Biology subjects

Lim, D.

Publications and source records attributed to Lim, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Diverse priming outcomes under conditions of very rare precursor B cells

Rare B cells can have special pathogen-recognition features giving them the potential to make outsized contributions to protective immunity. However, rare naive B cells infrequently participate in immune responses. We investigated how germline-targeting vaccine antigen delivery and adjuvant selection affect priming of exceptionally rare BG18-like HIV broadly neutralizing antibody-precursor B cells (~1 in 50 million) in non-human primates. Only escalating dose (ED) priming immunization using the saponin adjuvant SMNP elicited detectable BG18-like cells in germinal centers (GCs). All groups had strong GC responses, but only ED+SMNP and bolus+SMNP induced BG18-like memory B cells in >50% of animals. One group had vaccine-specific GC responses equivalent to ED+SMNP, but BG18-like memory B cells were rarely detected. Following homologous boosting, BG18-like memory B cells were more frequent in a bolus priming group, but had lower somatic hypermutation and affinities. This outcome was inversely associated with post-prime antibody titers, suggesting antibody feedback can significantly influence rare precursor B cell responses.

immunology↗

bioRxiv2024

Spatial 3D genome organization controls the activity of bivalent chromatin during human neurogenesis

The nuclear genome is spatially organized into a three-dimensional (3D) architecture by physical association of large chromosomal domains with subnuclear compartments including the nuclear lamina at the radial periphery and nuclear speckles within the nucleoplasm1-5. However, how spatial genome architecture regulates human brain development has been overlooked owing to technical limitations. Here, we generate high-resolution maps of genomic interactions with the lamina and speckles in cells of the neurogenic lineage isolated from midgestational human cortex, uncovering an intimate association between subnuclear genome compartmentalization, chromatin state and transcription. During cortical neurogenesis, spatial genome organization is extensively remodeled, relocating hundreds of neuronal genes from the lamina to speckles including key neurodevelopmental genes bivalent for H3K27me3 and H3K4me3. At the lamina, bivalent genes have exceptionally low expression, and relocation to speckles enhances resolution of bivalent chromatin to H3K4me3 and increases transcription >7-fold. We further demonstrate that proximity to the nuclear periphery - not the presence of H3K27me3 - is the dominant factor in maintaining the lowly expressed, poised state of bivalent genes embedded in the lamina. In addition to uncovering a critical role of subnuclear genome compartmentalization in neurogenic transcriptional regulation, our results establish a new paradigm in which knowing the spatial location of a gene is necessary to understanding its epigenomic regulation.

neuroscience↗

bioRxiv2024

ER-mitochondria distance is a critical parameter for efficient mitochondrial Ca2+ uptake and oxidative metabolism.

IP3 receptor (IP3R)-mediated Ca2+ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca2+ uptake and oxidative metabolism and is linked to different pathologies, including Parkinsons disease (PD). The dependence of Ca2+ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically-encoded Ca2+ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of [~]20 nm is optimal for Ca2+ transfer and mitochondrial oxidative metabolism due to enrichment of IP3R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced which correlated with a reduction of mitochondrial Ca2+ uptake. Our work determines with precision the optimal distance for Ca2+ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function.

cell biology↗

bioRxiv2024

Chromatin regulator Kdm6b is required for the establishment and maintenance of neural stem cells in mouse hippocampus

Neural stem cells (NSCs) in the mouse hippocampal dentate gyrus (DG) - a structure important to learning and memory - generate new neurons postnatally and throughout adult life. However, the regulators that enable this lifelong neurogenesis remain incompletely understood. Here we show that the chromatin regulator KDM6B is required for both the establishment and maintenance of NSCs in the mouse DG. Conditional deletion of Kdm6b in embryonic DG precursors results in an adult hippocampus that is essentially devoid of NSCs, and hippocampal-dependent behaviors are defective. Kdm6b-deletion causes precocious neuronal differentiation, and the NSC population fails to become established in the postnatal DG. Using single cell RNA sequencing (scRNA-seq), we observed that Kdm6b-deletion disrupts the transcriptomic signature of NSC maintenance. Furthermore, deleting Kdm6b in adult DG NSCs induces early neuronal differentiation, and the NSC population is not properly maintained. These data illustrate the critical role that Kdm6b plays in adult DG neurogenesis, which may help understand how mutations in this chromatin regulator result in cognitive disorders in human patients.

neuroscience↗

bioRxiv2023

Structure-Aware Annotation of Leucine-rich Repeat Domains

Protein domain annotation is typically done by predictive models such as HMMs trained on sequence motifs. However, sequence-based annotation methods are prone to error, particularly in calling domain boundaries and motifs within them. These methods are limited by a lack of structural information accessible to the model. With the advent of deep learning-based protein structure prediction, existing sequenced-based domain annotation methods can be improved by taking into account the geometry of protein structures. We develop dimensionality reduction methods to annotate repeat units of the Leucine Rich Repeat solenoid domain. The methods are able to correct mistakes made by existing machine learning-based annotation tools and enable the automated detection of hairpin loops and structural anomalies in the solenoid. The methods are applied to 127 predicted structures of LRR-containing intracellular innate immune proteins in the model plant Arabidopsis thaliana and validated against a benchmark dataset of 172 manually-annotated LRR domains. Author summaryIn immune receptors across various organisms, repeating protein structures play a crucial role in recognizing and responding to pathogen threats. These structures resemble the coils of a slinky toy, allowing these receptors to adapt and change over time. One particularly vital but challenging structure to study is the Leucine Rich Repeat (LRR). Traditional methods that rely just on analyzing the sequence of these proteins can miss subtle changes due to rapid evolution. With the introduction of protein structure prediction tools like AlphaFold 2, annotation methods can study the coarser geometric properties of the structure. In this study, we visualize LRR proteins in three dimensions and use a mathematical approach to flatten them into two dimensions, so that the coils form circles. We then used a mathematical concept called winding number to determine the number of repeats and where they are in a protein sequence. This process helps reveal their repeating patterns with enhanced clarity. When we applied this method to immune receptors from a model plant organism, we found that our approach could accurately identify coiling patterns. Furthermore, we detected errors made by previous methods and highlighted unique structural variations. Our research offers a fresh perspective on understanding immune receptors, potentially influencing studies on their evolution and function.

bioinformatics↗

bioRxiv2023

A Parasite Odyssey: An RNA virus concealed in Toxoplasma gondii

We are entering a "Platinum Age of Virus Discovery", an era marked by exponential growth in the discovery of virus biodiversity, and driven by advances in metagenomics and computational analysis. In the ecosystem of a human (or any animal) there are more species of viruses than simply those directly infecting the animal cells. Viruses can infect all organisms constituting the microbiome, including bacteria, fungi, and unicellular parasites. Thus the complexity of possible interactions between host, microbe, and viruses is unfathomable. To understand this interaction network we must employ computationally-assisted virology as a means of analyzing and interpreting the millions of available samples to make inferences about the ways in which viruses may intersect human health. From a computational viral screen of human neuronal datasets, we identified a novel narnavirus Apocryptovirus odysseus (Ao) which likely infects the neurotropic parasite Toxoplasma gondii. Previously, several parasitic protozoan viruses (PPVs) have been mechanistically established as triggers of host innate responses, and here we present in silico evidence that Ao is a plausible pro-inflammatory factor in human and mouse cells infected by T. gondii. T. gondii infects billions of people worldwide, yet the prognosis of toxoplasmosis disease is highly variable, and PPVs like Ao could function as a hitherto undescribed hypervirulence factor. In a broader screen of over 7.6 million samples, we explored phylogenetically-proximal viruses to Ao and discovered 19 Apocryptovirus species, all found in libraries annotated as vertebrate transcriptome or metatranscriptomes. While the Narnavirus samples making up this genus-like clade are derived from sheep, goat, bat, rabbit, chicken, and pigeon samples, the presence of virus is strongly predictive of parasitic (Apicomplexa) nucleic acid co-occurrence, supporting that these are a clade of parasite-infecting viruses. This is a computational proof-of-concept study in which we rapidly analyze millions of datasets from which we distilled a mechanistically, ecologically, and phylogenetically refined hypothesis. We predict this highly diverged Ao RNA virus is biologically a T. gondii infection, and that Ao, and other viruses like it, will modulate this disease which afflicts billions worldwide.

microbiology↗

bioRxiv2023

In vivo perturb-seq of cancer and immune cells dissects oncologic drivers and therapy response

Genetic perturbation screens with single cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking. Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery (CED) of sgRNA libraries into models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance, as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune/stromal cells following in vivo CRISPRi perturbations. In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application.

genomics↗

bioRxiv2023

Integration of thalamic and retrosplenial inputs in presubicular layer 3 gates cross-laminar bursting signal to upstream head direction circuit

Head-direction (HD) signals function as the brains internal compass. They are organized as an attractor, and anchor to the environment via visual landmarks. Here we examine how thalamic HD signals and visual information from the retrosplenial cortex combine in the presubiculum. We find that monosynaptic excitatory connections from anterior thalamic nucleus and from retrosplenial cortex converge on single layer 3 pyramidal neurons in the dorsal portion of mouse presubiculum. Independent dual wavelength photostimulation of these inputs in slices leads to action potential generation preferentially for near-coincident inputs, indicating that layer 3 neurons can transmit a visually matched HD signal to medial entorhinal cortex. Layer 4 neurons, which innervate the lateral mammillary nucleus, form a second step in the association of HD and landmark signals. They receive little direct input from thalamic and retrosplenial axons. We show that layer 4 cells are excited di-synaptically, transforming regular spiking activity into bursts of action potentials, and that their firing is enhanced by cholinergic agonists. Thus, a coherent sense of orientation involves projection specific translaminar processing in the presubiculum, where neuromodulation facilitates landmark updating of HD signals in the lateral mammillary nucleus.

neuroscience↗

bioRxiv2023

A molecular glue approach to control the half-life of CRISPR-based technologies

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latters rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

bioengineering↗

bioRxiv2023

FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. HighlightsO_LIFALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effects C_LIO_LIFALCON is applicable to many and diverse cell types C_LIO_LIA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidity C_LIO_LINew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for disease C_LIO_LIC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling C_LI

cell biology↗

bioRxiv2022

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimers disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2 and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2 without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2 and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2 and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2 levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

neuroscience↗

bioRxiv2020

Label-free bone marrow white blood cell classification using refractive index tomograms and deep learning

In this study, we report a label-free bone marrow white blood cell classification framework that captures the three-dimensional (3D) refractive index (RI) distributions of individual cells and analyzes with deep learning. Without using labeling or staining processes, 3D RI distributions of individual white blood cells were exploited for accurate profiling of their subtypes. Powered by deep learning, our method used the high-dimensional information of the WBC RI tomogram voxels and achieved high accuracy. The results show >99 % accuracy for the binary classification of myeloids and lymphoids and >96 % accuracy for the four-type classification of B, T lymphocytes, monocytes, and myelocytes. Furthermore, the feature learning of our approach was visualized via an unsupervised dimension reduction technique. We envision that this framework can be integrated into existing workflows for blood cell investigation, thereby providing cost-effective and rapid diagnosis of hematologic malignancy.

bioengineering↗

bioRxiv2020

Immortalised hippocampal astrocytes from 3xTG-AD mice fail to support BBB integrity in vitro: Role of extracellular vesicles in glial-endothelial communication

Impairments of the blood brain barrier (BBB) and vascular dysfunction contribute to Alzheimers disease (AD) from the earliest stages. However, the effects of AD-affected astrocytes on the BBB remain largely unexplored. In the present study we created an in vitro BBB using human immortalised endothelial cells in combination with immortalised astroglial cell lines from the hippocampus of 3xTG-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively). We found that co-culturing endothelial monolayers with WT-iAstro up-regulates expression of endothelial tight junction proteins (claudin-5, occludin, ZO-1) and increases the trans-endothelial electrical resistance (TEER). In contrast, co-culturing with 3Tg-iAstro does not affect expression of tight junction proteins and does not change the TEER of endothelial monolayers. The same in vitro model has been used to evaluate the effects of extracellular vesicles (EVs) derived from the WT-iAstro and 3Tg-iAstro. The EVs derived from WT-iAstro increased TEER and up-regulated expression of tight junction proteins, whereas EVs from 3Tg-iAstro were ineffective. In conclusion, we show for the first time that immortalised hippocampal astrocytes from 3xTG-AD mice exhibit impaired capacity to support BBB integrity in vitro through paracrine mechanisms and may represent an important factor underlying vascular abnormalities during development of AD.

neuroscience↗

bioRxiv2020

Deletion of calcineurin from astrocytes reproduces proteome signature of Alzheimer's disease and epilepsy and predisposes to seizures

In astrocytes, calcineurin (CaN) is involved in neuroinflammation and gliosis, while its role in healthy CNS or in early neuro-pathogenesis is poorly understood. Here we report that in astroglial CaN KO (ACN-KO) mice, at one month of age, proteome is deranged in hippocampus and cerebellum. Bioinformatic analysis reveals association with Alzheimers disease (AD) and epilepsy. We found significant overlap with the proteome of an AD mouse model and of human subjects with drug-resistant epilepsy. In Barnes maze ACN-KO mice learned the task but adopted serial search strategy. Strikingly, from five months of age ACN-KO mice develop spontaneous seizures with an inflammatory signature of epileptic brains. These results suggest that astroglial CaN KO impairs hippocampal connectivity, produces proteome features of neurological disorders and predisposes mice to seizures. We suggest that astroglial CaN may serve as a novel Ca2+-sensitive switch which regulates protein expression and homeostasis in the CNS.

neuroscience↗

bioRxiv2020

Presynaptic PTPσ organizes neurotransmitter release machinery at excitatory synapses

Leukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) are evolutionarily conserved presynaptic organizers. The synaptic role of vertebrate LAR-RPTPs in vivo, however, remains unclear. This study systematically analyzed the effects of genetic deletions of LAR-RPTP genes by generating single conditional knockout (cKO) mice targeting PTP{sigma} and PTP{delta}. Although the numbers of synapses were reduced in cultured neurons deficient in individual PTPs, abnormalities in synaptic transmission, synaptic ultrastructures, and vesicle localization were observed only in PTP{sigma}-deficient neurons. Strikingly, loss of presynaptic PTP{sigma} reduced neurotransmitter release prominently at excitatory synapses, concomitant with drastic reductions in excitatory innervations onto postsynaptic target areas in vivo. However, postsynaptic PTP{sigma} deletion had no effect on excitatory synaptic strength. Furthermore, conditional deletion of PTP{sigma} in ventral CA1 specifically altered anxiety-like behaviors. Taken together, these results demonstrate that PTP{sigma} is a bona fide presynaptic adhesion molecule that controls neurotransmitter release and excitatory inputs.

neuroscience↗

bioRxiv2019

Time-dynamics of mitochondrial membrane potential reveal an inhibition of ATP synthesis in mitosis

The energetic demands of a cell are believed to increase during mitosis 1-7. As cells transit from G2 into mitosis, mitochondrial electron transport chain (ETC) activity increases 4,8,9, and cellular ATP levels progressively decrease until the metaphase-anaphase transition 3,7,10, consistent with elevated consumption. The rates of ATP synthesis during mitosis, however, have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytes and demonstrate that cyclin-dependent kinase 1 (CDK1) activity causes mitochondrial hyperpolarization from G2/M until the metaphase-anaphase transition. By using an electrical circuit model of mitochondria, we quantify the time-dynamics of mitochondrial membrane potential under normal and perturbed conditions to extract mitochondrial ATP synthesis rates in mitosis. We found that mitochondrial ATP synthesis decreases by approximately 50 % during early mitosis, when CDK1 is active, and increases back to G2 levels during cytokinesis. Consistently, acute inhibition of mitochondrial ATP synthesis failed to delay cell division. Our results provide a quantitative understanding of mitochondrial bioenergetics in mitosis and challenge the traditional dogma that cell division is a highly energy demanding process.

cell biology↗

bioRxiv2019

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic {beta} cells to secrete protective immunomodulatory factor interleukin-10.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC=\"FIGDIR/small/732354v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@48a34aorg.highwire.dtl.DTLVardef@d886d0org.highwire.dtl.DTLVardef@17acc61org.highwire.dtl.DTLVardef@fffe44_HPS_FORMAT_FIGEXP M_FIG TOC GRAPHIC C_FIG

synthetic biology↗

bioRxiv2019

Deletion of calcineurin from GFAP-expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+/K+ ATPase.

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca2+/calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein (GFAP)-expressing astrocytes (astroglial calcineurin knock-out, ACN-KO). Here we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs). This impairment was associated with a decrease in after-hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K+ homeostasis. Indeed, blockade of Na+/K+-ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca2+-sensitive switch.

neuroscience↗