Astrocytes as Drivers and Disruptors of Behavior: New Advances in Basic Mechanisms and Therapeutic Targeting.

Astrocytes are emerging as key regulators of cognitive function and behavior. This review highlights some of the latest advances in the understanding of astrocyte roles in different behavioral domains across lifespan and in disease. We address specific molecular and circuit mechanisms by which astrocytes modulate behavior, discuss their functional diversity and versatility, and highlight emerging astrocyte-targeted treatment strategies that might alleviate behavioral and cognitive dysfunction in pathologic conditions. Converging evidence across different model systems and manipulations is revealing that astrocytes regulate behavioral processes in a precise and context-dependent manner. Improved understanding of these astrocytic functions may generate new therapeutic strategies for various conditions with cognitive and behavioral impairments.

Istradefylline reduces memory deficits in aging mice with amyloid pathology.

Adenosine A2A receptors are putative therapeutic targets for neurological disorders. The adenosine A2A receptor antagonist istradefylline is approved in Japan for Parkinson's disease and is being tested in clinical trials for this condition elsewhere. A2A receptors on neurons and astrocytes may contribute to Alzheimer's disease (AD) by impairing memory. However, it is not known whether istradefylline enhances cognitive function in aging animals with AD-like amyloid plaque pathology. Here, we show that elevated levels of Aß, C-terminal fragments of the amyloid precursor protein (APP), or amyloid plaques, but not overexpression of APP per se, increase astrocytic A2A receptor levels in the hippocampus and neocortex of aging mice. Moreover, in amyloid plaque-bearing mice, low-dose istradefylline treatment enhanced spatial memory and habituation, supporting the conclusion that, within a well-defined dose range, A2A receptor blockers might help counteract memory problems in patients with Alzheimer's disease.

Levetiracetam suppresses neuronal network dysfunction and reverses synaptic and cognitive deficits in an Alzheimer's disease model.

In light of the rising prevalence of Alzheimer's disease (AD), new strategies to prevent, halt, and reverse this condition are needed urgently. Perturbations of brain network activity are observed in AD patients and in conditions that increase the risk of developing AD, suggesting that aberrant network activity might contribute to AD-related cognitive decline. Human amyloid precursor protein (hAPP) transgenic mice simulate key aspects of AD, including pathologically elevated levels of amyloid-ß peptides in brain, aberrant neural network activity, remodeling of hippocampal circuits, synaptic deficits, and behavioral abnormalities. Whether these alterations are linked in a causal chain remains unknown. To explore whether hAPP/amyloid-ß-induced aberrant network activity contributes to synaptic and cognitive deficits, we treated hAPP mice with different antiepileptic drugs. Among the drugs tested, only levetiracetam (LEV) effectively reduced abnormal spike activity detected by electroencephalography. Chronic treatment with LEV also reversed hippocampal remodeling, behavioral abnormalities, synaptic dysfunction, and deficits in learning and memory in hAPP mice. Our findings support the hypothesis that aberrant network activity contributes causally to synaptic and cognitive deficits in hAPP mice. LEV might also help ameliorate related abnormalities in people who have or are at risk for AD.

Astrocytes in selective vulnerability to neurodegenerative disease.

Selective vulnerability of specific brain regions and cell populations is a hallmark of neurodegenerative disorders. Mechanisms of selective vulnerability involve neuronal heterogeneity, functional specializations, and differential sensitivities to stressors and pathogenic factors. In this review we discuss the growing body of literature suggesting that, like neurons, astrocytes are heterogeneous and specialized, respond to and integrate diverse inputs, and induce selective effects on brain function. In disease, astrocytes undergo specific, context-dependent changes that promote different pathogenic trajectories and functional outcomes. We propose that astrocytes contribute to selective vulnerability through maladaptive transitions to context-divergent phenotypes that impair specific brain regions and functions. Further studies on the multifaceted roles of astrocytes in disease may provide new therapeutic approaches to enhance resilience against neurodegenerative disorders.

Mitochondrial complex III-derived ROS amplify immunometabolic changes in astrocytes and promote dementia pathology.

Neurodegenerative disorders alter mitochondrial functions, including the production of reactive oxygen species (ROS). Mitochondrial complex III (CIII) generates ROS implicated in redox signaling, but its triggers, targets, and disease relevance are not clear. Using site-selective suppressors and genetic manipulations together with mitochondrial ROS imaging and multiomic profiling, we found that CIII is the dominant source of ROS production in astrocytes exposed to neuropathology-related stimuli. Astrocytic CIII-ROS production was dependent on nuclear factor-κB (NF-κB) and the mitochondrial sodium-calcium exchanger (NCLX) and caused oxidation of select cysteines within immune and metabolism-associated proteins linked to neurological disease. CIII-ROS amplified metabolomic and pathology-associated transcriptional changes in astrocytes, with STAT3 activity as a major mediator, and facilitated neuronal toxicity in a non-cell-autonomous manner. As proof-of-concept, suppression of CIII-ROS in mice decreased dementia-linked tauopathy and neuroimmune cascades and extended lifespan. Our findings establish CIII-ROS as an important immunometabolic signal transducer and tractable therapeutic target in neurodegenerative disease.

Hippocampal astrocytes induce sex-dimorphic effects on memory.

Astrocytic receptors influence cognitive function and can promote behavioral deficits in disease. These effects may vary based on variables such as biological sex, but it is not known if the effects of astrocytic receptors are dependent on sex. We leveraged in vivo gene editing and chemogenetics to examine the roles of astrocytic receptors in spatial memory and other processes. We show that reductions in metabotropic glutamate receptor 3 (mGluR3), the main astrocytic glutamate receptor in adults, impair memory in females but enhance memory in males. Similarly, increases in astrocytic mGluR3 levels have sex-dependent effects and enhance memory in females. mGluR3 manipulations also alter spatial search strategies during recall in a sex-specific manner. In addition, acute chemogenetic stimulation of Gi/o-coupled or Gs-coupled receptors in hippocampal astrocytes induces bidirectional and sex-dimorphic effects on memory. Thus, astrocytes are sex-dependent modulators of cognitive function and may promote sex differences in aging and disease.

Effects of adenosine A2A receptors on cognitive function in health and disease.

Adenosine A2A receptors have been studied extensively in the context of motor function and movement disorders such as Parkinson's disease. In addition to these roles, A2A receptors have also been increasingly implicated in cognitive function and cognitive impairments in diverse conditions, including Alzheimer's disease, schizophrenia, acute brain injury, and stress. We review the roles of A2A receptors in cognitive processes in health and disease, focusing primarily on the effects of reducing or enhancing A2A expression levels or activities in animal models. Studies reveal that A2A receptors in neurons and astrocytes modulate multiple aspects of cognitive function, including memory and motivation. Converging evidence also indicates that A2A receptor levels and activities are aberrantly increased in aging, acute brain injury, and chronic disorders, and these increases contribute to neurocognitive impairments. Therapeutically targeting A2A receptors with selective modulators may alleviate cognitive deficits in diverse neurological and neuropsychiatric conditions. Further research on the exact neural mechanisms of these effects as well as the efficacy of selective A2A modulators on cognitive alterations in humans are important areas for future investigation.

Astrocytic TDP-43 dysregulation impairs memory by modulating antiviral pathways and interferon-inducible chemokines.

Transactivating response region DNA binding protein 43 (TDP-43) pathology is prevalent in dementia, but the cell type-specific effects of TDP-43 pathology are not clear, and therapeutic strategies to alleviate TDP-43-linked cognitive decline are lacking. We found that patients with Alzheimer's disease or frontotemporal dementia have aberrant TDP-43 accumulation in hippocampal astrocytes. In mouse models, induction of widespread or hippocampus-targeted accumulation in astrocytic TDP-43 caused progressive memory loss and localized changes in antiviral gene expression. These changes were cell-autonomous and correlated with impaired astrocytic defense against infectious viruses. Among the changes, astrocytes had elevated levels of interferon-inducible chemokines, and neurons had elevated levels of the corresponding chemokine receptor CXCR3 in presynaptic terminals. CXCR3 stimulation altered presynaptic function and promoted neuronal hyperexcitability, akin to the effects of astrocytic TDP-43 dysregulation, and blockade of CXCR3 reduced this activity. Ablation of CXCR3 also prevented TDP-43-linked memory loss. Thus, astrocytic TDP-43 dysfunction contributes to cognitive impairment through aberrant chemokine-mediated astrocytic-neuronal interactions.

Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia.

BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.

Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

Branched Photoswitchable Tethered Ligands Enable Ultra-efficient Optical Control and Detection of G Protein-Coupled Receptors In Vivo.

The limitations of classical drugs have spurred the development of covalently tethered photoswitchable ligands to control neuromodulatory receptors. However, a major shortcoming of tethered photopharmacology is the inability to obtain optical control with an efficacy comparable with that of the native ligand. To overcome this, we developed a family of branched photoswitchable compounds to target metabotropic glutamate receptors (mGluRs). These compounds permit photo-agonism of Gi/o-coupled group II mGluRs with near-complete efficiency relative to glutamate when attached to receptors via a range of orthogonal, multiplexable modalities. Through a chimeric approach, branched ligands also allow efficient optical control of Gq-coupled mGluR5, which we use to probe the spatiotemporal properties of receptor-induced calcium oscillations. In addition, we report branched, photoswitch-fluorophore compounds for simultaneous receptor imaging and manipulation. Finally, we demonstrate this approach in vivo in mice, where photoactivation of SNAP-mGluR2 in the medial prefrontal cortex reversibly modulates working memory in normal and disease-associated states.

Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory.

Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer's disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice and increased the levels of Arc (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss.

Interleukin-1 mediates long-term hippocampal dentate granule cell loss following postnatal viral infection.

Viral infections of the developing CNS can cause long-term neuropathological sequela through undefined mechanisms. Proinflammatory cytokines such as IL-1beta have gained attention in mediating neurodegeneration in corticohippocampal structures due to a variety of insults in adults, though there is less information on the developing brain. Little is known concerning the spatial-temporal pattern of IL-1beta induction in the developing hippocampus following live virus infection, and there are few studies addressing the long-term consequences of this cytokine induction. We report that infection of rats with lymphocytic choriomeningitis virus on postnatal day 4 induces IL-1beta protein in select regions of the hippocampus on 6, 15, 21, and 45 days after infection. This infection resulted in a 71% reduction of dentate granule cell neurons by the time the rats reached mid-adulthood. We further investigated the causative role of IL-1 in this dentate granule cell loss by blocking IL-1 activity using an IL-1ra-expressing adenoviral vector administered at the time of infection. Blockade of IL-1 abrogated the infection-associated neuron loss in this vivo model. Considering that IL-1 can be triggered by multiple perinatal insults, our findings suggest that early therapy with anti-inflammatory agents that block IL-1 may be effective for reducing adulthood neuropathology.

A subunit-selective potentiator of NR2C- and NR2D-containing NMDA receptors.

NMDA receptors are tetrameric complexes of NR1 and NR2A-D subunits that mediate excitatory synaptic transmission and have a role in neurological disorders. In this article, we identify a novel subunit-selective potentiator of NMDA receptors containing the NR2C or NR2D subunit, which could allow selective modification of circuit function in regions expressing NR2C/D subunits. The substituted tetrahydroisoquinoline CIQ (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone) enhances receptor responses two-fold with an EC(50) of 3 µM by increasing channel opening frequency without altering mean open time or EC(50) values for glutamate or glycine. The actions of CIQ depend on a single residue in the M1 region (NR2D Thr592) and on the linker between the N-terminal domain and agonist binding domain. CIQ potentiates native NR2D-containing NMDA receptor currents from subthalamic neurons. Our identification of a subunit-selective NMDA receptor modulator reveals a new class of pharmacological tools with which to probe the role of NR2C- and NR2D-containing NMDA receptors in brain function and disease.

Differential regulation of microglial motility by ATP/ADP and adenosine.

Microglia are motile immune-competent cells of the central nervous system. They assume a highly branched morphology and monitor the brain parenchyma under physiological conditions. In the presence of injury, microglia retract their branching processes, migrate to the site of injury, and help clear cellular debris by phagocytosis. This response appears to be mediated in part by ATP released at the site of injury. Here, we review the evidence for the involvement of ATP and the purinergic P2Y(12) receptor in microglial process extension and chemoattraction to injury. We subsequently discuss recent findings regarding a switch of this chemotactic response to ATP in activated, or proinflammatory, microglia. Specifically, in LPS-activated microglia, ATP induces process retraction and repulsive migration, effects opposite to those seen in unstimulated cells. These repulsive effects of ATP are mediated by the G(s)-coupled adenosine A(2A) receptor and depend on the breakdown of ATP to adenosine. Thus, ATP-induced repulsion by activated microglia involves upregulation of the adenosine A(2A) receptor and coincident downregulation of the P2Y(12) receptor. The roles of the A(2A) receptor in brain pathologies such as Parkinson's disease and ischemia are also examined. We propose that the effects of A(2A) receptor antagonists on brain injury may be in part due to the inactivation of A(2A) on activated microglia.

Adenosine A(2A) receptor mediates microglial process retraction.

Cell motility drives many biological processes, including immune responses and embryonic development. In the brain, microglia are immune cells that survey and scavenge brain tissue using elaborate and motile cell processes. The motility of these processes is guided by the local release of chemoattractants. However, most microglial processes retract during prolonged brain injury or disease. This hallmark of brain inflammation remains unexplained. We identified a molecular pathway in mouse and human microglia that converted ATP-driven process extension into process retraction during inflammation. This chemotactic reversal was driven by upregulation of the A(2A) adenosine receptor coincident with P2Y(12) downregulation. Thus, A(2A) receptor stimulation by adenosine, a breakdown product of extracellular ATP, caused activated microglia to assume their characteristic amoeboid morphology during brain inflammation. Our results indicate that purine nucleotides provide an opportunity for context-dependent shifts in receptor signaling. Thus, we reveal an unexpected chemotactic switch that generates a hallmark feature of CNS inflammation.

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1.

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.