ABSTRACT
During cerebral cortex development, excitatory pyramidal neurons (PNs) establish specific projection patterns while receiving inputs from GABAergic inhibitory interneurons (INs). Whether these inhibitory inputs can shape PNs' projection patterns is, however, unknown. While layer 4 (L4) PNs of the primary somatosensory (S1) cortex are all born as long-range callosal projection neurons (CPNs), most of them acquire local connectivity upon activity-dependent elimination of their interhemispheric axons during postnatal development. Here, we demonstrate that precise developmental regulation of inhibition is key for the retraction of S1L4 PNs' callosal projections. Ablation of somatostatin INs leads to premature inhibition from parvalbumin INs onto S1L4 PNs and prevents them from acquiring their barrel-restricted local connectivity pattern. As a result, adult S1L4 PNs retain interhemispheric projections responding to tactile stimuli, and the mice lose whisker-based texture discrimination. Overall, we show that temporally ordered IN activity during development is key to shaping local ipsilateral S1L4 PNs' projection pattern, which is required for fine somatosensory processing.
Subject(s)
GABAergic Neurons , Interneurons , Somatosensory Cortex , Animals , Interneurons/metabolism , Interneurons/physiology , Interneurons/cytology , GABAergic Neurons/metabolism , GABAergic Neurons/physiology , GABAergic Neurons/cytology , Somatosensory Cortex/physiology , Somatosensory Cortex/metabolism , Somatosensory Cortex/cytology , Mice , Pyramidal Cells/metabolism , Pyramidal Cells/physiology , Parvalbumins/metabolismABSTRACT
This work introduces NeoMag, a system designed to enhance cell mechanics assays in substrate deformation studies. NeoMag uses multidomain magneto-active materials to mechanically actuate the substrate, transmitting reversible mechanical cues to cells. The system boasts full flexibility in alternating loading substrate deformation modes, seamlessly adapting to both upright and inverted microscopes. The multidomain substrates facilitate mechanobiology assays on 2D and 3D cultures. The integration of the system with nanoindenters allows for precise evaluation of cellular mechanical properties under varying substrate deformation modes. The system is used to study the impact of substrate deformation on astrocytes, simulating mechanical conditions akin to traumatic brain injury and ischemic stroke. The results reveal local heterogeneous changes in astrocyte stiffness, influenced by the orientation of subcellular regions relative to substrate strain. These stiffness variations, exceeding 50% in stiffening and softening, and local deformations significantly alter calcium dynamics. Furthermore, sustained deformations induce actin network reorganization and activate Piezo1 channels, leading to an initial increase followed by a long-term inhibition of calcium events. Conversely, fast and dynamic deformations transiently activate Piezo1 channels and disrupt the actin network, causing long-term cell softening. These findings unveil mechanical and functional alterations in astrocytes during substrate deformation, illustrating the multiple opportunities this technology offers.
Subject(s)
Astrocytes , Astrocytes/metabolism , Astrocytes/cytology , Animals , Calcium/metabolism , Calcium/chemistry , Biomechanical Phenomena , Mechanical Phenomena , Actins/metabolism , Ion Channels/metabolism , MiceABSTRACT
Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.
Subject(s)
Adaptor Proteins, Signal Transducing , Memory Disorders , Ubiquitin-Protein Ligases , Animals , Mice , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Mutation , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB1/metabolism , Memory Disorders/genetics , Memory Disorders/metabolismABSTRACT
Astrocytes play crucial roles in brain homeostasis and are regulatory elements of neuronal and synaptic physiology. Astrocytic alterations have been found in Major Depressive Disorder (MDD) patients; however, the consequences of astrocyte Ca2+ signaling in MDD are poorly understood. Here, we found that corticosterone-treated juvenile mice (Cort-mice) showed altered astrocytic Ca2+ dynamics in mPFC both in resting conditions and during social interactions, in line with altered mice behavior. Additionally, Cort-mice displayed reduced serotonin (5-HT)-mediated Ca2+ signaling in mPFC astrocytes, and aberrant 5-HT-driven synaptic plasticity in layer 2/3 mPFC neurons. Downregulation of astrocyte Ca2+ signaling in naïve animals mimicked the synaptic deficits found in Cort-mice. Remarkably, boosting astrocyte Ca2+ signaling with Gq-DREADDS restored to the control levels mood and cognitive abilities in Cort-mice. This study highlights the important role of astrocyte Ca2+ signaling for homeostatic control of brain circuits and behavior, but also reveals its potential therapeutic value for depressive-like states.
Subject(s)
Astrocytes , Depressive Disorder, Major , Humans , Mice , Animals , Astrocytes/physiology , Serotonergic Neurons , Serotonin , Signal Transduction/physiologyABSTRACT
Insulin-like growth factor-I (IGF-I) exerts multiple actions, yet the role of IGF-I from different sources is poorly understood. Here, we explored the functional and behavioral consequences of the conditional deletion of Igf-I in the nervous system (Igf-I Δ/Δ), and demonstrated that long-term potentiation was impaired in hippocampal slices. Moreover, Igf-I Δ/Δ mice showed spatial memory deficits in the Morris water maze, and the significant sex-dependent differences displayed by Igf-I Ctrl/Ctrl mice disappeared in Igf-I Δ/Δ mice in the open field and rota-rod tests. Brain Igf-I deletion disorganized the granule cell layer of the dentate gyrus (DG), and it modified the relative expressions of GAD and VGLUT1, which are preferentially localized to inhibitory and excitatory presynaptic terminals. Furthermore, Igf-I deletion altered protein modules involved in receptor trafficking, synaptic proteins, and proteins that functionally interact with estrogen and androgen metabolism. Our findings indicate that brain IGF-I is crucial for long-term potentiation, and that it is involved in the regulation of spatial memory and sexual dimorphic behaviors, possibly by maintaining the granule cell layer structure and the stability of synaptic-related protein modules.
Subject(s)
Insulin-Like Growth Factor I , Long-Term Potentiation , Animals , Mice , Brain/metabolism , Hippocampus/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Spatial MemoryABSTRACT
Here, we present a protocol to selectively downregulate GABAB receptor (GABABR) expression in astrocytes of mouse medial prefrontal cortex (mPFC). We first describe the procedure of surgeries and viral injections. We then detail genetic, histological, and functional characterizations of astrocytic GABABR ablation using RT-PCR, imaging, and behavioral assays. The use of GABAB flox mice can be easily adapted to generate astrocyte-selective GABABR ablation in different brain areas and postnatal stages, leading to local downregulation of GABAergic-astrocyte signaling without developmental issues. For complete details on the use and execution of this protocol, please refer to Mederos et al. (2021).
Subject(s)
Astrocytes , Receptors, GABA-B , Mice , Animals , Astrocytes/metabolism , Receptors, GABA-B/metabolism , Signal Transduction , Prefrontal Cortex/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
Brain slice preparations are widely used for research in neuroscience. However, a high-quality preparation is essential and there is no consensus regarding stable parameters that can be used to define the status of the brain slice preparation after its collection at different time points. Thus, it is critical to fully characterize the experimental conditions for ex vivo studies using brain slices for electrophysiological recording. In this study, we used a multiplatform (LC-MS and GC-MS) untargeted metabolomics-based approach to shed light on the metabolome and lipidome changes taking place at different time intervals during the brain slice preparation process. We have found significant modifications in the levels of 300 compounds, including several lipid classes and their derivatives, as well as metabolites involved in the GABAergic pathway and the TCA cycle. All these preparation-dependent changes in the brain biochemistry related to the time interval should be taken into consideration for future studies to facilitate non-biased interpretations of the experimental results.
Subject(s)
Brain/metabolism , Gas Chromatography-Mass Spectrometry/methods , Metabolome/physiology , Metabolomics/methods , Animals , Brain/cytology , Chromatography, Liquid/methods , Lipidomics/methods , Male , Mice , Mice, Inbred C57BL , Organ Culture Techniques/methods , Time FactorsABSTRACT
GABA interneurons play a critical role in higher brain functions. Astrocytic glial cells interact with synapses throughout the whole brain and are recognized as regulatory elements of excitatory synaptic transmission. However, it is largely unknown how GABAergic interneurons and astrocytes interact and contribute to stable performance of complex behaviors. Here, we found that genetic ablation of GABAB receptors in medial prefrontal cortex astrocytes altered low-gamma oscillations and firing properties of cortical neurons, which affected goal-directed behaviors. Remarkably, working memory deficits were restored by optogenetic stimulation of astrocytes with melanopsin. Furthermore, melanopsin-activated astrocytes in wild-type mice enhanced the firing rate of cortical neurons and gamma oscillations, as well as improved cognition. Therefore, our work identifies astrocytes as a hub for controlling inhibition in cortical circuits, providing a novel pathway for the behaviorally relevant midrange time-scale regulation of cortical information processing and consistent goal-directed behaviors.
Subject(s)
Astrocytes/physiology , Goals , Prefrontal Cortex/physiology , Signal Transduction/physiology , gamma-Aminobutyric Acid/physiology , Animals , Cognition/drug effects , Decision Making , GABAergic Neurons/physiology , Gamma Rhythm/physiology , Interneurons/physiology , Memory, Short-Term/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Optogenetics , Psychomotor Performance/physiology , Receptors, GABA-B/genetics , Receptors, GABA-B/physiology , Rod Opsins/pharmacologyABSTRACT
Melanopsin, a mammalian G-protein-coupled photopigment, is a novel optical tool which enables studying astrocyte-neuron networks. Here, we describe the required guidelines to take advantage of this promising optical tool for functional neuron-glia studies. The selective expression of melanopsin in astrocytes allows triggering astrocytic Ca2+ signaling, changes in synaptic transmission, and modifying behavioral responses.
Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Neurons/cytology , Neurons/metabolism , Rod Opsins/metabolism , Animals , Calcium/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BL , Rod Opsins/genetics , Signal Transduction/physiology , Synaptic Transmission/physiologyABSTRACT
BACKGROUND: The apolipoprotein E (APOE) gene exists in three isoforms in humans: APOE2, APOE3 and APOE4. APOE4 causes structural and functional alterations in normal brains, and is the strongest genetic risk factor of the sporadic form of Alzheimer's disease (LOAD). Research on APOE4 has mainly focused on the neuronal damage caused by defective cholesterol transport and exacerbated amyloid-ß and Tau pathology. The impact of APOE4 on non-neuronal cell functions has been overlooked. Astrocytes, the main producers of ApoE in the healthy brain, are building blocks of neural circuits, and Ca2+ signaling is the basis of their excitability. Because APOE4 modifies membrane-lipid composition, and lipids regulate Ca2+ channels, we determined whether APOE4 dysregulates Ca2+signaling in astrocytes. METHODS: Ca2+ signals were recorded in astrocytes in hippocampal slices from APOE3 and APOE4 gene targeted replacement male and female mice using Ca2+ imaging. Mechanistic analyses were performed in immortalized astrocytes. Ca2+ fluxes were examined with pharmacological tools and Ca2+ probes. APOE3 and APOE4 expression was manipulated with GFP-APOE vectors and APOE siRNA. Lipidomics of lysosomal and whole-membranes were also performed. RESULTS: We found potentiation of ATP-elicited Ca2+responses in APOE4 versus APOE3 astrocytes in male, but not female, mice. The immortalized astrocytes modeled the male response, and showed that Ca2+ hyperactivity associated with APOE4 is caused by dysregulation of Ca2+ handling in lysosomal-enriched acidic stores, and is reversed by the expression of APOE3, but not of APOE4, pointing to loss of function due to APOE4 malfunction. Moreover, immortalized APOE4 astrocytes are refractory to control of Ca2+ fluxes by extracellular lipids, and present distinct lipid composition in lysosomal and plasma membranes. CONCLUSIONS: Immortalized APOE4 versus APOE3 astrocytes present: increased Ca2+ excitability due to lysosome dysregulation, altered membrane lipidomes and intracellular cholesterol distribution, and impaired modulation of Ca2+ responses upon changes in extracellular lipids. Ca2+ hyperactivity associated with APOE4 is found in astrocytes from male, but not female, targeted replacement mice. The study suggests that, independently of Aß and Tau pathologies, altered astrocyte excitability might contribute to neural-circuit hyperactivity depending on APOE allele, sex and lipids, and supports lysosome-targeted therapies to rescue APOE4 phenotypes in LOAD.
Subject(s)
Apolipoprotein E3/genetics , Apolipoprotein E4/genetics , Astrocytes/metabolism , Calcium/metabolism , Lysosomes/metabolism , Alzheimer Disease/metabolism , Animals , Apolipoprotein E3/metabolism , Cholesterol/metabolism , Female , Hippocampus/metabolism , Male , Mice, Transgenic , Neurons/metabolismABSTRACT
Systems neuroscience is still mainly a neuronal field, despite the plethora of evidence supporting the fact that astrocytes modulate local neural circuits, networks, and complex behaviors. In this article, we sought to identify which types of studies are necessary to establish whether astrocytes, beyond their well-documented homeostatic and metabolic functions, perform computations implementing mathematical algorithms that sub-serve coding and higher-brain functions. First, we reviewed Systems-like studies that include astrocytes in order to identify computational operations that these cells may perform, using Ca2+ transients as their encoding language. The analysis suggests that astrocytes may carry out canonical computations in a time scale of subseconds to seconds in sensory processing, neuromodulation, brain state, memory formation, fear, and complex homeostatic reflexes. Next, we propose a list of actions to gain insight into the outstanding question of which variables are encoded by such computations. The application of statistical analyses based on machine learning, such as dimensionality reduction and decoding in the context of complex behaviors, combined with connectomics of astrocyte-neuronal circuits, is, in our view, fundamental undertakings. We also discuss technical and analytical approaches to study neuronal and astrocytic populations simultaneously, and the inclusion of astrocytes in advanced modeling of neural circuits, as well as in theories currently under exploration such as predictive coding and energy-efficient coding. Clarifying the relationship between astrocytic Ca2+ and brain coding may represent a leap forward toward novel approaches in the study of astrocytes in health and disease.
Subject(s)
Astrocytes/physiology , Brain/physiology , Neurosciences/methods , Systems Biology/methods , Animals , Astrocytes/chemistry , Brain/cytology , Brain Chemistry/physiology , Humans , Neurons/chemistry , Neurons/physiology , Neurosciences/trends , Optogenetics/methods , Systems Biology/trendsABSTRACT
The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 µm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.
Subject(s)
Brain/physiology , Optogenetics/methods , Photons , Receptors, Cell Surface/metabolism , Animals , Astrocytes/metabolism , Astrocytes/physiology , Brain/metabolism , Calcium/metabolism , Neurons/metabolism , Neurons/physiology , Rats , Rats, Sprague-Dawley , Receptor, Metabotropic Glutamate 5/metabolism , Receptor, Metabotropic Glutamate 5/physiology , Receptors, Cell Surface/physiologyABSTRACT
Interneurons play a critical role in precise control of network operation. Indeed, higher brain capabilities such as working memory, cognitive flexibility, attention, or social interaction rely on the action of GABAergic interneurons. Evidence from excitatory neurons and synapses has revealed astrocytes as integral elements of synaptic transmission. However, GABAergic interneurons can also engage astrocyte signaling; therefore, it is tempting to speculate about different scenarios where, based on particular interneuron cell type, GABAergic-astrocyte interplay would be involved in diverse outcomes of brain function. In this review, we will highlight current data supporting the existence of dynamic GABAergic-astrocyte communication and its impact on the inhibitory-regulated brain responses, bringing new perspectives on the ways astrocytes might contribute to efficient neuronal coding.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , GABAergic Neurons/metabolism , Interneurons/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Neural Inhibition , Synaptic TransmissionABSTRACT
Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.
Subject(s)
Astrocytes/metabolism , Nerve Net/metabolism , Neurons/metabolism , Optogenetics/methods , Rod Opsins/metabolism , 2-Amino-5-phosphonovalerate/pharmacology , Adenosine A2 Receptor Antagonists/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Azo Compounds/pharmacology , Channelrhodopsins/genetics , Channelrhodopsins/metabolism , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Hippocampus/cytology , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Light , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pyridoxal Phosphate/analogs & derivatives , Pyridoxal Phosphate/pharmacology , Pyrimidines/pharmacology , Rod Opsins/genetics , Synaptic Potentials/physiology , Triazoles/pharmacology , Xanthenes/pharmacologyABSTRACT
Whole-cell patch clamp allows the characterization of synaptic transmission in neurons. It is possible to manipulate astrocytic activity and record how these glial cells affect neuronal networks. Here we describe the methodology to monitor the endogenously activation of astrocytes by inhibitory synaptic activity. Afterward, such glial activation will let us study the consequences of interneuron-astrocyte signaling on excitatory neurotransmission at hippocampal synapses.
Subject(s)
Astrocytes/metabolism , Interneurons/metabolism , Signal Transduction , Synaptic Transmission , Calcium/metabolism , Calcium Signaling , Electrophysiological Phenomena , GABAergic Neurons/metabolism , Hippocampus/metabolism , Patch-Clamp Techniques , Pyramidal Cells/cytology , Pyramidal Cells/metabolismABSTRACT
Research on glial cells over the past 30 years has confirmed the critical role of astrocytes in pathophysiological brain states. However, most of our knowledge about astrocyte physiology and of the interactions between astrocytes and neurons is based on the premises that astrocytes constitute a homogeneous cell type, without considering the particular properties of the circuits or brain nuclei in which the astrocytes are located. Therefore, we argue that more-sophisticated experiments are required to elucidate the specific features of astrocytes in different brain regions, and even within different layers of a particular circuit. Thus, in addition to considering the diverse mechanisms used by astrocytes to communicate with neurons and synaptic partners, it is necessary to take into account the cellular heterogeneity that likely contributes to the outcomes of astrocyte-neuron signaling. In this review article, we briefly summarize the current data regarding the anatomical, molecular and functional properties of astrocyte-neuron communication, as well as the heterogeneity within this communication.
ABSTRACT
Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580.
Subject(s)
Aging , Astrocytes/physiology , Brain/cytology , Cell Communication/physiology , Neurons/physiology , Signal Transduction/physiology , Acetylcholine/pharmacology , Adenosine Triphosphate/pharmacology , Amyloid beta-Protein Precursor/deficiency , Amyloid beta-Protein Precursor/genetics , Animals , Astrocytes/drug effects , Brain/growth & development , Calcium/metabolism , Cell Communication/drug effects , Excitatory Amino Acid Agents/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/genetics , Methoxyhydroxyphenylglycol/analogs & derivatives , Methoxyhydroxyphenylglycol/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/drug effects , Presenilin-1/deficiency , Presenilin-1/genetics , Signal Transduction/drug effects , Synapses/drug effects , Synapses/physiology , Synaptic Potentials/drug effects , Synaptic Potentials/geneticsABSTRACT
The targeting of two independent but synergistic enzymatic activities, histone deacetylases (HDACs, class I and HDAC6) and phosphodiesterase 5 (PDE5), has recently been validated as a potentially novel therapeutic approach for Alzheimer's disease (AD). Here we report the discovery of a new first-in-class small-molecule (CM-414) that acts as a dual inhibitor of PDE5 and HDACs. We have used this compound as a chemical probe to validate this systems therapeutics strategy, where an increase in the activation of cAMP/cGMP-responsive element-binding protein (CREB) induced by PDE5 inhibition, combined with moderate HDAC class I inhibition, leads to efficient histone acetylation. This molecule rescued the impaired long-term potentiation evident in hippocampal slices from APP/PS1 mice. Chronic treatment of Tg2576 mice with CM-414 diminished brain Aß and tau phosphorylation (pTau) levels, increased the inactive form of GSK3ß, reverted the decrease in dendritic spine density on hippocampal neurons, and reversed their cognitive deficits, at least in part by inducing the expression of genes related to synaptic transmission. Thus, CM-414 may serve as the starting point to discover balanced dual inhibitors with an optimal efficacy and safety profile for clinical testing on AD patients.
Subject(s)
Alzheimer Disease/drug therapy , Hippocampus/drug effects , Histone Deacetylase Inhibitors/administration & dosage , Neuronal Plasticity/drug effects , Phosphodiesterase 5 Inhibitors/administration & dosage , Pyrazoles/therapeutic use , Pyrimidinones/therapeutic use , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/prevention & control , Animals , Behavior, Animal/drug effects , Disease Models, Animal , Female , Hippocampus/physiopathology , Histone Deacetylase Inhibitors/pharmacology , Mice , Mice, Transgenic , Motor Activity/drug effects , Phosphodiesterase 5 Inhibitors/pharmacology , Primary Cell Culture , Pyrazoles/administration & dosage , Pyrazoles/pharmacology , Pyrimidinones/administration & dosage , Pyrimidinones/pharmacologyABSTRACT
Brain activity requires a flux of glucose to active regions to sustain increased metabolic demands. Insulin, the main regulator of glucose handling in the body, has been traditionally considered not to intervene in this process. However, we now report that insulin modulates brain glucose metabolism by acting on astrocytes in concert with IGF-I. The cooperation of insulin and IGF-I is needed to recover neuronal activity after hypoglycemia. Analysis of underlying mechanisms show that the combined action of IGF-I and insulin synergistically stimulates a mitogen-activated protein kinase/protein kinase D pathway resulting in translocation of GLUT1 to the cell membrane through multiple protein-protein interactions involving the scaffolding protein GAIP-interacting protein C terminus and the GTPase RAC1. Our observations identify insulin-like peptides as physiological modulators of brain glucose handling, providing further support to consider the brain as a target organ in diabetes.
Subject(s)
Astrocytes/metabolism , Glucose/metabolism , Animals , Biological Transport/physiology , Glucose Transporter Type 1/metabolism , Glycogen/metabolism , Immunoassay , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Lactic Acid/metabolism , Male , Mice , Neurons/metabolism , Plasmids , Polymerase Chain Reaction , Positron-Emission TomographyABSTRACT
Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABAA receptors, potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay.