Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency.

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.

CBP and p300 Jointly Maintain Neural Progenitor Viability but Play Unique Roles in the Differentiation of Neural Lineages.

The paralogous lysine acetyltransferases 3 (KAT3), CBP and P300, play critical roles during neurodevelopment, but their specific roles in neural precursors maintenance and differentiation remain obscure. In fact, it is still unclear whether these proteins are individually or jointly essential in processes such as proliferation of neural precursors, differentiation to specific neural cell types, or both. Here, we use subventricular zone-derived neurospheres as a potential ex vivo developmental model to analyze the proliferation and differentiation of neural stem cells (NSCs) lacking CBP, p300, or both proteins. The results showed that CBP and p300 are not individually essential for maintenance and proliferation of NSCs, although their combined ablation seriously compromised cell division. In turn, the absence of either of the two proteins compromised the differentiation of NSC into the neuronal and astrocytic lineages. Single-nucleus RNA sequencing analysis of neural cell cultures derived from CBP or p300 mutant neurospheres revealed divergent trajectories of neural differentiation upon CBP or p300 ablation, confirming unique functions and nonredundant roles in neural development. These findings contribute to a better understanding of the shared and individual roles of KAT3 proteins in neural differentiation and the etiology of neurodevelopmental disorders caused by their deficiency.

CBP Is Required for Establishing Adaptive Gene Programs in the Adult Mouse Brain.

Environmental factors and life experiences impinge on brain circuits triggering adaptive changes. Epigenetic regulators contribute to this neuroadaptation by enhancing or suppressing specific gene programs. The paralogous transcriptional coactivators and lysine acetyltransferases CREB binding protein (CBP) and p300 are involved in brain plasticity and stimulus-dependent transcription, but their specific roles in neuroadaptation are not fully understood. Here we investigated the impact of eliminating either CBP or p300 in excitatory neurons of the adult forebrain of mice from both sexes using inducible and cell type-restricted knock-out strains. The elimination of CBP, but not p300, reduced the expression and chromatin acetylation of plasticity genes, dampened activity-driven transcription, and caused memory deficits. The defects became more prominent in elderly mice and in paradigms that involved enduring changes in transcription, such as kindling and environmental enrichment, in which CBP loss interfered with the establishment of activity-induced transcriptional and epigenetic changes in response to stimulus or experience. These findings further strengthen the link between CBP deficiency in excitatory neurons and etiopathology in the nervous system.SIGNIFICANCE STATEMENT How environmental conditions and life experiences impinge on mature brain circuits to elicit adaptive responses that favor the survival of the organism remains an outstanding question in neurosciences. Epigenetic regulators are thought to contribute to neuroadaptation by initiating or enhancing adaptive gene programs. In this article, we examined the role of CREB binding protein (CBP) and p300, two paralogous transcriptional coactivators and histone acetyltransferases involved in cognitive processes and intellectual disability, in neuroadaptation in adult hippocampal circuits. Our experiments demonstrate that CBP, but not its paralog p300, plays a highly specific role in the epigenetic regulation of neuronal plasticity gene programs in response to stimulus and provide unprecedented insight into the molecular mechanisms underlying neuroadaptation.

A protocol to extract cell-type-specific signatures from differentially expressed genes in bulk-tissue RNA-seq.

Bulk-tissue RNA-seq is widely used to dissect variation in gene expression levels across tissues and under different experimental conditions. Here, we introduce a protocol that leverages existing single-cell expression data to deconvolve patterns of cell-type-specific gene expression in differentially expressed gene lists from highly heterogeneous tissue. We apply this protocol to interrogate cell-type-specific gene expression and variation in cell type composition between the distinct sublayers of the hippocampal CA1 region of the brain in a rodent model of epilepsy. For complete details on the use and execution of this protocol, please refer to Cid et al. (2021).

Secondary loss of miR-3607 reduced cortical progenitor amplification during rodent evolution.

The evolutionary expansion and folding of the mammalian cerebral cortex resulted from amplification of progenitor cells during embryonic development. This process was reversed in the rodent lineage after splitting from primates, leading to smaller and smooth brains. Genetic mechanisms underlying this secondary loss in rodent evolution remain unknown. We show that microRNA miR-3607 is expressed embryonically in the large cortex of primates and ferret, distant from the primate-rodent lineage, but not in mouse. Experimental expression of miR-3607 in embryonic mouse cortex led to increased Wnt/ß-catenin signaling, amplification of radial glia cells (RGCs), and expansion of the ventricular zone (VZ), via blocking the ß-catenin inhibitor APC (adenomatous polyposis coli). Accordingly, loss of endogenous miR-3607 in ferret reduced RGC proliferation, while overexpression in human cerebral organoids promoted VZ expansion. Our results identify a gene selected for secondary loss during mammalian evolution to limit RGC amplification and, potentially, cortex size in rodents.

Transcriptional regulation of chemokine network by biologic monotherapy in ileum of patients with Crohn's disease.

BACKGROUND: Crohn's disease (CD) exacerbation is marked by an intense cellular trafficking. We set out to determine the specific impact of biologic therapies on regulating chemokine network gene expression in healthy, mildly and severely inflamed tissue of CD patients. METHODS: Twenty CD patients on biologics (adalimumab, ustekinumab, vedolizumab) or untreated undergoing colonoscopy due to clinical symptoms of flare. Healthy, mildly and severely inflamed ileum biopsies from each patient were collected. Chemokines and receptors gene expression was analyzed and a STRING analysis for functional enrichment was performed. RESULTS: The chemokine network exhibited wide transcriptional differences among tissues in active untreated patients, whereas all biologic treatments reduced these differences and homogenized their transcriptional activity. In mildly inflamed tissue, all treatments showed gene upregulation while ustekinumab additionally maintained the downregulation of genes such as CCL2, CCL3, CCL17 or CCL23, involved in T cell chemotaxis, inflammatory monocyte and NK trafficking. In severely inflamed tissue, all treatments shared a downregulatory effect on chemokines controlling T cell response (i.e. CXCL16, CXCR3). Adalimumab and vedolizumab significantly reduced the expression of genes promoting antigen presentation by DCs and the initiation of leukocyte extravasation (i.e. CXCL12, CCL25, CCR7). Ustekinumab significantly reduced genes positively regulating Th1 cytokine production and IL-8 mediated signaling (i.e. IL1B, XCL1, CXCR1, CXCR2). CONCLUSION: Biologic therapies differentially target the chemokine network gene expression profile in the ileal tissue of active CD patients. These results may contribute to better understanding cell homing and to defining future personalized therapeutic strategies for CD patients.

SFRP1 modulates astrocyte-to-microglia crosstalk in acute and chronic neuroinflammation.

Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.

Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis.

Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type-specific vulnerability and their progression and histopathological classification remain controversial. Using single-cell electrophysiology in vivo and immediate-early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rodents. Bulk tissue and single-nucleus expression profiling disclose sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage channels, synaptic signaling, and cell adhesion are deregulated differently by epilepsy across sublayers, whereas neurodegenerative signatures primarily involve superficial cells. Pseudotime analysis of gene expression in single nuclei and in situ validation reveal separated trajectories from health to epilepsy across cell types and identify a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate that sublayer- and cell-type-specific changes associated with selective CA1 neuronal damage contribute to progression of hippocampal sclerosis.

Astrocytes and neurons share region-specific transcriptional signatures that confer regional identity to neuronal reprogramming.

Neural cell diversity is essential to endow distinct brain regions with specific functions. During development, progenitors within these regions are characterized by specific gene expression programs, contributing to the generation of diversity in postmitotic neurons and astrocytes. While the region-specific molecular diversity of neurons and astrocytes is increasingly understood, whether these cells share region-specific programs remains unknown. Here, we show that in the neocortex and thalamus, neurons and astrocytes express shared region-specific transcriptional and epigenetic signatures. These signatures not only distinguish cells across these two brain regions but are also detected across substructures within regions, such as distinct thalamic nuclei, where clonal analysis reveals the existence of common nucleus-specific progenitors for neurons and astrocytes. Consistent with their shared molecular signature, regional specificity is maintained following astrocyte-to-neuron reprogramming. A detailed understanding of these regional-specific signatures may thus inform strategies for future cell-based brain repair.

A Zic2-regulated switch in a noncanonical Wnt/ßcatenin pathway is essential for the formation of bilateral circuits.

The Wnt pathway is involved in a wide array of biological processes during development and is deregulated in many pathological scenarios. In neurons, Wnt proteins promote both axon extension and repulsion, but the molecular mechanisms underlying these opposing axonal responses are unknown. Here, we show that Wnt5a is expressed at the optic chiasm midline and promotes the crossing of retinal axons by triggering an alternative Wnt pathway that depends on the accumulation of ßcatenin but does not activate the canonical pathway. In ipsilateral neurons, the transcription factor Zic2 switches this alternative Wnt pathway by regulating the expression of a set of Wnt receptors and intracellular proteins. In combination with this alternative Wnt pathway, the asymmetric activation of EphB1 receptors at the midline phosphorylates ßcatenin and elicits a repulsive response. This alternative Wnt pathway and its Zic2-triggered switch may operate in other contexts that require a two-way response to Wnt ligands.

Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium.

Structural integrity and cellular homeostasis of the embryonic stem cell niche are critical for normal tissue development. In the telencephalic neuroepithelium, this is controlled in part by cell adhesion molecules and regulators of progenitor cell lineage, but the specific orchestration of these processes remains unknown. Here, we studied the role of microRNAs in the embryonic telencephalon as key regulators of gene expression. By using the early recombiner Rx-Cre mouse, we identify novel and critical roles of miRNAs in early brain development, demonstrating they are essential to preserve the cellular homeostasis and structural integrity of the telencephalic neuroepithelium. We show that Rx-Cre;DicerF/F mouse embryos have a severe disruption of the telencephalic apical junction belt, followed by invagination of the ventricular surface and formation of hyperproliferative rosettes. Transcriptome analyses and functional experiments in vivo show that these defects result from upregulation of Irs2 upon loss of let-7 miRNAs in an apoptosis-independent manner. Our results reveal an unprecedented relevance of miRNAs in early forebrain development, with potential mechanistic implications in pediatric brain cancer.

KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain.

The lysine acetyltransferases type 3 (KAT3) family members CBP and p300 are important transcriptional co-activators, but their specific functions in adult post-mitotic neurons remain unclear. Here, we show that the combined elimination of both proteins in forebrain excitatory neurons of adult mice resulted in a rapidly progressing neurological phenotype associated with severe ataxia, dendritic retraction and reduced electrical activity. At the molecular level, we observed the downregulation of neuronal genes, as well as decreased H3K27 acetylation and pro-neural transcription factor binding at the promoters and enhancers of canonical neuronal genes. The combined deletion of CBP and p300 in hippocampal neurons resulted in the rapid loss of neuronal molecular identity without de- or transdifferentiation. Restoring CBP expression or lysine acetylation rescued neuronal-specific transcription in cultured neurons. Together, these experiments show that KAT3 proteins maintain the excitatory neuron identity through the regulation of histone acetylation at cell type-specific promoter and enhancer regions.

Ecdysone-Induced 3D Chromatin Reorganization Involves Active Enhancers Bound by Pipsqueak and Polycomb.

Evidence suggests that Polycomb (Pc) is present at chromatin loop anchors in Drosophila. Pc is recruited to DNA through interactions with the GAGA binding factors GAF and Pipsqueak (Psq). Using HiChIP in Drosophila cells, we find that the psq gene, which has diverse roles in development and tumorigenesis, encodes distinct isoforms with unanticipated roles in genome 3D architecture. The BR-C, ttk, and bab domain (BTB)-containing Psq isoform (PsqL) colocalizes genome-wide with known architectural proteins. Conversely, Psq lacking the BTB domain (PsqS) is consistently found at Pc loop anchors and at active enhancers, including those that respond to the hormone ecdysone. After stimulation by this hormone, chromatin 3D organization is altered to connect promoters and ecdysone-responsive enhancers bound by PsqS. Our findings link Psq variants lacking the BTB domain to Pc-bound active enhancers, thus shedding light into their molecular function in chromatin changes underlying the response to hormone stimulus.

Cbp-dependent histone acetylation mediates axon regeneration induced by environmental enrichment in rodent spinal cord injury models.

After a spinal cord injury, axons fail to regenerate in the adult mammalian central nervous system, leading to permanent deficits in sensory and motor functions. Increasing neuronal activity after an injury using electrical stimulation or rehabilitation can enhance neuronal plasticity and result in some degree of recovery; however, the underlying mechanisms remain poorly understood. We found that placing mice in an enriched environment before an injury enhanced the activity of proprioceptive dorsal root ganglion neurons, leading to a lasting increase in their regenerative potential. This effect was dependent on Creb-binding protein (Cbp)-mediated histone acetylation, which increased the expression of genes associated with the regenerative program. Intraperitoneal delivery of a small-molecule activator of Cbp at clinically relevant times promoted regeneration and sprouting of sensory and motor axons, as well as recovery of sensory and motor functions in both the mouse and rat model of spinal cord injury. Our findings showed that the increased regenerative capacity induced by enhancing neuronal activity is mediated by epigenetic reprogramming in rodent models of spinal cord injury. Understanding the mechanisms underlying activity-dependent neuronal plasticity led to the identification of potential molecular targets for improving recovery after spinal cord injury.

Development and maintenance of the brain's immune toolkit: Microglia and non-parenchymal brain macrophages.

Microglia and non-parenchymal macrophages located in the perivascular space, the meninges and the choroid plexus are independent immune populations that play vital roles in brain development, homeostasis, and tissue healing. Resident macrophages account for a significant proportion of cells in the brain and their density remains stable throughout the lifespan thanks to constant turnover. Microglia develop from yolk sac progenitors, later evolving through intermediate progenitors in a fine-tuned process in which intrinsic factors and external stimuli combine to progressively sculpt their cell type-specific transcriptional profiles. Recent evidence demonstrates that non-parenchymal macrophages are also generated during early embryonic development. In recent years, the development of powerful fate mapping approaches combined with novel genomic and transcriptomic methodologies have greatly expanded our understanding of how brain macrophages develop and acquire specialized functions, and how cell population dynamics are regulated. Here, we review the transcription factors, epigenetic remodeling, and signaling pathways orchestrating the embryonic development of microglia and non-parenchymal macrophages. Next, we describe the dynamics of the macrophage populations of the brain and discuss the role of progenitor cells, to gain a better understanding of their functions in the healthy and diseased brain. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 561-579, 2018.

Loss of Kdm5c Causes Spurious Transcription and Prevents the Fine-Tuning of Activity-Regulated Enhancers in Neurons.

During development, chromatin-modifying enzymes regulate both the timely establishment of cell-type-specific gene programs and the coordinated repression of alternative cell fates. To dissect the role of one such enzyme, the intellectual-disability-linked lysine demethylase 5C (Kdm5c), in the developing and adult brain, we conducted parallel behavioral, transcriptomic, and epigenomic studies in Kdm5c-null and forebrain-restricted inducible knockout mice. Together, genomic analyses and functional assays demonstrate that Kdm5c plays a critical role as a repressor responsible for the developmental silencing of germline genes during cellular differentiation and in fine-tuning activity-regulated enhancers during neuronal maturation. Although the importance of these functions declines after birth, Kdm5c retains an important genome surveillance role preventing the incorrect activation of non-neuronal and cryptic promoters in adult neurons.

Lack of IL-1R8 in neurons causes hyperactivation of IL-1 receptor pathway and induces MECP2-dependent synaptic defects.

Inflammation modifies risk and/or severity of a variety of brain diseases through still elusive molecular mechanisms. Here we show that hyperactivation of the interleukin 1 pathway, through either ablation of the interleukin 1 receptor 8 (IL-1R8, also known as SIGIRR or Tir8) or activation of IL-1R, leads to up-regulation of the mTOR pathway and increased levels of the epigenetic regulator MeCP2, bringing to disruption of dendritic spine morphology, synaptic plasticity and plasticity-related gene expression. Genetic correction of MeCP2 levels in IL-1R8 KO neurons rescues the synaptic defects. Pharmacological inhibition of IL-1R activation by Anakinra corrects transcriptional changes, restores MeCP2 levels and spine plasticity and ameliorates cognitive defects in IL-1R8 KO mice. By linking for the first time neuronal MeCP2, a key player in brain development, to immune activation and demonstrating that synaptic defects can be pharmacologically reversed, these data open the possibility for novel treatments of neurological diseases through the immune system modulation.

Specific promoter deacetylation of histone H3 is conserved across mouse models of Huntington's disease in the absence of bulk changes.

Defective epigenetic regulation has been postulated as a possible cause for the extensive and premature transcriptional dysregulation observed in experimental models of Huntington's disease (HD). In this study, we extended our observations in the N171-82Q mouse strain relating to the limited impact of polyQ pathology on the global histone acetylation to other animal and cellular models of HD, namely the R6/1 and YAC128 strains, striatal-electroporated mice, primary neuronal cultures and stably transfected PC12 cells. In the absence of bulk chromatin changes, we nonetheless documented histone deacetylation events at the transcription start sites (TSS) of genes relevant to neuronal functions (e.g., Rin1, Plk5, Igfbp5, Eomes, and Fos). In some instances, these local deficits were associated with an increased susceptibility to transcriptional dysregulation (e.g., Camk1g and Rasl11b) and the defective trimethylation of histone H3 at lysine 4 (H3K4me3), another covalent modification of histone tails that is related to active transcription and is also altered in HD. Overall, this study provides further insight into the nature and extent of epigenetic dysregulation in HD pathology.

Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.

The RNase Dicer is essential for the maturation of most microRNAs, a molecular system that plays an essential role in fine-tuning gene expression. To gain molecular insight into the role of Dicer and the microRNA system in brain function, we conducted 2 complementary RNA-seq screens in the hippocampus of inducible forebrain-restricted Dicer1 mutants aimed at identifying the microRNAs primarily affected by Dicer loss and their targets, respectively. Functional genomics analyses predicted the main biological processes and phenotypes associated with impaired microRNA maturation, including categories related to microRNA biology, signal transduction, seizures, and synaptic transmission and plasticity. Consistent with these predictions, we found that, soon after recombination, Dicer-deficient mice exhibited an exaggerated seizure response, enhanced induction of immediate early genes in response to different stimuli, stronger and more stable fear memory, hyperphagia, and increased excitability of CA1 pyramidal neurons. In the long term, we also observed slow and progressive excitotoxic neurodegeneration. Overall, our results indicate that interfering with microRNA biogenesis causes an increase in neuronal responsiveness and disrupts homeostatic mechanisms that protect the neuron against overactivation, which may explain both the initial and late phenotypes associated with the loss of Dicer in excitatory neurons.

Brain size regulations by cbp haploinsufficiency evaluated by in-vivo MRI based volumetry.

The Rubinstein-Taybi Syndrome (RSTS) is a congenital disease that affects brain development causing severe cognitive deficits. In most cases the disease is associated with dominant mutations in the gene encoding the CREB binding protein (CBP). In this work, we present the first quantitative analysis of brain abnormalities in a mouse model of RSTS using magnetic resonance imaging (MRI) and two novel self-developed automated algorithms for image volumetric analysis. Our results quantitatively confirm key syndromic features observed in RSTS patients, such as reductions in brain size (-16.31%, p < 0.05), white matter volume (-16.00%, p < 0.05), and corpus callosum (-12.40%, p < 0.05). Furthermore, they provide new insight into the developmental origin of the disease. By comparing brain tissues in a region by region basis between cbp(+/-) and cbp(+/+) littermates, we found that cbp haploinsufficiency is specifically associated with significant reductions in prosencephalic tissue, such us in the olfactory bulb and neocortex, whereas regions evolved from the embryonic rhombencephalon were spared. Despite the large volume reductions, the proportion between gray-, white-matter and cerebrospinal fluid were conserved, suggesting a role of CBP in brain size regulation. The commonalities with holoprosencephaly and arhinencephaly conditions suggest the inclusion of RSTS in the family of neuronal migration disorders.