Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

The astrocyte α1A-adrenoreceptor is a key component of the neuromodulatory system in mouse visual cortex.

Noradrenaline (norepinephrine) is known to modulate many physiological functions and behaviors. In this study, we tested to what extent astrocytes, a type of glial cell, participate in noradrenergic signaling in mouse primary visual cortex (V1). Astrocytes are essential partners of neurons in the central nervous system. They are central to brain homeostasis, but also dynamically regulate neuronal activity, notably by relaying and regulating neuromodulator signaling. Indeed, astrocytes express receptors for multiple neuromodulators, including noradrenaline, but the extent to which astrocytes are involved in noradrenergic signaling remains unclear. To test whether astrocytes are involved in noradrenergic neuromodulation in mice, we employed both short hairpin RNA mediated knockdown as well as pharmacological manipulation of the major noradrenaline receptor in astrocytes, the α1A-adrenoreceptor. Using acute brain slices, we found that the astrocytic α1A-adrenoreceptor subtype contributes to the generation of large intracellular Ca2+ signals in visual cortex astrocytes, which are generally thought to underlie astrocyte function. To test if reduced α1A-adrenoreceptor signaling in astrocytes affected the function of neuronal circuits in V1, we used both patch-clamp and field potential recordings. These revealed that noradrenergic signaling through the astrocyte α1A-adrenoreceptor is important to not only modulate synaptic activity but also to regulate plasticity in V1, through the potentiation of synaptic responses in circuits involved in visual information processing.

Astrocyte structural heterogeneity in the mouse hippocampus.

Astrocytes are integral components of brain circuits, where they sense, process, and respond to surrounding activity, maintaining homeostasis and regulating synaptic transmission, the sum of which results in behavior modulation. These interactions are possible due to their complex morphology, composed of a tree-like structure of processes to cover defined territories ramifying in a mesh-like system of fine leaflets unresolved by conventional optic microscopy. While recent reports devoted more attention to leaflets and their dynamic interactions with synapses, our knowledge about the tree-like "backbone" structure in physiological conditions is incomplete. Recent transcriptomic studies described astrocyte molecular diversity, suggesting structural heterogeneity in regions such as the hippocampus, which is crucial for cognitive and emotional behaviors. In this study, we carried out the structural analysis of astrocytes across the hippocampal subfields of Cornu Ammonis area 1 (CA1) and dentate gyrus in the dorsoventral axis. We found that astrocytes display heterogeneity across the hippocampal subfields, which is conserved along the dorsoventral axis. We further found that astrocytes appear to contribute in an exocytosis-dependent manner to a signaling loop that maintains the backbone structure. These findings reveal astrocyte heterogeneity in the hippocampus, which appears to follow layer-specific cues and depend on the neuro-glial environment.

Astrocytes shape the plastic response of adult cortical neurons to vision loss.

Astrocytes are vital for preserving correct brain functioning by continuously sustaining neuronal activity and survival. They are in contact with multiple synapses at once allowing the expansion of local synaptic events into activity changes in neuronal networks. Furthermore, cortical astrocytes integrate local sensory inputs and behavioral state. From an anatomical, molecular, and functional perspective, astrocytes are thus ideal candidates to influence complex large-scale brain mechanisms such as plasticity. We collected evidence for the astrocytic potential for plasticity modulation, using the monocular enucleation (ME) mouse model of visual cortex plasticity. The impact of one-eyed vision involves the functional recruitment of the deprived visual cortex by the spared senses within a 7-week time frame, reflecting a substantial change in sensory information processing. In visually deprived cortex, a swift upregulation in Aldh1l1-positive astrocyte density lasts until maximal functional recovery is reached. Transient metabolic silencing of visual cortex astrocytes at the time of ME induction, through intracranial fluorocitrate injections, reveals that astrocytes are required on site to achieve adequate long-term neuronal reactivation. In addition, chronic stimulation by Gi but not Gq G-protein coupled receptor activation of local astrocytes boosts the cortical plasticity phenomenon. Hence, functional manipulation of protoplasmic astrocytes has long-lasting effects on the functional recovery of cortical neurons upon sensory loss, possibly by influencing the neuronal threshold to reactivate. Together, our results highlight an integral role for astrocytes in mediating adult cortical plasticity and unmask astrocyte specific Gi signaling as an interesting therapeutic pathway for brain plasticity regulation.

Favouring inhibitory synaptic drive mediated by GABA(A) receptors in the basolateral nucleus of the amygdala efficiently reduces pain symptoms in neuropathic mice.

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. The central nucleus of the amygdala is often called the 'nociceptive amygdala', but little is known about the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the basolateral nucleus of the amygdala (BLA) via chronically-implanted cannulas. We found that an N-methyl-D-aspartate-type glutamate receptor antagonist (MK-801) exerted a potent antiallodynic effect, whereas a transient allodynia was induced after perfusion of bicuculline, a GABA(A) receptor antagonist. Potentiating GABA(A) receptor function using diazepam or etifoxine (a non-benzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the antiallodynic effect of etifoxine disappeared in animals that were incapable of producing 3α-steroids. Diazepam had a similar effect but of shorter duration. As indicated by patch-clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABA(A) receptor-mediated synaptic transmission. Together with a presynaptic elevation of miniature inhibitory postsynaptic current frequency, the duration and amplitude of GABA(A) miniature inhibitory postsynaptic currents were also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of the BLA and the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore, the BLA could be a novel interesting target for the development of pharmacological or non-pharmacological therapies.

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's disease.

Dysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimer's disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate local neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex several years before amyloid deposition. We find the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astrocytic calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral disruptions. In conclusion, we show that astrocytes mediate initial features of AD and drive clinically relevant phenotypes.

AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

Astrocytes, Noradrenaline, α1-Adrenoreceptors, and Neuromodulation: Evidence and Unanswered Questions.

Noradrenaline is a major neuromodulator in the central nervous system (CNS). It is released from varicosities on neuronal efferents, which originate principally from the main noradrenergic nuclei of the brain - the locus coeruleus - and spread throughout the parenchyma. Noradrenaline is released in response to various stimuli and has complex physiological effects, in large part due to the wide diversity of noradrenergic receptors expressed in the brain, which trigger diverse signaling pathways. In general, however, its main effect on CNS function appears to be to increase arousal state. Although the effects of noradrenaline have been researched extensively, the majority of studies have assumed that noradrenaline exerts its effects by acting directly on neurons. However, neurons are not the only cells in the CNS expressing noradrenaline receptors. Astrocytes are responsive to a range of neuromodulators - including noradrenaline. In fact, noradrenaline evokes robust calcium transients in astrocytes across brain regions, through activation of α1-adrenoreceptors. Crucially, astrocytes ensheath neurons at synapses and are known to modulate synaptic activity. Hence, astrocytes are in a key position to relay, or amplify, the effects of noradrenaline on neurons, most notably by modulating inhibitory transmission. Based on a critical appraisal of the current literature, we use this review to argue that a better understanding of astrocyte-mediated noradrenaline signaling is therefore essential, if we are ever to fully understand CNS function. We discuss the emerging concept of astrocyte heterogeneity and speculate on how this might impact the noradrenergic modulation of neuronal circuits. Finally, we outline possible experimental strategies to clearly delineate the role(s) of astrocytes in noradrenergic signaling, and neuromodulation in general, highlighting the urgent need for more specific and flexible experimental tools.

Star power: the emerging role of astrocytes as neuronal partners during cortical plasticity.

Plasticity is a fundamental property of neuronal circuits, allowing them to adapt to alterations in activation. Generally speaking, plasticity has been viewed from a 'neuron-centric' perspective, with changes in circuit function attributed to alterations in neuronal excitability, synaptic strength or neuronal connectivity. However, it is now clear that glial cells, in particular astrocytes, are key regulators of neuronal plasticity. This article reviews recent progress made in understanding astrocyte function and attempts to summarize these functions into a coherent framework that positions astrocytes as central players in the plasticity process.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Identification of region-specific astrocyte subtypes at single cell resolution.

Astrocytes, a major cell type found throughout the central nervous system, have general roles in the modulation of synapse formation and synaptic transmission, blood-brain barrier formation, and regulation of blood flow, as well as metabolic support of other brain resident cells. Crucially, emerging evidence shows specific adaptations and astrocyte-encoded functions in regions, such as the spinal cord and cerebellum. To investigate the true extent of astrocyte molecular diversity across forebrain regions, we used single-cell RNA sequencing. Our analysis identifies five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Validation of our data in situ reveals distinct spatial positioning of defined subtypes, reflecting the distribution of morphologically and physiologically distinct astrocyte populations. Our findings are evidence for specialized astrocyte subtypes between and within brain regions. The data are available through an online database (https://holt-sc.glialab.org/), providing a resource on which to base explorations of local astrocyte diversity and function in the brain.

A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System.

Oxytocin (OT) release by axonal terminals onto the central nucleus of the amygdala exerts anxiolysis. To investigate which subpopulation of OT neurons contributes to this effect, we developed a novel method: virus-delivered genetic activity-induced tagging of cell ensembles (vGATE). With the vGATE method, we identified and permanently tagged a small subpopulation of OT cells, which, by optogenetic stimulation, strongly attenuated contextual fear-induced freezing, and pharmacogenetic silencing of tagged OT neurons impaired context-specific fear extinction, demonstrating that the tagged OT neurons are sufficient and necessary, respectively, to control contextual fear. Intriguingly, OT cell terminals of fear-experienced rats displayed enhanced glutamate release in the amygdala. Furthermore, rats exposed to another round of fear conditioning displayed 5-fold more activated magnocellular OT neurons in a novel environment than a familiar one, possibly for a generalized fear response. Thus, our results provide first evidence that hypothalamic OT neurons represent a fear memory engram.

A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing.

Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.