Gliovascular transcriptional perturbations in Alzheimer's disease reveal molecular mechanisms of blood brain barrier dysfunction.

To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer's disease, we performed single nucleus RNA sequencing in 24 Alzheimer's disease and control brains and focused on vascular and astrocyte clusters as main cell types of blood-brain-barrier gliovascular-unit. The majority of the vascular transcriptional changes were in pericytes. Of the vascular molecular targets predicted to interact with astrocytic ligands, SMAD3, upregulated in Alzheimer's disease pericytes, has the highest number of ligands including VEGFA, downregulated in Alzheimer's disease astrocytes. We validated these findings with external datasets comprising 4,730 pericyte and 150,664 astrocyte nuclei. Blood SMAD3 levels are associated with Alzheimer's disease-related neuroimaging outcomes. We determined inverse relationships between pericytic SMAD3 and astrocytic VEGFA in human iPSC and zebrafish models. Here, we detect vast transcriptome changes in Alzheimer's disease at the gliovascular-unit, prioritize perturbed pericytic SMAD3-astrocytic VEGFA interactions, and validate these in cross-species models to provide a molecular mechanism of blood-brain-barrier disintegrity in Alzheimer's disease.

ABCA7-dependent Neuropeptide-Y signalling is a resilience mechanism required for synaptic integrity in Alzheimer's disease.

Alzheimer's disease (AD) remains a complex challenge characterized by cognitive decline and memory loss. Genetic variations have emerged as crucial players in the etiology of AD, enabling hope for a better understanding of the disease mechanisms; yet the specific mechanism of action for those genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Using CRISPR/Cas9 gene editing, we generated a knockout model for abca7, orthologous to human ABCA7 - an established AD-risk gene. The abca7 +/- zebrafish showed reduced astroglial proliferation, synaptic density, and microglial abundance in response to amyloid beta 42 (Aß42). Single-cell transcriptomics revealed abca7 -dependent neuronal and glial cellular crosstalk through neuropeptide Y (NPY) signaling. The abca7 knockout reduced the expression of npy, bdnf and ngfra , which are required for synaptic integrity and astroglial proliferation. With clinical data in humans, we showed reduced NPY in AD correlates with elevated Braak stage, predicted regulatory interaction between NPY and BDNF , identified genetic variants in NPY associated with AD, found segregation of variants in ABCA7, BDNF and NGFR in AD families, and discovered epigenetic changes in the promoter regions of NPY, NGFR and BDNF in humans with specific single nucleotide polymorphisms in ABCA7 . These results suggest that ABCA7-dependent NPY signaling is required for synaptic integrity, the impairment of which generates a risk factor for AD through compromised brain resilience.

The forkhead transcription factor Foxj1 controls vertebrate olfactory cilia biogenesis and sensory neuron differentiation.

In vertebrates, olfactory receptors localize on multiple cilia elaborated on dendritic knobs of olfactory sensory neurons (OSNs). Although olfactory cilia dysfunction can cause anosmia, how their differentiation is programmed at the transcriptional level has remained largely unexplored. We discovered in zebrafish and mice that Foxj1, a forkhead domain-containing transcription factor traditionally linked with motile cilia biogenesis, is expressed in OSNs and required for olfactory epithelium (OE) formation. In keeping with the immotile nature of olfactory cilia, we observed that ciliary motility genes are repressed in zebrafish, mouse, and human OSNs. Strikingly, we also found that besides ciliogenesis, Foxj1 controls the differentiation of the OSNs themselves by regulating their cell type-specific gene expression, such as that of olfactory marker protein (omp) involved in odor-evoked signal transduction. In line with this, response to bile acids, odors detected by OMP-positive OSNs, was significantly diminished in foxj1 mutant zebrafish. Taken together, our findings establish how the canonical Foxj1-mediated motile ciliogenic transcriptional program has been repurposed for the biogenesis of immotile olfactory cilia, as well as for the development of the OSNs.

sox1a:eGFP transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons.

Sox transcription factors are crucial for vertebrate nervous system development. In zebrafish embryo, sox1 genes are expressed in neural progenitor cells and neurons of ventral spinal cord. Our recent study revealed that the loss of sox1a and sox1b function results in a significant decline of V2 subtype neurons (V2s). Using single-cell RNA sequencing, we analyzed the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four time points during embryonic development, employing the Tg(sox1a:eGFP) line. In addition to previously characterized sox1a-expressing neurons, we discovered the expression of sox1a in late-developing intraspinal serotonergic neurons (ISNs). Developmental trajectory analysis suggests that ISNs arise from lateral floor plate (LFP) progenitor cells. Pharmacological inhibition of the Notch signaling pathway revealed its role in negatively regulating LFP progenitor cell differentiation into ISNs. Our findings highlight the zebrafish LFP as a progenitor domain for ISNs, alongside known Kolmer-Agduhr (KA) and V3 interneurons.

Nerve growth factor receptor (Ngfr) induces neurogenic plasticity by suppressing reactive astroglial Lcn2/Slc22a17 signaling in Alzheimer's disease.

Neurogenesis, crucial for brain resilience, is reduced in Alzheimer's disease (AD) that induces astroglial reactivity at the expense of the pro-neurogenic potential, and restoring neurogenesis could counteract neurodegenerative pathology. However, the molecular mechanisms promoting pro-neurogenic astroglial fate despite AD pathology are unknown. In this study, we used APP/PS1dE9 mouse model and induced Nerve growth factor receptor (Ngfr) expression in the hippocampus. Ngfr, which promotes neurogenic fate of astroglia during the amyloid pathology-induced neuroregeneration in zebrafish brain, stimulated proliferative and neurogenic outcomes. Histological analyses of the changes in proliferation and neurogenesis, single-cell transcriptomics, spatial proteomics, and functional knockdown studies showed that the induced expression of Ngfr reduced the reactive astrocyte marker Lipocalin-2 (Lcn2), which we found was sufficient to reduce neurogenesis in astroglia. Anti-neurogenic effects of Lcn2 was mediated by Slc22a17, blockage of which recapitulated the pro-neurogenicity by Ngfr. Long-term Ngfr expression reduced amyloid plaques and Tau phosphorylation. Postmortem human AD hippocampi and 3D human astroglial cultures showed elevated LCN2 levels correlate with reactive gliosis and reduced neurogenesis. Comparing transcriptional changes in mouse, zebrafish, and human AD brains for cell intrinsic differential gene expression and weighted gene co-expression networks revealed common altered downstream effectors of NGFR signaling, such as PFKP, which can enhance proliferation and neurogenesis in vitro when blocked. Our study suggests that the reactive non-neurogenic astroglia in AD can be coaxed to a pro-neurogenic fate and AD pathology can be alleviated with Ngfr. We suggest that enhancing pro-neurogenic astroglial fate may have therapeutic ramifications in AD.

The Caribbean-Hispanic Alzheimer's disease brain transcriptome reveals ancestry-specific disease mechanisms.

Identifying ancestry-specific molecular profiles of late-onset Alzheimer's Disease (LOAD) in brain tissue is crucial to understand novel mechanisms and develop effective interventions in non-European, high-risk populations. We performed gene differential expression (DE) and consensus network-based analyses in RNA-sequencing data of postmortem brain tissue from 39 Caribbean Hispanics (CH). To identify ancestry-concordant and -discordant expression profiles, we compared our results to those from two independent non-Hispanic White (NHW) samples (n = 731). In CH, we identified 2802 significant DE genes, including several LOAD known-loci. DE effects were highly concordant across ethnicities, with 373 genes transcriptome-wide significant in all three cohorts. Cross-ancestry meta-analysis found NPNT to be the top DE gene. We replicated over 82% of meta-analyses genome-wide signals in single-nucleus RNA-seq data (including NPNT and LOAD known-genes SORL1, FBXL7, CLU, ABCA7). Increasing representation in genetic studies will allow for deeper understanding of ancestry-specific mechanisms and improving precision treatment options in understudied groups.

RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation.

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AßO). Notably, AßO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.

Single Cell/Nucleus Transcriptomics Comparison in Zebrafish and Humans Reveals Common and Distinct Molecular Responses to Alzheimer's Disease.

Neurogenesis is significantly reduced in Alzheimer's disease (AD) and is a potential therapeutic target. Contrary to humans, a zebrafish can regenerate its diseased brain, and thus is ideal for studying neurogenesis. To compare the AD-related molecular pathways between humans and zebrafish, we compared single cell or nuclear transcriptomic data from a zebrafish amyloid toxicity model and its controls (N = 12) with the datasets of two human adult brains (N = 10 and N = 48 (Microglia)), and one fetal brain (N = 10). Approximately 95.4% of the human and zebrafish cells co-clustered. Within each cell type, we identified differentially expressed genes (DEGs), enriched KEGG pathways, and gene ontology terms. We studied synergistic and non-synergistic DEGs to point at either common or uniquely altered mechanisms across species. Using the top DEGs, a high concordance in gene expression changes between species was observed in neuronal clusters. On the other hand, the molecular pathways affected by AD in zebrafish astroglia differed from humans in favor of the neurogenic pathways. The integration of zebrafish and human transcriptomes shows that the zebrafish can be used as a tool to study the cellular response to amyloid proteinopathies. Uniquely altered pathways in zebrafish could highlight the specific mechanisms underlying neurogenesis, which are absent in humans, and could serve as potential candidates for therapeutic developments.

Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.

Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.

KYNA/Ahr Signaling Suppresses Neural Stem Cell Plasticity and Neurogenesis in Adult Zebrafish Model of Alzheimer's Disease.

Neurogenesis decreases in Alzheimer's disease (AD) patients, suggesting that restoring the normal neurogenic response could be a disease modifying intervention. To study the mechanisms of pathology-induced neuro-regeneration in vertebrate brains, zebrafish is an excellent model due to its extensive neural regeneration capacity. Here, we report that Kynurenic acid (KYNA), a metabolite of the amino acid tryptophan, negatively regulates neural stem cell (NSC) plasticity in adult zebrafish brain through its receptor, aryl hydrocarbon receptor 2 (Ahr2). The production of KYNA is suppressed after amyloid-toxicity through reduction of the levels of Kynurenine amino transferase 2 (KAT2), the key enzyme producing KYNA. NSC proliferation is enhanced by an antagonist for Ahr2 and is reduced with Ahr2 agonists or KYNA. A subset of Ahr2-expressing zebrafish NSCs do not express other regulatory receptors such as il4r or ngfra, indicating that ahr2-positive NSCs constitute a new subset of neural progenitors that are responsive to amyloid-toxicity. By performing transcriptome-wide association studies (TWAS) in three late onset Alzheimer disease (LOAD) brain autopsy cohorts, we also found that several genes that are components of KYNA metabolism or AHR signaling are differentially expressed in LOAD, suggesting a strong link between KYNA/Ahr2 signaling axis to neurogenesis in LOAD.

Functional properties of habenular neurons are determined by developmental stage and sequential neurogenesis.

The developing brain undergoes drastic alterations. Here, we investigated developmental changes in the habenula, a brain region that mediates behavioral flexibility during learning, social interactions, and aversive experiences. We showed that developing habenular circuits exhibit multiple alterations that lead to an increase in the structural and functional diversity of cell types, inputs, and functional modules. As the habenula develops, it sequentially transforms into a multisensory brain region that can process visual, olfactory, mechanosensory, and aversive stimuli. Moreover, we observed that the habenular neurons display spatiotemporally structured spontaneous activity that shows prominent alterations and refinement with age. These alterations in habenular activity are accompanied by sequential neurogenesis and the integration of distinct neural clusters across development. Last, we revealed that habenular neurons with distinct functional properties are born sequentially at distinct developmental time windows. Our results highlight a strong link between the functional properties of habenular neurons and their precise birthdate.

Protocol for Dissection and Dissociation of Zebrafish Telencephalon for Single-Cell Sequencing.

Single-cell sequencing (sc-Seq) is a powerful tool to investigate the molecular signatures of cell types in a complex mixture of cells. A critical step in sc-Seq is preparing a single-cell suspension with a high number of viable cells. Here, we show how to dissect zebrafish telencephalon and how to dissociate it into a single-cell suspension. This is followed by flow cytometry-based sorting to enrich for neural progenitor stem cells. Our technique typically yields 70,000 live cells from one zebrafish telencephalon. For complete details on the use and execution of this protocol, please refer to Cosacak et al. (2019).

Type 1 Interleukin-4 Signaling Obliterates Mouse Astroglia in vivo but Not in vitro.

Recent findings suggest that reduced neurogenesis could be one of the underlying reasons for the exacerbated neuropathology in humans, thus restoring the neural stem cell proliferation and neurogenesis could help to circumvent some pathological aspects of Alzheimer's disease. We recently identified Interleukin-4/STAT6 signaling as a neuron-glia crosstalk mechanism that enables glial proliferation and neurogenesis in adult zebrafish brain and 3D cultures of human astroglia, which manifest neurogenic properties. In this study, by using single cell sequencing in the APP/PS1dE9 mouse model of AD, we found that IL4 receptor (Il4r) is not expressed in mouse astroglia and IL4 signaling is not active in these cells. We tested whether activating IL4/STAT6 signaling would enhance cell proliferation and neurogenesis in healthy and disease conditions. Lentivirus-mediated expression of IL4R or constitutively active STAT6VT impaired the survival capacity of mouse astroglia in vivo but not in vitro. These results suggest that the adult mouse brain generates a non-permissive environment that dictates a negative effect of IL4 signaling on astroglial survival and neurogenic properties in contrast to zebrafish brains and in vitro mammalian cell cultures. Our findings that IL4R signaling in dentate gyrus (DG) of adult mouse brain impinges on the survival of DG cells implicate an evolutionary mechanism that might underlie the loss of neuroregenerative ability of the brain, which might be utilized for basic and clinical aspects for neurodegenerative diseases.

Neuron-glia interaction through Serotonin-BDNF-NGFR axis enables regenerative neurogenesis in Alzheimer's model of adult zebrafish brain.

It was recently suggested that supplying the brain with new neurons could counteract Alzheimer's disease (AD). This provocative idea requires further testing in experimental models in which the molecular basis of disease-induced neuronal regeneration could be investigated. We previously found that zebrafish stimulates neural stem cell (NSC) plasticity and neurogenesis in AD and could help to understand the mechanisms to be harnessed for developing new neurons in diseased mammalian brains. Here, by performing single-cell transcriptomics, we found that amyloid toxicity-induced interleukin-4 (IL4) promotes NSC proliferation and neurogenesis by suppressing the tryptophan metabolism and reducing the production of serotonin. NSC proliferation was suppressed by serotonin via down-regulation of brain-derived neurotrophic factor (BDNF)-expression in serotonin-responsive periventricular neurons. BDNF enhances NSC plasticity and neurogenesis via nerve growth factor receptor A (NGFRA)/ nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFkB) signaling in zebrafish but not in rodents. Collectively, our results suggest a complex neuron-glia interaction that regulates regenerative neurogenesis after AD conditions in zebrafish.

Alzheimer's disease, neural stem cells and neurogenesis: cellular phase at single-cell level.

Alzheimer's disease cannot be cured as of yet. Our current understanding on the causes of Alzheimer's disease is limited. To develop treatments, experimental models that represent a particular cellular phase of the disease and more rigorous scrutiny of the cellular pathological mechanisms are crucial. In recent years, Alzheimer's disease research underwent a paradigm shift. According to this tendency, Alzheimer's disease is increasingly being conceived of a disease where not only neurons but also multiple cell types synchronously partake to manifest the pathology. Knowledge on every cell type adds an alternative approach and hope for the efforts towards the treatment. Neural stem cells and their neurogenic ability are making an appearance as a new aspect of the disease manifestation based on the recent findings that neurogenesis reduces dramatically in Alzheimer's disease patients compared to healthy individuals. Therefore, understanding how neural stem cells can form new neurons in Alzheimer's disease brains holds an immense potential for clinics. However, this provocative idea requires further evidence and tools for investigation. Recently, single cell sequencing appeared as a revolutionary tool to understand cellular programs in unprecedented resolution and it will undoubtedly facilitate comprehensive investigation of different cell types in Alzheimer's disease. In this mini-review, we will touch upon recent studies that use single cell sequencing for investigating cellular response in Alzheimer's disease and some consideration pertaining to the utilization of neural regeneration for Alzheimer's disease research.

Glia-neuron interactions underlie state transitions to generalized seizures.

Brain activity and connectivity alter drastically during epileptic seizures. The brain networks shift from a balanced resting state to a hyperactive and hypersynchronous state. It is, however, less clear which mechanisms underlie the state transitions. By studying neural and glial activity in zebrafish models of epileptic seizures, we observe striking differences between these networks. During the preictal period, neurons display a small increase in synchronous activity only locally, while the gap-junction-coupled glial network was highly active and strongly synchronized across large distances. The transition from a preictal state to a generalized seizure leads to an abrupt increase in neural activity and connectivity, which is accompanied by a strong alteration in glia-neuron interactions and a massive increase in extracellular glutamate. Optogenetic activation of glia excites nearby neurons through the action of glutamate and gap junctions, emphasizing a potential role for glia-glia and glia-neuron connections in the generation of epileptic seizures.

Single-Cell Transcriptomics Analyses of Neural Stem Cell Heterogeneity and Contextual Plasticity in a Zebrafish Brain Model of Amyloid Toxicity.

The neural stem cell (NSC) reservoir can be harnessed for stem cell-based regenerative therapies. Zebrafish remarkably regenerate their brain by inducing NSC plasticity in a Amyloid-ß-42 (Aß42)-induced experimental Alzheimer's disease (AD) model. Interleukin-4 (IL-4) is also critical for AD-induced NSC proliferation. However, the mechanisms of this response have remained unknown. Using single-cell transcriptomics in the adult zebrafish brain, we identify distinct subtypes of NSCs and neurons and differentially regulated pathways and their gene ontologies and investigate how cell-cell communication is altered through ligand-receptor pairs in AD conditions. Our results propose the existence of heterogeneous and spatially organized stem cell populations that react distinctly to amyloid toxicity. This resource article provides an extensive database for the molecular basis of NSC plasticity in the AD model of the adult zebrafish brain. Further analyses of stem cell heterogeneity and neuro-regenerative ability at single-cell resolution could yield drug targets for mobilizing NSCs for endogenous neuro-regeneration in humans.

GATA3 Promotes the Neural Progenitor State but Not Neurogenesis in 3D Traumatic Injury Model of Primary Human Cortical Astrocytes.

Astrocytes are abundant cell types in the vertebrate central nervous system and can act as neural stem cells in specialized niches where they constitutively generate new neurons. Outside the stem cell niches, however, these glial cells are not neurogenic. Although injuries in the mammalian central nervous system lead to profound proliferation of astrocytes, which cluster at the lesion site to form a gliotic scar, neurogenesis does not take place. Therefore, a plausible regenerative therapeutic option is to coax the endogenous reactive astrocytes to a pre-neurogenic progenitor state and use them as an endogenous reservoir for repair. However, little is known on the mechanisms that promote the neural progenitor state after injuries in humans. Gata3 was previously found to be a mechanism that zebrafish brain uses to injury-dependent induction of neural progenitors. However, the effects of GATA3 in human astrocytes after injury are not known. Therefore, in this report, we investigated how overexpression of GATA3 in primary human astrocytes would affect the neurogenic potential before and after injury in 2D and 3D cultures. We found that primary human astrocytes are unable to induce GATA3 after injury. Lentivirus-mediated overexpression of GATA3 significantly increased the number of GFAP/SOX2 double positive astrocytes and expression of pro-neural factor ASCL1, but failed to induce neurogenesis, suggesting that GATA3 is required for enhancing the neurogenic potential of primary human astrocytes and is not sufficient to induce neurogenesis alone.

Small RNA data set that includes tRNA-derived fragments from Jurkat cells treated with camptothecin.

In this article, we report a small RNA data set obtained from human T cell acute leukemia Jurkat cells, which were treated with the universal apoptotic agent camptothecin. Based on the Annexin-V labeling pattern, we sorted two Jurkat subpopulations in treated cells: one that is sensitive to the drug and the other being relatively more resistant. We report new original data that include the frequency of tRNA-derived fragments (tRF) in drug-sensitive and resistant cells. We also present partially analyzed data to show the origin of reads on tRNAs as well as the borders of the fragments. We believe that this data can benefit the science community working in the field of tRF and/or apoptosis.

Re-Arrangements in the Cytoplasmic Distribution of Small RNAs Following the Maternal-to-Zygotic Transition in Drosophila Embryos.

Small ribonucleic acids (RNAs) are known to regulate gene expression during early development. However, the dynamics of interaction between small RNAs and polysomes during this process is largely unknown. To investigate this phenomenon, 0-1 h and 7-8 h Drosophila melanogaster embryos were fractionated on sucrose density gradients into four fractions based on A254 reading (1) translationally inactive messenger ribonucleoprotein (mRNP), (2) 60S, (3) monosome, and (4) polysome. Comparative analysis of deep-sequencing reads from fractionated and un-fractionated 0-1 h and 7-8 h embryos revealed development-specific co-sedimentation pattern of small RNAs with the cellular translation machinery. Although most micro RNAs (miRNAs) did not have a specific preference for any state of the translational machinery, we detected fraction-specific enrichment of a few miRNAs such as dme-miR-1-3p, -184-3p, 5-5p and 263-5p. More interestingly, we observed changes in the subcellular location of a subset of miRNAs in fractionated embryos despite no measurable difference in their amount in unfractionated embryos. Transposon-derived endo small interfering RNAs (siRNAs) were over-expressed in 7-8 h embryos and associated mainly with the mRNP fraction. In contrast, transposon-derived PIWI-interacting RNAs (piRNA), which were more abundant in 0-1 h embryos, co-sedimented primarily with the polysome fractions. These results suggest that there appears to be a complex interplay among the small RNAs with respect to their polysome-cosedimentation pattern during early development in Drosophila melanogaster.