Astrocytes tile the central nervous system, but their functions in neural microcircuits in vivo and their roles in mammalian behavior remain incompletely defined. We used two-photon laser scanning microscopy, electrophysiology, MINIscopes, RNA-seq, and a genetic approach to explore the effects of reduced striatal astrocyte Ca signaling in vivo. In wild-type mice, reducing striatal astrocyte Ca-dependent signaling increased repetitive self-grooming behaviors by altering medium spiny neuron (MSN) activity. The mechanism involved astrocyte-mediated neuromodulation facilitated by ambient GABA and was corrected by blocking astrocyte GABA transporter 3 (GAT-3). Furthermore, in a mouse model of Huntington's disease, dysregulation of GABA and astrocyte Ca signaling accompanied excessive self-grooming, which was relieved by blocking GAT-3. Assessments with RNA-seq revealed astrocyte genes and pathways regulated by Ca signaling in a cell-autonomous and non-cell-autonomous manner, including Rab11a, a regulator of GAT-3 functional expression. Thus, striatal astrocytes contribute to neuromodulation controlling mouse obsessive-compulsive-like behavior.

Highlights d Striatal MSNs release GABA to activate astrocyte Gi-coupled GABA B receptors d Astrocyte Gi pathway activation results in hyperactivity with disrupted attention d Astrocyte Gi pathway activation increases fast synaptic excitation and MSN firing d Behavioral and synaptic effects are due to reactivation of TSP1 in astrocytes

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The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species. Using two substrate proteins, dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate here that HSP90 newly acquires a chaperone activity when incubated at temperatures higher than 46°C, which is coupled with self-oligomerization of HSP90. While chemically denatured DHFR refolds spontaneously upon dilution from denaturant, oligomerized HSP90 bound DHFR during the process of refolding and prevented it from renaturation. DHFR was released from the complex with HSP90 by incubating with GroEL/ES complexes in an ATP-dependent manner and refolded into the native form. ␣-Casein inhibited the binding of DHFR to HSP90 and also chased DHFR from the complex with HSP90. These results suggest that HSP90 binds substrates to maintain them in a folding-competent structure. Furthermore, we found that HSP90 prevents luciferase from irreversible thermal denaturation and enables it to refold when postincubated with reticulocyte lysates. This heat-induced chaperone activity of HSP90 associated with its oligomerization may have a pivotal role in protection of cells from thermal damages.Stress response and acquired stress tolerance are commonly seen in all organisms from bacteria to higher vertebrates and are considered to be essential defense mechanisms of the cell against various environmental stresses (1). When cultured mammalian cells are exposed to 45°C or higher temperatures, they are killed rapidly. However, if they are preheated at nonlethally high temperatures, they can survive the subsequent exposure to 45°C (2, 3). These results were interpreted as indications that the acquisition of thermotolerance is attributed to the accumulation of stress proteins. This has been supported by the fact that overexpression of stress proteins such as HSP90 (3), HSP70 (4), and HSP27 (5-8) rendered cells stress-tolerant. In the case of budding yeast, HSP104 plays a crucial role in stress tolerance, and mutations in this gene drastically reduced survival at extreme temperatures (9).It is now accepted that stress proteins prevent catastrophic protein aggregation induced by heat and other stresses and also assist refolding of damaged proteins during recovery from stress (10 -13). For instance, Escherichia coli molecular chaperones, DnaK/DnaJ/GrpE (14 -16), GroEL/ES (16, 17), and ClpA (18), mitochondrial HSP60 (19), and HSP90 (20) protect substrate proteins from thermally induced aggregation. Furthermore it was revealed that HSP104 does not prevent protein aggregation but rather disaggregates and reactivates stressdamaged proteins (21,22). DnaK/DnaJ/GrpE and GroEL/ES are also proposed to have reactivation activities (14,16).HSP90 interacts with a specialized class of proteins such as steroid hormone receptors, protein kinases, and cytoskeletal proteins, and regulates their functions (11,23). Since the mode of action of HSP90 has not been fully investigated, its function as a molecular chaperone remains elusive. It has been shown r...

Adenosine 5′ triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.