Molecular signatures underlying neurofibrillary tangle susceptibility in Alzheimer's disease.

Tau aggregation in neurofibrillary tangles (NFTs) is closely associated with neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the molecular signatures that distinguish between aggregation-prone and aggregation-resistant cell states are unknown. We developed methods for the high-throughput isolation and transcriptome profiling of single somas with NFTs from the human AD brain, quantified the susceptibility of 20 neocortical subtypes for NFT formation and death, and identified both shared and cell-type-specific signatures. NFT-bearing neurons shared a marked upregulation of synaptic transmission-related genes, including a core set of 63 genes enriched for synaptic vesicle cycling. Oxidative phosphorylation and mitochondrial dysfunction were highly cell-type dependent. Apoptosis was only modestly enriched, and the susceptibilities of NFT-bearing and NFT-free neurons for death were highly similar. Our analysis suggests that NFTs represent cell-type-specific responses to stress and synaptic dysfunction. We provide a resource for biomarker discovery and the investigation of tau-dependent and tau-independent mechanisms of neurodegeneration.

Single-nucleus transcriptome analysis reveals disease- and regeneration-associated endothelial cells in white matter vascular dementia.

BACKGROUND: Vascular dementia (VaD) is the accumulation of vascular lesions in the subcortical white matter of the brain. These lesions progress and there is no direct medical therapy. AIMS: To determine the specific cellular responses in VaD so as to provide molecular targets for therapeutic development. MATERIALS AND METHODS: Single-nucleus transcriptome analysis was performed in human periventricular white matter (PVWM) samples of VaD and normal control (NC) subjects. RESULTS: Differential analysis shows that cell type-specific transcriptomic changes in VaD are associated with the disruption of specific biological processes, including angiogenesis, immune activation, axonal injury and myelination. Each cell type in the neurovascular unit within white matter has a specific alteration in gene expression in VaD. In a central cell type for this disease, subcluster analysis of endothelial cells (EC) indicates that VaD contains a disease-associated EC subcluster that expresses genes associated with programmed cell death and a response to protein folding. Two other subpopulations of EC in VaD express molecular systems associated with regenerative processes in angiogenesis, and in axonal sprouting and oligodendrocyte progenitor cell maturation. CONCLUSION: This comprehensive molecular profiling of brain samples from patients with VaD reveals previously unknown molecular changes in cells of the neurovascular niche, and an attempt at regeneration in injured white matter.

Defining a Role for G-Protein Coupled Receptor/cAMP/CRE-Binding Protein Signaling in Hair Follicle Stem Cell Activation.

Manipulation of adrenergic signaling has been shown experimentally and clinically to affect hair follicle growth. In this study, we provide direct evidence that canonical cAMP/CRE-binding protein signaling through adrenergic receptors can regulate hair follicle stem cell (HFSC) activation and hair cycle. We found that CRE-binding protein activation is regulated through the hair cycle and coincides with HFSC activation. Both isoproterenol and procaterol, agonists of adrenergic receptors, show the capacity to activate the hair cycle in mice. Furthermore, deletion of ADRB2 receptor, which is thought to mediate sympathetic nervous system regulation of HFSCs, was sufficient to block HFSC activation. Downstream, stimulation of adenylyl cyclase with forskolin or inhibition of phosphodiesterase to increase cAMP accumulation or direct application of cAMP was each sufficient to promote HFSC activation and accelerate initiation of hair cycle. Genetic induction of a Designer Receptors Exclusively Activated by Designer Drug allele showed that G-protein coupled receptor/GαS stimulation, specifically in HFSCs, promoted the activation of the hair cycle. Finally, we provide evidence that G-protein coupled receptor/CRE-binding protein signaling can potentially act on HFSCs by promoting glycolytic metabolism, which was previously shown to stimulate HFSC activation. Together, these data provide mechanistic insights into the role of sympathetic innervation on HFSC function.

Defining the nature of human pluripotent stem cell-derived interneurons via single-cell analysis.

The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes.

Identification of neural oscillations and epileptiform changes in human brain organoids.

Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.

Reliable generation of glial enriched progenitors from human fibroblast-derived iPSCs.

White matter stroke (WMS) occurs as small infarcts in deep penetrating blood vessels in the brain and affects the regions of the brain that carry connections, termed the subcortical white matter. WMS progresses over years and has devastating clinical consequences. Unlike large grey matter strokes, WMS disrupts the axonal architecture of the brain and depletes astrocytes, oligodendrocyte lineage cells, axons and myelinating cells, resulting in abnormalities of gait and executive function. An astrocytic cell-based therapy is positioned as a strong therapeutic candidate after WMS. In this study we report, the reliable generation of a novel stem cell-based therapeutic product, glial enriched progenitors (GEPs) derived from human induced pluripotent stem cells (hiPSCs). By transient treatment of hiPSC derived neural progenitors (hiPSC-NPCs) with the small molecule deferoxamine, a prolyl hydroxylase inhibitor, for three days hiPSC-NPCs become permanently biased towards an astrocytic fate, producing hiPSC-GEPs. In preparation for clinical application, we have developed qualification assays to ensure identity, safety, purity, and viability of the cells prior to manufacture. Using tailored q-RT-PCR-based assays, we have demonstrated the lack of pluripotency in our final therapeutic candidate cells (hiPSC-GEPs) and we have identified the unique genetic profile of hiPSC-GEPs that is clearly distinct from the parent lines, hiPSCs and iPSC-NPCs. After completion of the viability assay, we have stablished the therapeutic window of use for hiPSC-GEPs in future clinical applications (7 h). Lastly, we were able to reliably and consistently produce a safe therapeutic final product negative for contamination by any human or murine viral pathogens, selected bacteria, common laboratory mycoplasmas, growth of any aerobes, anaerobes, yeast, or fungi and 100 times less endotoxin levels than the maximum acceptable value. This study demonstrates the reliable and safe generation of patient derived hiPSC-GEPs that are clinically ready as a cell-based therapeutic approach for WMS.

Patient-derived glial enriched progenitors repair functional deficits due to white matter stroke and vascular dementia in rodents.

Subcortical white matter stroke (WMS) accounts for up to 30% of all stroke events. WMS damages primarily astrocytes, axons, oligodendrocytes, and myelin. We hypothesized that a therapeutic intervention targeting astrocytes would be ideally suited for brain repair after WMS. We characterize the cellular properties and in vivo tissue repair activity of glial enriched progenitor (GEP) cells differentiated from human-induced pluripotent stem cells, termed hiPSC-derived GEPs (hiPSC-GEPs). hiPSC-GEPs are derived from hiPSC-neural progenitor cells via an experimental manipulation of hypoxia inducible factor activity by brief treatment with a prolyl hydroxylase inhibitor, deferoxamine. This treatment permanently biases these cells to further differentiate toward an astrocyte fate. hiPSC-GEPs transplanted into the brain in the subacute period after WMS in mice migrated widely, matured into astrocytes with a prorepair phenotype, induced endogenous oligodendrocyte precursor proliferation and remyelination, and promoted axonal sprouting. hiPSC-GEPs enhanced motor and cognitive recovery compared to other hiPSC-differentiated cell types. This approach establishes an hiPSC-derived product with easy scale-up capabilities that might be effective for treating WMS.

Inhibition of pyruvate oxidation as a versatile stimulator of the hair cycle in models of alopecia.

Hair follicle stem cells (HFSCs) are known to be responsible for the initiation of a new hair cycle, but typically remain quiescent for very long periods. In alopecia, or hair loss disorders, follicles can be refractory to activation for years or even permanently. Alopecia can be triggered by autoimmunity, age, chemotherapeutic treatment, stress, disrupted circadian rhythm or other environmental insults. We previously showed that hair follicle stem cells and the hair cycle can be manipulated by regulation of pyruvate entry into mitochondria for subsequent oxidation to fuel the TCA cycle in normal adult mice with typical hair cycling. Here, we present new data from our efforts to develop murine models of alopecia based on environmental triggers that have been shown to do the same in human skin. We found that inhibition of pyruvate transport into mitochondria can accelerate the hair cycle even during refractory hair cycling due to age, repeated chemotherapeutic treatment and stress. Hair cycle acceleration in these alopecia models led to the formation of histologically normal hair follicles within 30-40 days of treatment without any overt signs of toxicity or deleterious effects. Therefore, we propose inhibition of pyruvate entry into mitochondria as a versatile treatment strategy for alopecia in humans.

Development of Novel Mitochondrial Pyruvate Carrier Inhibitors to Treat Hair Loss.

Herein, we report the synthesis and evaluation of novel analogues of UK-5099 both in vitro and in vivo for the development of mitochondrial pyruvate carrier (MPC) inhibitors to treat hair loss. A comprehensive understanding of the structure-activity relationship was obtained by varying four positions of the hit compound, namely, the alkyl group on the N1 position, substituents on the indole core, various aromatic and heteroaromatic core structures, and various Michael acceptors. The major discovery was that the inhibitors with a 3,5-bis(trifluoromethyl)benzyl group at the N1 position were shown to have much better activity than JXL001 (UK-5099) to increase cellular lactate production. Additionally, analogue JXL069, possessing a 7-azaindole heterocycle, was also shown to have significant MPC inhibition activity, which further increases the chemical space for drug design. Finally, more than 10 analogues were tested on shaved mice by topical treatment and promoted obvious hair growth on mice.

Differential Gene Set Enrichment Analysis: a statistical approach to quantify the relative enrichment of two gene sets.

MOTIVATION: Gene Set Enrichment Analysis (GSEA) is an algorithm widely used to identify statistically enriched gene sets in transcriptomic data. However, GSEA cannot examine the enrichment of two gene sets or pathways relative to one another. Here we present Differential Gene Set Enrichment Analysis (DGSEA), an adaptation of GSEA that quantifies the relative enrichment of two gene sets. RESULTS: After validating the method using synthetic data, we demonstrate that DGSEA accurately captures the hypoxia-induced coordinated upregulation of glycolysis and downregulation of oxidative phosphorylation. We also show that DGSEA is more predictive than GSEA of the metabolic state of cancer cell lines, including lactate secretion and intracellular concentrations of lactate and AMP. Finally, we demonstrate the application of DGSEA to generate hypotheses about differential metabolic pathway activity in cellular senescence. Together, these data demonstrate that DGSEA is a novel tool to examine the relative enrichment of gene sets in transcriptomic data. AVAILABILITY AND IMPLEMENTATION: DGSEA software and tutorials are available at https://jamesjoly.github.io/DGSEA/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Defining Transcriptional Signatures of Human Hair Follicle Cell States.

The epidermis and its appendage, the hair follicle, represent an elegant developmental system in which cells are replenished with regularity because of controlled proliferation, lineage specification, and terminal differentiation. Although transcriptome data exists for human epidermal and dermal cells, the hair follicle remains poorly characterized. Through single-cell resolution profiling of the epidermis and anagen hair follicle, we characterized the anatomical, transcriptional, functional, and pathological profiles of distinct epidermal, hair follicle, and hair follicle-associated cell subpopulations including melanocytes, endothelial cells, and immune cells. We additionally traced the differentiation trajectory of interfollicular and matrix cell progenitors and explored the association of specific cell subpopulations to known molecular signatures of common skin conditions. These data simultaneously corroborate prior murine and human studies while offering new insights into epidermal and hair follicle differentiation and pathogenesis.

A Single-Cell Transcriptomic Atlas of Human Neocortical Development during Mid-gestation.

We performed RNA sequencing on 40,000 cells to create a high-resolution single-cell gene expression atlas of developing human cortex, providing the first single-cell characterization of previously uncharacterized cell types, including human subplate neurons, comparisons with bulk tissue, and systematic analyses of technical factors. These data permit deconvolution of regulatory networks connecting regulatory elements and transcriptional drivers to single-cell gene expression programs, significantly extending our understanding of human neurogenesis, cortical evolution, and the cellular basis of neuropsychiatric disease. We tie cell-cycle progression with early cell fate decisions during neurogenesis, demonstrating that differentiation occurs on a transcriptomic continuum; rather than only expressing a few transcription factors that drive cell fates, differentiating cells express broad, mixed cell-type transcriptomes before telophase. By mapping neuropsychiatric disease genes to cell types, we implicate dysregulation of specific cell types in ASD, ID, and epilepsy. We developed CoDEx, an online portal to facilitate data access and browsing.

Correction to: Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

The original article [1] contains an error in the legend of Fig 5 whereby the descriptions for panels 5d and 5e are incorrect; as such, the corrected legend can be viewed below with its respective figure images.

The WNT10B Network Is Associated with Survival and Metastases in Chemoresistant Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for ß-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for ß-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on ß-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg.

Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

Apparent bias toward long gene misregulation in MeCP2 syndromes disappears after controlling for baseline variations.

Recent studies have suggested that genes longer than 100 kb are more likely to be misregulated in neurological diseases associated with synaptic dysfunction, such as autism and Rett syndrome. These length-dependent transcriptional changes are modest in MeCP2-mutant samples, but, given the low sensitivity of high-throughput transcriptome profiling technology, here we re-evaluate the statistical significance of these results. We find that the apparent length-dependent trends previously observed in MeCP2 microarray and RNA-sequencing datasets disappear after estimating baseline variability from randomized control samples. This is particularly true for genes with low fold changes. We find no bias with NanoString technology, so this long gene bias seems to be particular to polymerase chain reaction amplification-based platforms. In contrast, authentic long gene effects, such as those caused by topoisomerase inhibition, can be detected even after adjustment for baseline variability. We conclude that accurate characterization of length-dependent (or other) trends requires establishing a baseline from randomized control samples.

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence.

To determine the role for mutations of MECP2 in Rett syndrome, we generated isogenic lines of human induced pluripotent stem cells, neural progenitor cells, and neurons from patient fibroblasts with and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal-specific gene expression changes, including induction of a senescence-associated secretory phenotype (SASP) program. Patient-derived neurons made without MECP2 showed signs of stress, including induction of P53, and senescence. The induction of P53 appeared to affect dendritic branching in Rett neurons, as P53 inhibition restored dendritic complexity. The induction of P53 targets was also detectable in analyses of human Rett patient brain, suggesting that this disease-in-a-dish model can provide relevant insights into the human disorder.