Single-Cell Detection of Secreted Aß and sAPPα from Human IPSC-Derived Neurons and Astrocytes.

Secreted factors play a central role in normal and pathological processes in every tissue in the body. The brain is composed of a highly complex milieu of different cell types and few methods exist that can identify which individual cells in a complex mixture are secreting specific analytes. By identifying which cells are responsible, we can better understand neural physiology and pathophysiology, more readily identify the underlying pathways responsible for analyte production, and ultimately use this information to guide the development of novel therapeutic strategies that target the cell types of relevance. We present here a method for detecting analytes secreted from single human induced pluripotent stem cell (iPSC)-derived neural cells and have applied the method to measure amyloid ß (Aß) and soluble amyloid precursor protein-alpha (sAPPα), analytes central to Alzheimer's disease pathogenesis. Through these studies, we have uncovered the dynamic range of secretion profiles of these analytes from single iPSC-derived neuronal and glial cells and have molecularly characterized subpopulations of these cells through immunostaining and gene expression analyses. In examining Aß and sAPPα secretion from single cells, we were able to identify previously unappreciated complexities in the biology of APP cleavage that could not otherwise have been found by studying averaged responses over pools of cells. This technique can be readily adapted to the detection of other analytes secreted by neural cells, which would have the potential to open new perspectives into human CNS development and dysfunction. SIGNIFICANCE STATEMENT: We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid ß (Aß) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single-cell analytical platform. First, we identify a previously unappreciated subpopulation that secretes high levels of Aß in the absence of detectable sAPPα. Further, we show that multiple cell types secrete high levels of Aß and sAPPα, but cells expressing GABAergic neuronal markers are overrepresented. Finally, we show that astrocytes are competent to secrete high levels of Aß and therefore may be a significant contributor to Aß accumulation in the brain.

C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death.

Recent evidence suggests that tau aggregation may spread via extracellular release and subsequent uptake by synaptically connected neurons, but little is known about the processes by which tau is released or the molecular forms of extracellular tau. To gain insight into the nature of extracellular tau, we used highly sensitive ELISAs, which, when used in tandem, are capable of differentiating between full-length (FL) tau, mid-region-bearing fragments, and C-terminal (CT) fragments. We applied these assays to the systematic study of the conditioned media of N2a cells, induced pluripotent stem cell-derived human cortical neurons, and primary rat cortical neurons, each of which was carefully assessed for viability. In all three neuronal models, the bulk of extracellular tau was free-floating and unaggregated and <0.2% was encapsulated in exosomes. Although most intracellular tau was FL, the majority of extracellular tau was CT truncated and appeared to be released both actively by living neurons and passively by dead cells. In contrast, only a small amount of extracellular tau was aggregation-competent tau (i.e., contained the microtubule-binding regions) and this material appears to be released solely due to a low level of cell death that occurs in all cell culture systems. Importantly, amyloid ß-protein (Aß)-induced neuronal compromise significantly increased the quantity of all forms of extracellular tau, but the presence of Aß before detectable cell compromise did not increase extracellular tau. Collectively, these results suggest that factors that induce neuronal death are likely to be necessary to initiate the extracellular spread of tau aggregation. SIGNIFICANCE STATEMENT: Recent studies suggest that the transfer of tau between neurons underlies the characteristic spatiotemporal progression of neurofibrillary pathology. We searched for tau in the conditioned medium of N2a cells, induced pluripotent stem cell-derived human cortical neurons, and primary rat cortical neurons and analyzed the material present using four different tau ELISAs. We demonstrate that the majority of tau released from healthy neurons is C-terminally truncated and lacks the microtubule-binding region (MTBR) thought necessary for self-aggregation. A small amount of MTBR-containing tau is present outside of cells, but this appears to be solely due to cell death. Therefore, if propagation of tau aggregation is mediated by extracellular tau, our findings suggest that neuronal compromise is required to facilitate this process.

The familial Alzheimer's disease APPV717I mutation alters APP processing and Tau expression in iPSC-derived neurons.

Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by extracellular plaques containing amyloid ß (Aß)-protein and intracellular tangles containing hyperphosphorylated Tau protein. Here, we describe the generation of inducible pluripotent stem cell lines from patients harboring the London familial AD (fAD) amyloid precursor protein (APP) mutation (V717I). We examine AD-relevant phenotypes following directed differentiation to forebrain neuronal fates vulnerable in AD. We observe that over differentiation time to mature neuronal fates, APP expression and levels of Aß increase dramatically. In both immature and mature neuronal fates, the APPV717I mutation affects both ß- and γ-secretase cleavage of APP. Although the mutation lies near the γ-secretase cleavage site in the transmembrane domain of APP, we find that ß-secretase cleavage of APP is elevated leading to generation of increased levels of both APPsß and Aß. Furthermore, we find that this mutation alters the initial cleavage site of γ-secretase, resulting in an increased generation of both Aß42 and Aß38. In addition to altered APP processing, an increase in levels of total and phosphorylated Tau is observed in neurons with the APPV717I mutation. We show that treatment with Aß-specific antibodies early in culture reverses the phenotype of increased total Tau levels, implicating altered Aß production in fAD neurons in this phenotype. These studies use human neurons to reveal previously unrecognized effects of the most common fAD APP mutation and provide a model system for testing therapeutic strategies in the cell types most relevant to disease processes.

Small molecule regulators of microRNAs identified by high-throughput screen coupled with high-throughput sequencing.

MicroRNAs (miRNAs) regulate fundamental biological processes by silencing mRNA targets and are dysregulated in many diseases. Therefore, miRNA replacement or inhibition can be harnessed as potential therapeutics. However, existing strategies for miRNA modulation using oligonucleotides and gene therapies are challenging, especially for neurological diseases, and none have yet gained clinical approval. We explore a different approach by screening a biodiverse library of small molecule compounds for their ability to modulate hundreds of miRNAs in human induced pluripotent stem cell-derived neurons. We demonstrate the utility of the screen by identifying cardiac glycosides as potent inducers of miR-132, a key neuroprotective miRNA downregulated in Alzheimer's disease and other tauopathies. Coordinately, cardiac glycosides downregulate known miR-132 targets, including Tau, and protect rodent and human neurons against various toxic insults. More generally, our dataset of 1370 drug-like compounds and their effects on the miRNome provides a valuable resource for further miRNA-based drug discovery.

Parkinson-causing mutations in LRRK2 impair the physiological tetramerization of endogenous α-synuclein in human neurons.

α-Synuclein (αSyn) aggregation in Lewy bodies and neurites defines both familial and 'sporadic' Parkinson's disease. We previously identified α-helically folded αSyn tetramers, in addition to the long-known unfolded monomers, in normal cells. PD-causing αSyn mutations decrease the tetramer:monomer (T:M) ratio, associated with αSyn hyperphosphorylation and cytotoxicity in neurons and a motor syndrome of tremor and gait deficits in transgenic mice that responds in part to L-DOPA. Here, we asked whether LRRK2 mutations, the most common genetic cause of cases previously considered sporadic PD, also alter tetramer homeostasis. Patient neurons carrying G2019S, the most prevalent LRRK2 mutation, or R1441C each had decreased T:M ratios and pSer129 hyperphosphorylation of their endogenous αSyn along with increased phosphorylation of Rab10, a widely reported substrate of LRRK2 kinase activity. Two LRRK2 kinase inhibitors normalized the T:M ratio and the hyperphosphorylation in the G2019S and R1441C patient neurons. An inhibitor of stearoyl-CoA desaturase, the rate-limiting enzyme for monounsaturated fatty acid synthesis, also restored the αSyn T:M ratio and reversed pSer129 hyperphosphorylation in both mutants. Coupled with the recent discovery that PD-causing mutations of glucocerebrosidase in Gaucher's neurons also decrease T:M ratios, our findings indicate that three dominant genetic forms of PD involve life-long destabilization of αSyn physiological tetramers as a common pathogenic mechanism that can occur upstream of progressive neuronal synucleinopathy. Based on αSyn's finely-tuned interaction with certain vesicles, we hypothesize that the fatty acid composition and fluidity of membranes regulate αSyn's correct binding to highly curved membranes and subsequent assembly into metastable tetramers.

Lipase regulation of cellular fatty acid homeostasis as a Parkinson's disease therapeutic strategy.

Synucleinopathy (Parkinson's disease (PD); Lewy body dementia) disease-modifying treatments represent a huge unmet medical need. Although the PD-causing protein α-synuclein (αS) interacts with lipids and fatty acids (FA) physiologically and pathologically, targeting FA homeostasis for therapeutics is in its infancy. We identified the PD-relevant target stearoyl-coA desaturase: inhibiting monounsaturated FA synthesis reversed PD phenotypes. However, lipid degradation also generates FA pools. Here, we identify the rate-limiting lipase enzyme, LIPE, as a candidate target. Decreasing LIPE in human neural cells reduced αS inclusions. Patient αS triplication vs. corrected neurons had increased pSer129 and insoluble αS and decreased αS tetramer:monomer ratios. LIPE inhibition rescued all these and the abnormal unfolded protein response. LIPE inhibitors decreased pSer129 and restored tetramer:monomer equilibrium in αS E46K-expressing human neurons. LIPE reduction in vivo alleviated αS-induced dopaminergic neurodegeneration in Caenorhabditis elegans. Co-regulating FA synthesis and degradation proved additive in rescuing PD phenotypes, signifying co-targeting as a therapeutic strategy.

Stem cell-derived neurons reflect features of protein networks, neuropathology, and cognitive outcome of their aged human donors.

We have generated a controlled and manipulable resource that captures genetic risk for Alzheimer's disease: iPSC lines from 53 individuals coupled with RNA and proteomic profiling of both iPSC-derived neurons and brain tissue of the same individuals. Data collected for each person include genome sequencing, longitudinal cognitive scores, and quantitative neuropathology. The utility of this resource is exemplified here by analyses of neurons derived from these lines, revealing significant associations between specific Aß and tau species and the levels of plaque and tangle deposition in the brain and, more importantly, with the trajectory of cognitive decline. Proteins and networks are identified that are associated with AD phenotypes in iPSC neurons, and relevant associations are validated in brain. The data presented establish this iPSC collection as a resource for investigating person-specific processes in the brain that can aid in identifying and validating molecular pathways underlying AD.

Shared effects of DISC1 disruption and elevated WNT signaling in human cerebral organoids.

The development of three-dimensional culture methods has allowed for the study of developing cortical morphology in human cells. This provides a new tool to study the neurodevelopmental consequences of disease-associated mutations. Here, we study the effects of isogenic DISC1 mutation in cerebral organoids. DISC1 has been implicated in psychiatric disease based on genetic studies, including its interruption by a balanced translocation that increases the risk of major mental illness. Isogenic wild-type and DISC1-disrupted human-induced pluripotent stem cells were used to generate cerebral organoids, which were then examined for morphology and gene expression. We show that DISC1-mutant cerebral organoids display disorganized structural morphology and impaired proliferation, which is phenocopied by WNT agonism and rescued by WNT antagonism. Furthermore, there are many shared changes in gene expression with DISC1 disruption and WNT agonism, including in neural progenitor and cell fate markers, regulators of neuronal migration, and interneuron markers. These shared gene expression changes suggest mechanisms for the observed morphologic dysregulation with DISC1 disruption and points to new avenues for future studies. The shared changes in three-dimensional cerebral organoid morphology and gene expression with DISC1 interruption and WNT agonism further strengthens the link between DISC1 mutation, abnormalities in WNT signaling, and neuropsychiatric disease.

Cell-type Dependent Alzheimer's Disease Phenotypes: Probing the Biology of Selective Neuronal Vulnerability.

Alzheimer's disease (AD) induces memory and cognitive impairment in the absence of motor and sensory deficits during its early and middle course. A major unresolved question is the basis for this selective neuronal vulnerability. Aß, which plays a central role in AD pathogenesis, is generated throughout the brain, yet some regions outside of the limbic and cerebral cortices are relatively spared from Aß plaque deposition and synapse loss. Here, we examine neurons derived from iPSCs of patients harboring an amyloid precursor protein mutation to quantify AD-relevant phenotypes following directed differentiation to rostral fates of the brain (vulnerable) and caudal fates (relatively spared) in AD. We find that both the generation of Aß and the responsiveness of TAU to Aß are affected by neuronal cell type, with rostral neurons being more sensitive than caudal neurons. Thus, cell-autonomous factors may in part dictate the pattern of selective regional vulnerability in human neurons in AD.

Genomic DISC1 Disruption in hiPSCs Alters Wnt Signaling and Neural Cell Fate.

Genetic and clinical association studies have identified disrupted in schizophrenia 1 (DISC1) as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11) translocation in a Scottish family in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We show that disruption of DISC1 near the site of the translocation results in decreased DISC1 protein levels because of nonsense-mediated decay of long splice variants. This results in an increased level of canonical Wnt signaling in neural progenitor cells and altered expression of fate markers such as Foxg1 and Tbr2. These gene expression changes are rescued by antagonizing Wnt signaling in a critical developmental window, supporting the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types generated during cortical development.

Comparison and optimization of hiPSC forebrain cortical differentiation protocols.

Several protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual SMAD inhibition differentiation protocols, manual vs. AggreWell aggregate formation, plating substrates, neural progenitor cell (NPC) isolation methods, NPC maintenance and expansion, and astrocyte co-culture. The embryoid aggregate protocol, using a Matrigel substrate, consistently generates a high yield and purity of neurons. NPC isolation by manual selection, enzymatic rosette selection, or FACS all are efficient, but exhibit some differences in resulting cell populations. Expansion of NPCs as neural aggregates yields higher cell purity than expansion in a monolayer. Finally, co-culture of iPSC-derived neurons with astrocytes increases neuronal maturity by day 40. This study directly compares commonly employed methods for neuronal differentiation of iPSCs, and can be used as a resource for choosing between various differentiation protocols.

Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

The folate and vitamin B12-dependent enzyme methionine synthase (MS) is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin) from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.