Neighbour-induced changes in root exudation patterns of buckwheat results in altered root architecture of redroot pigweed.

Roots are crucial in plant adaptation through the exudation of various compounds which are influenced and modified by environmental factors. Buckwheat root exudate and root system response to neighbouring plants (buckwheat or redroot pigweed) and how these exudates affect redroot pigweed was investigated. Characterising root exudates in plant-plant interactions presents challenges, therefore a split-root system which enabled the application of differential treatments to parts of a single root system and non-destructive sampling was developed. Non-targeted metabolome profiling revealed that neighbour presence and identity induces systemic changes. Buckwheat and redroot pigweed neighbour presence upregulated 64 and 46 metabolites, respectively, with an overlap of only 7 metabolites. Root morphology analysis showed that, while the presence of redroot pigweed decreased the number of root tips in buckwheat, buckwheat decreased total root length and volume, surface area, number of root tips, and forks of redroot pigweed. Treatment with exudates (from the roots of buckwheat and redroot pigweed closely interacting) on redroot pigweed decreased the total root length and number of forks of redroot pigweed seedlings when compared to controls. These findings provide understanding of how plants modify their root exudate composition in the presence of neighbours and how this impacts each other's root systems.

Astrocyte-secreted neurocan controls inhibitory synapse formation and function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. Several astrocyte-secreted synaptogenic proteins controlling excitatory synapse development were identified; however, those that induce inhibitory synaptogenesis remain elusive. Here, we identify neurocan as an astrocyte-secreted inhibitory synaptogenic protein. After secretion from astrocytes, neurocan is cleaved into N- and C-terminal fragments. We found that these fragments have distinct localizations in the extracellular matrix. The neurocan C-terminal fragment localizes to synapses and controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic domain have reduced inhibitory synapse numbers and function. Through super-resolution microscopy, in vivo proximity labeling by secreted TurboID, and astrocyte-specific rescue approaches, we discovered that the synaptogenic domain of neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

SynBot: An open-source image analysis software for automated quantification of synapses.

The formation of precise numbers of neuronal connections, known as synapses, is crucial for brain function. Therefore, synaptogenesis mechanisms have been one of the main focuses of cellular and molecular neuroscience. Immunohistochemistry is a common tool for labeling and visualization of synapses. Thus, quantifying the numbers of synapses from light microscopy images enables screening the impacts of experimental manipulations on synapse development. Despite its utility, this approach is paired with low throughput image analysis methods that are challenging to learn, and results are variable between experimenters. We developed a new open-source ImageJ-based software, SynBot, to address these technical bottlenecks by automating several stages of the analysis. SynBot incorporates the advanced algorithms ilastik and SynQuant for accurate thresholding for synaptic puncta identification, and the code can easily be modified by users. The use of this software will allow for rapid and reproducible screening of synaptic phenotypes in healthy and diseased nervous systems. Motivation: Light microscopy imaging of pre- and post-synaptic proteins from neurons in tissue or in vitro allows for the effective identification of synaptic structures. Previous methods for quantitative analysis of these images were time-consuming, required extensive user training, and the source code could not be easily modified. Here, we describe SynBot, a new open-source tool that automates the synapse quantification process, decreases the requirement for user training, and allows for easy modifications to the code.

Astrocyte signaling and interactions in Multiple Sclerosis.

Multiple Sclerosis (MS) is a common cause of impairment in working-aged adults. MS is characterized by neuroinflammation and infiltration of peripheral immune cells to the brain, which cause myelin loss and death of oligodendrocytes and neurons. Many studies on MS have focused on the peripheral immune sources of demyelination and repair. However, recent studies revealed that a glial cell type, the astrocytes, undergo robust morphological and transcriptomic changes that contribute significantly to demyelination and myelin repair. Here, we discuss recent findings elucidating signaling modalities that astrocytes acquire or lose in MS and how these changes alter the interactions of astrocytes with other nervous system cell types.

δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte-neuron cadherin interactions.

Astrocytes control the formation of specific synaptic circuits via cell adhesion and secreted molecules. Astrocyte synaptogenic functions are dependent on the establishment of their complex morphology. However, it is unknown if distinct neuronal cues differentially regulate astrocyte morphogenesis. δ-Catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. We found δ-catenin is also highly expressed by astrocytes and required both in astrocytes and neurons for astrocyte morphogenesis. δ-Catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and biochemical analyses to reveal that δ-catenin interacts with the N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte-neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

Training-induced circuit-specific excitatory synaptogenesis in mice is required for effort control.

Synaptogenesis is essential for circuit development; however, it is unknown whether it is critical for the establishment and performance of goal-directed voluntary behaviors. Here, we show that operant conditioning via lever-press for food reward training in mice induces excitatory synapse formation onto a subset of anterior cingulate cortex neurons projecting to the dorsomedial striatum (ACC→DMS). Training-induced synaptogenesis is controlled by the Gabapentin/Thrombospondin receptor α2δ-1, which is an essential neuronal protein for proper intracortical excitatory synaptogenesis. Using germline and conditional knockout mice, we found that deletion of α2δ-1 in the adult ACC→DMS circuit diminishes training-induced excitatory synaptogenesis. Surprisingly, this manipulation does not impact learning but results in a significant increase in effort exertion without affecting sensitivity to reward value or changing contingencies. Bidirectional optogenetic manipulation of ACC→DMS neurons rescues or phenocopies the behaviors of the α2δ-1 cKO mice, highlighting the importance of synaptogenesis within this cortico-striatal circuit in regulating effort exertion.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Astrocyte-Secreted Neurocan Controls Inhibitory Synapse Formation and Function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. To date, several astrocyte-secreted synaptogenic proteins controlling different stages of excitatory synapse development have been identified. However, the identities of astrocytic signals that induce inhibitory synapse formation remain elusive. Here, through a combination of in vitro and in vivo experiments, we identified Neurocan as an astrocyte-secreted inhibitory synaptogenic protein. Neurocan is a chondroitin sulfate proteoglycan that is best known as a protein localized to the perineuronal nets. However, Neurocan is cleaved into two after secretion from astrocytes. We found that the resulting N- and C-terminal fragments have distinct localizations in the extracellular matrix. While the N-terminal fragment remains associated with perineuronal nets, the Neurocan C-terminal fragment localizes to synapses and specifically controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic region have reduced inhibitory synapse numbers and function. Through super-resolution microscopy and in vivo proximity labeling by secreted TurboID, we discovered that the synaptogenic domain of Neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

GEARBOCS: An Adeno Associated Virus Tool for In Vivo Gene Editing in Astrocytes.

In the mammalian central nervous system (CNS), astrocytes are indispensable for brain development, function, and health. However, non-invasive tools to study astrocyte biology and function in vivo have been limited to genetically modified mice. CRISPR/Cas9-based genome engineering enables rapid and precise gene manipulations in the CNS. Here, we developed a non-invasive astrocyte-specific method utilizing a single AAV vector, GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System). We verified GEARBOCS' specificity to mouse cortical astrocytes and demonstrated its utility for three types of gene manipulations: knockout (KO); tagging (TagIN); and reporter gene knock-in (Gene-TRAP) strategies. We deployed GEARBOCS to determine whether cortical astrocytes express Vamp2 protein. The presence of Vamp2-positive vesicles in cultured astrocytes is well-established, however, Vamp2 protein expression in astrocytes in vivo has proven difficult to ascertain due to its overwhelming abundance in neurons. Using GEARBOCS, we delineated the in vivo astrocytic Vamp2 expression and found that it is required for maintaining excitatory and inhibitory synapse numbers in the visual cortex. GEARBOCS strategy provides fast and efficient means to study astrocyte biology in vivo.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

Hevin/Sparcl1 drives pathological pain through spinal cord astrocyte and NMDA receptor signaling.

High endothelial venule protein/SPARC-like 1 (hevin/Sparcl1) is an astrocyte-secreted protein that regulates synapse formation in the brain. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared with WT mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than WT mice from neuropathic pain after nerve injury. Intrathecal injection of WT hevin was sufficient to induce persistent mechanical allodynia in naive mice. In hevin-null mice with nerve injury, adeno-associated-virus-mediated (AAV-mediated) re-expression of hevin in glial fibrillary acidic protein-expressing (GFAP-expressing) spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin-potentiated N-Methyl-D-aspartic acid (NMDA) currents are mediated by GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing Ab against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin that was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal Ab may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.

Prenatal environmental stressors impair postnatal microglia function and adult behavior in males.

Gestational exposure to environmental toxins and socioeconomic stressors is epidemiologically linked to neurodevelopmental disorders with strong male bias, such as autism. We model these prenatal risk factors in mice by co-exposing pregnant dams to an environmental pollutant and limited-resource stress, which robustly activates the maternal immune system. Only male offspring display long-lasting behavioral abnormalities and alterations in the activity of brain networks encoding social interactions. Cellularly, prenatal stressors diminish microglial function within the anterior cingulate cortex, a central node of the social coding network, in males during early postnatal development. Precise inhibition of microglial phagocytosis within the anterior cingulate cortex (ACC) of wild-type (WT) mice during the same critical period mimics the impact of prenatal stressors on a male-specific behavior, indicating that environmental stressors alter neural circuit formation in males via impairing microglia function during development.

Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability.

Hevin is a secreted extracellular matrix protein that is encoded by the SPARCL1 gene. Recent studies have shown that Hevin plays an important role in regulating synaptogenesis and synaptic plasticity. Mutations in the SPARCL1 gene increase the risk of autism spectrum disorder (ASD). However, the molecular basis of how mutations in SPARCL1 increase the risk of ASD is not been fully understood. In this study, we show that one of the SPARCL1 mutations associated with ASD impairs normal Hevin secretion. We identified Hevin mutants lacking the EF-hand motif through analyzing ASD-related mice with vulnerable spliceosome functions. Hevin deletion mutants accumulate in the endoplasmic reticulum (ER), leading to the activation of unfolded protein responses. We also found that a single amino acid substitution of Trp647 with Arg in the EF-hand motif associated with a familial case of ASD causes a similar phenotype in the EF-hand deletion mutant. Importantly, molecular dynamics (MD) simulation revealed that this single amino acid substitution triggers exposure of a hydrophobic amino acid to the surface, increasing the binding of Hevin with molecular chaperons, BIP. Taken together, these data suggest that the integrity of the EF-hand motif in Hevin is crucial for proper folding and that ASD-related mutations impair the export of Hevin from the ER. Our data provide a novel mechanism linking a point mutation in the SPARCL1 gene to the molecular and cellular characteristics involved in ASD.

The role of astrocyte structural plasticity in regulating neural circuit function and behavior.

Brain circuits undergo substantial structural changes during development, driven by the formation, stabilization, and elimination of synapses. Synaptic connections continue to undergo experience-dependent structural rearrangements throughout life, which are postulated to underlie learning and memory. Astrocytes, a major glial cell type in the brain, are physically in contact with synaptic circuits through their structural ensheathment of synapses. Astrocytes strongly contribute to the remodeling of synaptic structures in healthy and diseased central nervous systems by regulating synaptic connectivity and behaviors. However, whether structural plasticity of astrocytes is involved in their critical functions at the synapse is unknown. This review will discuss the emerging evidence linking astrocytic structural plasticity to synaptic circuit remodeling and regulation of behaviors. Moreover, we will survey possible molecular and cellular mechanisms regulating the structural plasticity of astrocytes and their non-cell-autonomous effects on neuronal plasticity. Finally, we will discuss how astrocyte morphological changes in different physiological states and disease conditions contribute to neuronal circuit function and dysfunction.

Building and destroying synaptic bridges: How do Hevin/Sparcl1, SPARC, and MDGAs modify trans-synaptic neurexin-neuroligin interactions?

Astrocyte-secreted Hevin induces synapse formation by bridging the trans-synaptic organizers, neuroligins and neurexins. In this issue of Structure, Fan et al. (2021) elucidate the structural basis of Hevin's interaction with these molecules and reveal the competitive interplay between Hevin, SPARC, and MDGAs.

HepaCAM controls astrocyte self-organization and coupling.

Astrocytes extensively infiltrate the neuropil to regulate critical aspects of synaptic development and function. This process is regulated by transcellular interactions between astrocytes and neurons via cell adhesion molecules. How astrocytes coordinate developmental processes among one another to parse out the synaptic neuropil and form non-overlapping territories is unknown. Here we identify a molecular mechanism regulating astrocyte-astrocyte interactions during development to coordinate astrocyte morphogenesis and gap junction coupling. We show that hepaCAM, a disease-linked, astrocyte-enriched cell adhesion molecule, regulates astrocyte competition for territory and morphological complexity in the developing mouse cortex. Furthermore, conditional deletion of Hepacam from developing astrocytes significantly impairs gap junction coupling between astrocytes and disrupts the balance between synaptic excitation and inhibition. Mutations in HEPACAM cause megalencephalic leukoencephalopathy with subcortical cysts in humans. Therefore, our findings suggest that disruption of astrocyte self-organization mechanisms could be an underlying cause of neural pathology.

Cell adhesion molecules regulating astrocyte-neuron interactions.

A tripartite synapse comprises a neuronal presynaptic axon and a postsynaptic dendrite, which are closely ensheathed by a perisynaptic astrocyte process. Through their structural and functional association with thousands of neuronal synapses, astrocytes regulate synapse formation and function. Recent work revealed a diverse range of cell adhesion-based mechanisms that mediate astrocyte-synapse interactions at tripartite synapses. Here, we will review some of these findings unveiling a highly dynamic bidirectional signaling between astrocytes and synapses, which orchestrates astrocyte morphological maturation and synapse development. Moreover, we will discuss the roles of these newly discovered molecular pathways in brain physiology and function both in health and disease.

Role of astrocytes in synapse formation and maturation.

Astrocytes are the most abundant glial cells in the mammalian brain and directly participate in the proper functioning of the nervous system by regulating ion homeostasis, controlling glutamate reuptake, and maintaining the blood-brain barrier. In the last two decades, a growing body of work also identified critical roles for astrocytes in regulating synaptic connectivity. Stemming from the observation that functional and morphological development of astrocytes occur concurrently with synapse formation and maturation, these studies revealed that both developmental processes are directly linked. In fact, astrocytes both physically contact numerous synaptic structures and actively instruct many aspects of synaptic development and function via a plethora of secreted and adhesion-based molecular signals. The complex astrocyte-to-neuron signaling modalities control different stages of synaptic development such as regulating the initial formation of structural synapses as well as their functional maturation. Furthermore, the synapse-modulating functions of astrocytes are evolutionarily conserved and contribute to the development and plasticity of diverse classes of synapses and circuits throughout the central nervous system. Importantly, because impaired synapse formation and function is a hallmark of many neurodevelopmental disorders, deficits in astrocytes are likely to be major contributors to disease pathogenesis. In this chapter, we review our current understanding of the cellular and molecular mechanisms by which astrocytes contribute to synapse development and discuss the bidirectional secretion-based and contact-mediated mechanisms responsible for these essential developmental processes.

Acute post-injury blockade of α2δ-1 calcium channel subunits prevents pathological autonomic plasticity after spinal cord injury.

After spinal cord injury (SCI), normally innocuous visceral or somatic stimuli can trigger uncontrolled reflex activation of sympathetic circuitry, causing pathological dysautonomia. We show that remarkable structural remodeling and plasticity occur within spinal autonomic circuitry, creating abnormal sympathetic reflexes that promote dysautonomia. However, when mice are treated early after SCI with human-equivalent doses of the US Food and Drug Administration (FDA)-approved drug gabapentin (GBP), it is possible to block multi-segmental excitatory synaptogenesis and abolish sprouting of autonomic neurons that innervate immune organs and sensory afferents that trigger pain and autonomic dysreflexia (AD). This "prophylactic GBP" regimen decreases the frequency and severity of AD and protects against SCI-induced immune suppression. These benefits persist even 1 month after stopping treatment. GBP could be repurposed to prevent dysautonomia in at-risk individuals with high-level SCI.