Microglial dysfunction is increasingly recognized as a key contributor to the pathogenesis of Alzheimer's disease (AD). Environmental enrichment (EE) is well documented to enhance neuronal form and function, but almost nothing is known about whether and how it alters the brain's innate immune system. Here we found that prolonged exposure of naive wild-type mice to EE significantly altered microglial density and branching complexity in the dentate gyrus of hippocampus. In wild-type mice injected intraventricularly with soluble Aβ oligomers (oAβ) from hAPP-expressing cultured cells, EE prevented several morphological features of microglial inflammation and consistently prevented oAβ-mediated mRNA changes in multiple inflammatory genes both in vivo and in primary microglia cultured from the mice. Microdialysis in behaving mice confirmed that EE normalized increases in the extracellular levels of the key cytokines (CCL3, CCL4, TNFα) identified by the mRNA analysis. Moreover, EE prevented the changes in microglial gene expression caused by ventricular injection of oAβ extracted directly from AD cerebral cortex. We conclude that EE potently alters the form and function of microglia in a way that prevents their inflammatory response to human oAβ, suggesting that prolonged environmental enrichment could protect against AD by modulating the brain's innate immune system.

The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo applications due in part to the introduction of potentially oncogenic transcription factors via viral vectors. Recently developed lentiviral vectors allow the excision of viral reprogramming factors and the development of transgene-free iPS lines. However it is unclear if reprogramming strategy has an impact on the differentiation potential and the in vivo behavior of hiPS progeny. Here we subject viral factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines to a further challenge, by analyzing their differentiation potential along the 3 neural lineages and over extended periods of time in vitro, as well as by interrogating their ability to respond to local environmental cues by grafting into the striatum. We demonstrate similar and efficient differentiation into neurons, astrocytes and oligodendrocytes among all hiPS and human ES line controls. Upon intracranial grafting in the normal rat (Sprague Dawley), precursors derived from all hiPS lines exhibited good survival and response to environmental cues by integrating into the subventricular zone, acquiring phenotypes typical of type A, B or C cells and migrating along the rostral migratory stream into the olfactory bulb. There was no teratoma or other tumor formation 12 weeks after grafting in any of the 26 animals used in the study. Thus neither factor excision nor persistence of c-myc impact the behavior of hiPS lines in vivo.

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson's disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells--unfolded monomer or α-helically folded oligomer--is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs β-synuclein (βS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-β component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in βS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that βS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased βS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer the uniique potential to advance our understanding of PD etiology by providing disease-relevant cell-types carrying patient mutations along with isogenic control cells. To facilitate this experimental approach, we generated a collection of 55 cell lines genetically engineered to harbor mutations in genes associated with monogenic PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G+FS, SYNJ1 R258Q/FS, VPS13C A444P, VPS13C W395C, GBA1 IVS2+1). All mutations were generated in a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using CRISPR/Cas9 or prime editing-based approaches. We implemented rigorous quality controls, including high density genotyping to detect structural variants and confirm the genomic integrity of each cell line. This systematic approach ensures the high quality of our stem cell collection, highlights differences between conventional CRISPR/Cas9 and prime editing and provides a roadmap for how to generate gene-edited hPSCs collections at scale in an academic setting. We expect that our isogenic stem cell collection will become an accessible platform for the study of PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.

Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS "3K" (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.

Genetic and biochemical evidence attributes neuronal loss in Parkinson's disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS '3K' (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

Despite ongoing debate, the amyloid β-protein (Aβ) remains the prime therapeutic target for the treatment of Alzheimer's disease (AD). However, rational drug design has been hampered by a lack of knowledge about neuroactive Aβ. To help address this deficit, we developed live-cell imaging of iPSC-derived human neurons (iNs) to study the effects of the most disease relevant form of Aβ-oligomeric assemblies (oAβ) extracted from AD brain. Of ten brains studied, extracts from nine caused neuritotoxicity, and in eight cases this was abrogated by Aβ immunodepletion. Here we show that activity in this bioassay agrees relatively well with disruption of hippocampal long-term potentiation, a correlate of learning and memory, and that measurement of neurotoxic oAβ can be obscured by more abundant non-toxic forms of Aβ. These findings indicate that the development of novel Aβ targeting therapeutics may benefit from unbiased activity-based discovery. To test this principle, we directly compared 5 clinical antibodies (aducanumab, bapineuzumab,  BAN2401, gantenerumab, and SAR228810) together with an in-house aggregate-preferring antibody (1C22) and established relative EC50s in protecting human neurons from human Aβ. The results yielded objective numerical data on the potency of each antibody in neutralizing human oAβ neuritotoxicity. Their relative efficacies in this morphological assay were paralleled by their functional ability to rescue oAβ-induced inhibition of hippocampal synaptic plasticity. This novel paradigm provides an unbiased, all-human system for selecting candidate antibodies for advancement to human immunotherapy.

As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field.

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.

Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases, but mechanistic insights are impeded by a lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale epigenetic studies have highlighted the enrichment of GWAS-identified variants in regulatory DNA elements of disease-relevant cell types. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells. By generating a genetically precisely controlled experimental system, we identify a common Parkinson's disease associated risk variant in a non-coding distal enhancer element that regulates the expression of α-synuclein (SNCA), a key gene implicated in the pathogenesis of Parkinson's disease. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease-relevant phenotypes.

Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes of these cells have similar pluripotent states. Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs. Genome-wide maps of nucleosomes with histone H3K4me3 and H3K27me3 modifications indicate that there is little difference between ESCs and iPSCs with respect to these marks. Gene expression profiles confirm that the transcriptional programs of ESCs and iPSCs show very few consistent differences. Although some variation in chromatin structure and gene expression was observed in these cell lines, these variations did not serve to distinguish ESCs from iPSCs.

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.