoblasts is rescued by overexpression of miR-410 and miR-433, two miRNAs in the Gtl2-Dio3 locus that repress sFRP2, or by treatment with recombinant WNT3A and WNT5A. Thus, miRNA-mediated modulation of WNT signaling by MEF2A is a requisite step for proper muscle regeneration, and represents an attractive pathway for enhancing regeneration of diseased muscle.

Functional output of the hippocampus, a brain region subserving memory function, depends on highly orchestrated cellular and molecular processes that regulate synaptic plasticity throughout life. The structural requirements of such plasticity and molecular events involved in this regulation are poorly understood. Specific molecules, including tissue inhibitor of metalloproteinases-2 (TIMP2) have been implicated in plasticity processes in the hippocampus, a role that decreases with brain aging as expression is lost. Here, we report that TIMP2 is highly expressed by neurons within the hippocampus and its loss drives changes in cellular programs related to adult neurogenesis and dendritic spine turnover with corresponding impairments in hippocampus-dependent memory. Consistent with the accumulation of extracellular matrix (ECM) in the hippocampus we observe with aging, we find that TIMP2 acts to reduce accumulation of ECM around synapses in the hippocampus. Moreover, its deletion results in hindrance of newborn neuron migration through a denser ECM network. A novel conditional TIMP2 knockout (KO) model reveals that neuronal TIMP2 regulates adult neurogenesis, accumulation of ECM, and ultimately hippocampus-dependent memory. Our results define a mechanism whereby hippocampus-dependent function is regulated by TIMP2 and its interactions with the ECM to regulate diverse processes associated with synaptic plasticity.

Parkinson's disease (PD) is characterized pathologically by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Whether cell types beyond DA neurons in the SN show vulnerability in PD remains unclear. Through transcriptomic profiling of 315,867 high-quality single nuclei in the SN from individuals with and without PD, we identified cell clusters representing various neuron types, glia, endothelial cells, pericytes, fibroblasts, and T cells and investigated cell type-dependent alterations in gene expression in PD. Notably, a unique neuron cluster marked by the expression of RIT2, a PD risk gene, also displayed vulnerability in PD. We validated RIT2-enriched neurons in midbrain organoids and the mouse SN. Our results demonstrated distinct transcriptomic signatures of the RIT2-enriched neurons in the human SN and implicated reduced RIT2 expression in the pathogenesis of PD. Our study sheds light on the diversity of cell types, including DA neurons, in the SN and the complexity of molecular and cellular changes associated with PD pathogenesis.

Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that has significant overlap with frontotemporal dementia (FTD). Mutations in specific genes have been identified that can cause and/or predispose patients to ALS. However, the clinical variability seen in ALS patients suggests that additional genes impact pathology, susceptibility, severity, and/or progression of the disease. To identify molecular pathways involved in ALS, we undertook a meta-analysis of published genetic modifiers both in patients and in model organisms, and undertook bioinformatic pathway analysis. From 72 published studies, we generated a list of 946 genes whose perturbation (1) impacted ALS in patient populations, (2) altered defects in laboratory models, or (3) modified defects caused by ALS gene ortholog loss of function. Herein, these are all called modifier genes. We found 727 modifier genes that encode proteins with human orthologs. Of these, 43 modifier genes were identified as modifiers of more than one ALS gene/model, consistent with the hypothesis that shared genes and pathways may underlie ALS. Further, we used a gene ontology-based bioinformatic analysis to identify pathways and associated genes that may be important in ALS. To our knowledge this is the first comprehensive survey of ALS modifier genes. This work suggests that shared molecular mechanisms may underlie pathology caused by different ALS disease genes. Surprisingly, few ALS modifier genes have been tested in more than one disease model. Understanding genes that modify ALS-associated defects will help to elucidate the molecular pathways that underlie ALS and provide additional targets for therapeutic intervention.

Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy.

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.

Motor neuron degeneration, the defining feature of amyotrophic lateral sclerosis (ALS), is a primary example of cell-type specificity in neurodegenerative diseases. Using isogenic pairs of induced pluripotent stem cells (iPSCs) harboring different familial ALS mutations, we assess the capacity of iPSC-derived lower motor neurons, sensory neurons, astrocytes, and superficial cortical neurons to capture disease features including transcriptional and splicing dysregulation observed in human postmortem neurons. At early time points, differentially regulated genes in iPSC-derived lower motor neurons, but not other cell types, overlap with one-third of the differentially regulated genes in laser-dissected motor neurons from ALS compared with control postmortem spinal cords. For genes altered in both the iPSC model and bona fide human lower motor neurons, expression changes correlate between the two populations. In iPSC-derived lower motor neurons, but not other derived cell types, we detect the downregulation of genes affected by TDP-43-dependent splicing. This reduction takes place exclusively within genotypes known to involve TDP-43 pathology.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

The concept of the neurovascular unit emphasizes the importance of cell-cell signaling between neural, glial, and vascular compartments. In neurogenesis, for example, brain endothelial cells play a key role by supplying trophic support to neural progenitors. Here, we describe a surprising phenomenon where brain endothelial cells may release trans-differentiation signals that convert astrocytes into neural progenitor cells in male mice after stroke. After oxygen-glucose deprivation, brain endothelial cells release microvesicles containing pro-neural factor Ascl1 that enter into astrocytes to induce their trans-differentiation into neural progenitors. In mouse models of focal cerebral ischemia, Ascl1 is upregulated in endothelium prior to astrocytic conversion into neural progenitor cells. Injecting brain endothelial-derived microvesicles amplifies the process of astrocyte trans-differentiation. Endothelial-specific overexpression of Ascl1 increases the local conversion of astrocytes into neural progenitors and improves behavioral recovery. Our findings describe an unexpected vascular-regulated mechanism of neuroplasticity that may open up therapeutic opportunities for improving outcomes after stroke.

Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain. For complete details on the use and execution of this protocol, please refer to Ahfeldt et al. (2020).

Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

Hundreds of proteins determine the function of synapses, and synapses define the neuronal circuits that subserve myriad brain, cognitive, and behavioral functions. It is thus necessary to precisely manipulate specific proteins at specific sub-cellular locations and times to elucidate the roles of particular proteins and synapses in brain function. We developed PHOtochemically TArgeting Chimeras (PHOTACs) as a strategy to optically degrade specific proteins with high spatial and temporal precision. PHOTACs are small molecules that, upon wavelength-selective illumination, catalyze ubiquitylation and degradation of target proteins through endogenous proteasomes. Here, we describe the design and chemical properties of a PHOTAC that targets Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα), which is abundant and crucial for the baseline synaptic function of excitatory neurons. We validate the PHOTAC strategy, showing that the CaMKIIα-PHOTAC is effective in mouse brain tissue. Light activation of CaMKIIα-PHOTAC removed CaMKIIα from regions of the mouse hippocampus only within 25 μm of the illuminated brain surface. The optically controlled degradation decreases synaptic function within minutes of light activation, measured by the light-initiated attenuation of evoked field excitatory postsynaptic potential (fEPSP) responses to physiological stimulation. The PHOTACs methodology should be broadly applicable to other key proteins implicated in synaptic function, especially for evaluating their precise roles in the maintenance of long-term potentiation and memory within subcellular dendritic domains.

Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.

Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES). The penta-EF-hand (PEF) protein apoptosis-linked gene 2 (ALG-2) stabilizes sec31A at ER exit sites (ERES) and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK) cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport-peflin is a negative regulator of transport.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in the United States. Recent studies suggest that pericardial adipose tissue (PCAT) secretes inflammatory factors that contribute to the development of CVD. To better characterize the role of PCAT in the pathogenesis of disease, we performed a large-scale unbiased analysis of the transcriptional differences between PCAT and subcutaneous adipose tissue, analysing 53 microarrays across 19 individuals. As it was unknown whether PCAT-secreted factors are produced by adipocytes or cells in the supporting stromal fraction, we also sought to identify differentially expressed genes in isolated pericardial adipocytes vs. isolated subcutaneous adipocytes. Using microarray analysis, we found that: 1) pericardial adipose tissue and isolated pericardial adipocytes both overexpress atherosclerosis-promoting chemokines and 2) pericardial and subcutaneous fat depots, as well as isolated pericardial adipocytes and subcutaneous adipocytes, express specific patterns of homeobox genes. In contrast, a core set of lipid processing genes showed no significant overlap with differentially expressed transcripts. These depot-specific homeobox signatures and transcriptional profiles strongly suggest different functional roles for the pericardial and subcutaneous adipose depots. Further characterization of these inter-depot differences should be a research priority.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease for which the origin and underlying cellular defects are not fully understood. Although motor neuron degeneration is the signature feature of ALS, it is not clear whether motor neurons or other cells of the motor circuit are the site of disease initiation. To better understand the contribution of multiple cell types in ALS, we made use of a Drosophila Sod1G85R knock-in model, in which all cells harbor the disease allele. End-stage dSod1G85R animals of both sexes exhibit severe motor deficits with clear degeneration of motor neurons. Interestingly, earlier in dSod1G85R larvae, motor function is also compromised, but their motor neurons exhibit only subtle morphological and electrophysiological changes that are unlikely to cause the observed decrease in locomotion. We analyzed the intact motor circuit and identified a defect in sensory feedback that likely accounts for the altered motor activity of dSod1G85R We found cell-autonomous activation of bone morphogenetic protein signaling in proprioceptor sensory neurons which are critical for the relay of the contractile status of muscles back to the central nerve cord, completely rescues early-stage motor defects and partially rescue late-stage motor function to extend lifespan. Identification of a defect in sensory feedback as a potential initiating event in ALS motor dysfunction, coupled with the ability of modified proprioceptors to alleviate such motor deficits, underscores the critical role that nonmotor neurons play in disease progression and highlights their potential as a site to identify early-stage ALS biomarkers and for therapeutic intervention.SIGNIFICANCE STATEMENT At diagnosis, many cellular processes are already disrupted in the amyotrophic lateral sclerosis (ALS) patient. Identifying the initiating cellular events is critical for achieving an earlier diagnosis to slow or prevent disease progression. Our findings indicate that neurons relaying sensory information underlie early stage motor deficits in a Drosophila knock-in model of ALS that best replicates gene dosage in familial ALS (fALS). Importantly, studies on intact motor circuits revealed defects in sensory feedback before evidence of motor neuron degeneration. These findings strengthen our understanding of how neural circuit dysfunctions lead to neurodegeneration and, coupled with our demonstration that the activation of bone morphogenetic protein signaling in proprioceptors alleviates both early and late motor dysfunction, underscores the importance of considering nonmotor neurons as therapeutic targets.

Human induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.