APP antisense oligonucleotides reduce Aß aggregation and rescue endolysosomal dysfunction in Alzheimer's disease.

APP gene dosage is strongly associated with Alzheimer's disease (AD) pathogenesis. Genomic duplication of the APP locus leads to autosomal dominant early-onset AD. Individuals with Down syndrome (trisomy of chromosome 21) harbor 3 copies of the APP gene and invariably develop progressive AD with highly characteristic neuropathological features. Restoring expression of APP to the equivalent of that of two gene copies, or lower, is a rational therapeutic strategy, as it would restore physiological levels of neuronal APP protein without the potentially deleterious consequences of inadvertently inducing loss of APP function. Here we find that antisense oligonucleotides (ASOs) targeting APP are an effective approach to reduce APP protein levels and rescue endolysosome and autophagy dysfunction in APP duplication human induced pluripotent stem cell (hiPSC)-derived cortical neurons. Importantly, using ultrasensitive single-aggregate imaging techniques, we show that APP targeting ASOs significantly reduce both intracellular and extracellular Aß-containing aggregates. Our results highlight the potential of APP ASOs as a therapeutic approach for forms of AD caused by duplication of the APP gene, including monogenic AD and AD related to Down syndrome.

Loss of CNTNAP2 Alters Human Cortical Excitatory Neuron Differentiation and Neural Network Development.

BACKGROUND: Loss-of-function mutations in the contactin-associated protein-like 2 (CNTNAP2) gene are causal for neurodevelopmental disorders, including autism, schizophrenia, epilepsy, and intellectual disability. CNTNAP2 encodes CASPR2, a single-pass transmembrane protein that belongs to the neurexin family of cell adhesion molecules. These proteins have a variety of functions in developing neurons, including connecting presynaptic and postsynaptic neurons, and mediating signaling across the synapse. METHODS: To study the effect of loss of CNTNAP2 function on human cerebral cortex development, and how this contributes to the pathogenesis of neurodevelopmental disorders, we generated human induced pluripotent stem cells from one neurotypical control donor null for full-length CNTNAP2, modeling cortical development from neurogenesis through to neural network formation in vitro. RESULTS: CNTNAP2 is particularly highly expressed in the first two populations of early-born excitatory cortical neurons, and loss of CNTNAP2 shifted the relative proportions of these two neuronal types. Live imaging of excitatory neuronal growth showed that loss of CNTNAP2 reduced neurite branching and overall neuronal complexity. At the network level, developing cortical excitatory networks null for CNTNAP2 had complex changes in activity compared with isogenic controls: an initial period of relatively reduced activity compared with isogenic controls, followed by a lengthy period of hyperexcitability, and then a further switch to reduced activity. CONCLUSIONS: Complete loss of CNTNAP2 contributes to the pathogenesis of neurodevelopmental disorders through complex changes in several aspects of human cerebral cortex excitatory neuron development that culminate in aberrant neural network formation and function.

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry.

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

The role of contactin-associated protein-like 2 in neurodevelopmental disease and human cerebral cortex evolution.

The contactin-associated protein-like 2 (CNTNAP2) gene is associated with multiple neurodevelopmental disorders, including autism spectrum disorder (ASD), intellectual disability (ID), and specific language impairment (SLI). Experimental work has shown that CNTNAP2 is important for neuronal development and synapse formation. There is also accumulating evidence for the differential use of CNTNAP2 in the human cerebral cortex compared with other primates. Here, we review the current literature on CNTNAP2, including what is known about its expression, disease associations, and molecular/cellular functions. We also review the evidence for its role in human brain evolution, such as the presence of eight human accelerated regions (HARs) within the introns of the gene. While progress has been made in understanding the function(s) of CNTNAP2, more work is needed to clarify the precise mechanisms through which CNTNAP2 acts. Such information will be crucial for developing effective treatments for CNTNAP2 patients. It may also shed light on the longstanding question of what makes us human.

Endolysosome and autophagy dysfunction in Alzheimer disease.

Abnormalities of the neuronal endolysosome and macroautophagy/autophagy system are an early and prominent feature of Alzheimer disease (AD). SORL1 is notable as a gene in which mutations are causal for a rare, autosomal dominant form of AD, and also variants that increase the risk of developing the common form of late-onset AD. In our recent study, we used patient-derived stem cells and CRISPR engineering to study the effects of SORL1 mutations on the endolysosome and autophagy system in human forebrain neurons. SORL1 mutations causal for monogenic AD are typically truncating mutations, and we found, using stem cells generated from an individual with dementia due to a heterozygous SORL1 truncation mutation, that this class of mutation results in SORL1 haploinsufficiency. Reducing SORL1 protein by half results in disrupted endosomal trafficking in patient-derived neurons, which we confirmed by studying the endolysosomal system in isogenic CRISPR-engineered SORL1 heterozygous null neurons. We also found that SORL1 homozygous null neurons develop more severe phenotypes, with endosome abnormalities, lysosome dysfunction and defects in the degradative phase of autophagy. Endolysosome and autophagy defects in SORL1 mutant neurons are dependent on APP, a key AD gene, as they are rescued by extracellular antisense oligonucleotides that reduce APP protein.

SORL1 deficiency in human excitatory neurons causes APP-dependent defects in the endolysosome-autophagy network.

Dysfunction of the endolysosomal-autophagy network is emerging as an important pathogenic process in Alzheimer's disease. Mutations in the sorting receptor-encoding gene SORL1 cause autosomal-dominant Alzheimer's disease, and SORL1 variants increase risk for late-onset AD. To understand the contribution of SORL1 mutations to AD pathogenesis, we analyze the effects of a SORL1 truncating mutation on SORL1 protein levels and endolysosome function in human neurons. We find that truncating mutation results in SORL1 haploinsufficiency and enlarged endosomes in human neurons. Analysis of isogenic SORL1 wild-type, heterozygous, and homozygous null neurons demonstrates that, whereas SORL1 haploinsufficiency results in endosome dysfunction, complete loss of SORL1 leads to additional defects in lysosome function and autophagy. Neuronal endolysosomal dysfunction caused by loss of SORL1 is relieved by extracellular antisense oligonucleotide-mediated reduction of APP protein, demonstrating that PSEN1, APP, and SORL1 act in a common pathway regulating the endolysosome system, which becomes dysfunctional in AD.

Variable Outcomes in Neural Differentiation of Human PSCs Arise from Intrinsic Differences in Developmental Signaling Pathways.

Directed differentiation of human pluripotent stem cells varies in specificity and efficiency. Stochastic, genetic, intracellular, and environmental factors affect maintenance of pluripotency and differentiation into early embryonic lineages. However, factors affecting variation in in vitro differentiation to defined cell types are not well understood. To address this, we focused on a well-established differentiation process to cerebral cortex neural progenitor cells and their neuronal progeny from human pluripotent stem cells. Analysis of 162 differentiation outcomes of 61 stem cell lines derived from 37 individuals showed that most variation occurs along gene expression axes reflecting dorsoventral and rostrocaudal spatial expression during in vivo brain development. Line-independent and line-dependent variations occur, with the latter driven largely by differences in endogenous Wnt signaling activity. Tuning Wnt signaling during a specific phase early in the differentiation process reduces variability, demonstrating that cell-line/genome-specific differentiation outcome biases can be corrected by controlling extracellular signaling.

Tumour necrosis factor induces increased production of extracellular amyloid-ß- and α-synuclein-containing aggregates by human Alzheimer's disease neurons.

In addition to increased aberrant protein aggregation, inflammation has been proposed as a key element in the pathogenesis and progression of Alzheimer's disease. How inflammation interacts with other disease pathways and how protein aggregation increases during disease are not clear. We used single-molecule imaging approaches and membrane permeabilization assays to determine the effect of chronic exposure to tumour necrosis factor, a master proinflammatory cytokine, on protein aggregation in human-induced pluripotent stem cell-derived neurons harbouring monogenic Alzheimer's disease mutations. We report that exposure of Alzheimer's disease neurons, but not control neurons, to tumour necrosis factor induces substantial production of extracellular protein aggregates. Aggregates from Alzheimer's disease neurons are composed of amyloid-ß and α-synuclein and induce significant permeabilization of lipid membranes in an assay of pathogenicity. These findings provide support for a causal relationship between two crucial processes in Alzheimer's disease pathogenesis and suggest that targeting inflammation, particularly tumour necrosis factor, may have beneficial downstream effects on ameliorating aberrant protein aggregation and accumulation.

Mutations in thyroid hormone receptor α1 cause premature neurogenesis and progenitor cell depletion in human cortical development.

Mutations in the thyroid hormone receptor α 1 gene (THRA) have recently been identified as a cause of intellectual deficit in humans. Patients present with structural abnormalities including microencephaly, reduced cerebellar volume and decreased axonal density. Here, we show that directed differentiation of THRA mutant patient-derived induced pluripotent stem cells to forebrain neural progenitors is markedly reduced, but mutant progenitor cells can generate deep and upper cortical layer neurons and form functional neuronal networks. Quantitative lineage tracing shows that THRA mutation-containing progenitor cells exit the cell cycle prematurely, resulting in reduced clonal output. Using a micropatterned chip assay, we find that spatial self-organization of mutation-containing progenitor cells in vitro is impaired, consistent with down-regulated expression of cell-cell adhesion genes. These results reveal that thyroid hormone receptor α1 is required for normal neural progenitor cell proliferation in human cerebral cortical development. They also exemplify quantitative approaches for studying neurodevelopmental disorders using patient-derived cells in vitro.

Microtubules Deform the Nuclear Membrane and Disrupt Nucleocytoplasmic Transport in Tau-Mediated Frontotemporal Dementia.

The neuronal microtubule-associated protein tau, MAPT, is central to the pathogenesis of many dementias. Autosomal-dominant mutations in MAPT cause inherited frontotemporal dementia (FTD), but the underlying pathogenic mechanisms are unclear. Using human stem cell models of FTD due to MAPT mutations, we find that tau becomes hyperphosphorylated and mislocalizes to cell bodies and dendrites in cortical neurons, recapitulating a key early event in FTD. Mislocalized tau in the cell body leads to abnormal microtubule movements in FTD-MAPT neurons that grossly deform the nuclear membrane. This results in defective nucleocytoplasmic transport, which is corrected by microtubule depolymerization. Neurons in the post-mortem human FTD-MAPT cortex have a high incidence of nuclear invaginations, indicating that tau-mediated nuclear membrane dysfunction is an important pathogenic process in FTD. Defects in nucleocytoplasmic transport in FTD point to important commonalities in the pathogenic mechanisms of tau-mediated dementias and ALS-FTD due to TDP-43 and C9orf72 mutations.

Altered γ-Secretase Processing of APP Disrupts Lysosome and Autophagosome Function in Monogenic Alzheimer's Disease.

Abnormalities of the endolysosomal and autophagy systems are found in Alzheimer's disease, but it is not clear whether defects in these systems are a cause or consequence of degenerative processes in the disease. In human neuronal models of monogenic Alzheimer's disease, APP and PSEN1 mutations disrupt lysosome function and autophagy, leading to impaired lysosomal proteolysis and defective autophagosome clearance. Processing of APP by γ-secretase is central to the pathogenic changes in the lysosome-autophagy system caused by PSEN1 and APP mutations: reducing production of C-terminal APP by inhibition of BACE1 rescued these phenotypes in both APP and PSEN1 mutant neurons, whereas inhibition of γ-secretase induced lysosomal and autophagic pathology in healthy neurons. Defects in lysosomes and autophagy due to PSEN1 mutations are rescued by CRISPR-knockout of APP. These data demonstrate a key role for proteolysis of the C-terminal of APP by γ-secretase in neuronal dysfunction in monogenic Alzheimer's disease.

In vivo modeling of human neuron dynamics and Down syndrome.

Harnessing the potential of human stem cells for modeling the physiology and diseases of cortical circuitry requires monitoring cellular dynamics in vivo. We show that human induced pluripotent stem cell (iPSC)-derived cortical neurons transplanted into the adult mouse cortex consistently organized into large (up to ~100 mm3) vascularized neuron-glia territories with complex cytoarchitecture. Longitudinal imaging of >4000 grafted developing human neurons revealed that neuronal arbors refined via branch-specific retraction; human synaptic networks substantially restructured over 4 months, with balanced rates of synapse formation and elimination; and oscillatory population activity mirrored the patterns of fetal neural networks. Lastly, we found increased synaptic stability and reduced oscillations in transplants from two individuals with Down syndrome, demonstrating the potential of in vivo imaging in human tissue grafts for patient-specific modeling of cortical development, physiology, and pathogenesis.

Publisher Correction: Guided self-organization and cortical plate formation in human brain organoids.

This corrects the article DOI: 10.1038/nbt.3906.

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Chromatin Accessibility Impacts Transcriptional Reprogramming in Oocytes.

Oocytes have a remarkable ability to reactivate silenced genes in somatic cells. However, it is not clear how the chromatin architecture of somatic cells affects this transcriptional reprogramming. Here, we investigated the relationship between the chromatin opening and transcriptional activation. We reveal changes in chromatin accessibility and their relevance to transcriptional reprogramming after transplantation of somatic nuclei into Xenopus oocytes. Genes that are silenced, but have pre-existing open transcription start sites in donor cells, are prone to be activated after nuclear transfer, suggesting that the chromatin signature of somatic nuclei influences transcriptional reprogramming. There are also activated genes associated with new open chromatin sites, and transcription factors in oocytes play an important role in transcriptional reprogramming from such genes. Finally, we show that genes resistant to reprogramming are associated with closed chromatin configurations. We conclude that chromatin accessibility is a central factor for successful transcriptional reprogramming in oocytes.

Extracellular Forms of Aß and Tau from iPSC Models of Alzheimer's Disease Disrupt Synaptic Plasticity.

The early stages of Alzheimer's disease are associated with synaptic dysfunction prior to overt loss of neurons. To identify extracellular molecules that impair synaptic plasticity in the brain, we studied the secretomes of human iPSC-derived neuronal models of Alzheimer's disease. When introduced into the rat brain, secretomes from human neurons with either a presenilin-1 mutation, amyloid precursor protein duplication, or trisomy of chromosome 21 all strongly inhibit hippocampal long-term potentiation. Synaptic dysfunction caused by presenilin-1 mutant and amyloid precusor protein duplication secretomes is mediated by Aß peptides, whereas trisomy of chromosome 21 (trisomy 21) neuronal secretomes induce dysfunction through extracellular tau. In all cases, synaptotoxicity is relieved by antibody blockade of cellular prion protein. These data indicate that human models of Alzheimer's disease generate distinct proteins that converge at the level of cellular prion protein to induce synaptic dysfunction in vivo.

Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia.

The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2), which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations.

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.

In Alzheimer's disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon.

Filopodyan: An open-source pipeline for the analysis of filopodia.

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.

Guided self-organization and cortical plate formation in human brain organoids.

Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells or used bioengineered constructs to arrange cells in an organ-like configuration. While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.