The role of astrocyte-secreted matricellular proteins in central nervous system development and function

Matricellular proteins, such as thrombospondins (TSPs1-4), SPARC, SPARC-like1 (hevin) and tenascin C are expressed by astrocytes in the central nervous system (CNS) of rodents. The spatial and temporal expression patterns of these proteins suggest that they may be involved in important developmental processes such as cell proliferation and maturation, cell migration, axonal guidance and synapse formation. In addition, upon injury to the nervous system the expression of these proteins is upregulated, suggesting that they play a role in tissue remodeling and repair in the adult CNS. The genes encoding these proteins have been disrupted in mice. Interestingly, none of these proteins are required for survival, and furthermore, there are no evident abnormalities at the gross anatomical level in the CNS. However, detailed analyses of some of these mice in the recent years have revealed interesting CNS phenotypes. Here we will review the expression of these proteins in the CNS. We will discuss a newly described function for thrombospondins in synapse formation in the CNS in detail, and speculate whether other matricellular proteins could play similar roles in nervous system development and function

Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4.

Postnatal/adult neural stem cells (NSCs) within the rodent subventricular zone (SVZ; also called subependymal zone) generate doublecortin (Dcx)(+) neuroblasts that migrate and integrate into olfactory bulb circuitry. Continuous production of neuroblasts is controlled by the SVZ microenvironmental niche. It is generally thought that enhancing the neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear whether there are conditions that favour astrogenesis over neurogenesis in the SVZ niche, and whether astrocytes produced there have different properties compared with astrocytes produced elsewhere in the brain. Here we show in mice that SVZ-generated astrocytes express high levels of thrombospondin 4 (Thbs4), a secreted homopentameric glycoprotein, in contrast to cortical astrocytes, which express low levels of Thbs4. We found that localized photothrombotic/ischaemic cortical injury initiates a marked increase in Thbs4(hi) astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-creER(tm)4 lineage tracing demonstrated that it is these SVZ-generated Thbs4(hi) astrocytes, and not Dcx(+) neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production. Consequently, Thbs4 homozygous knockout mice (Thbs4(KO/KO)) showed severe defects in cortical-injury-induced SVZ astrogenesis, instead producing cells expressing Dcx migrating from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular haemorrhage into the brain parenchyma of Thbs4(KO/KO) mice. Taken together, these findings have important implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members have important roles

Circuit-Selective Striatal Synaptic Dysfunction in the Sapap3 Knockout Mouse Model of Obsessive-Compulsive Disorder

Background: Synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3) is an excitatory postsynaptic protein implicated in the pathogenesis of obsessive-compulsive behaviors. In mice, genetic deletion of Sapap3 causes obsessive-compulsive disorder (OCD)-like behaviors that are rescued by striatal expression of Sapap3, demonstrating the importance of striatal neurotransmission for the OCD-like behaviors. In the striatum, there are two main excitatory synaptic circuits, corticostriatal and thalamostriatal. Neurotransmission defects in either or both of these circuits could potentially contribute to the OCD-like behaviors of Sapap3 knockout (KO) mice. Previously, we reported that Sapap3 deletion reduces corticostriatal alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-type glutamate receptor-mediated synaptic transmission. Methods: Whole-cell electrophysiological recording techniques in acute brain slices were used to measure synaptic transmission in the corticostriatal and thalamostriatal circuits of Sapap3 KO mice and littermate control animals. Transgenic fluorescent reporters identified striatopallidal and striatonigral projection neurons. SAPAP isoforms at corticostriatal and thalamostriatal synapses were detected using immunostaining techniques. Results: I n contrast to corticostriatal synapses, thalamostriatal synaptic activity is unaffected by Sapap3 deletion. At the molecular level, we find that another SAPAP family member, SAPAP4, is present at thalamostriatal, but not corticostriatal, synapses. This finding provides a molecular rationale for the functional divergence we observe between thalamic and cortical striatal circuits in Sapap3 KO mice. Conclusions: These findings define the circuit-level neurotransmission defects in a genetic mouse model for OCD-related behaviors, focusing attention on the corticostriatal circuit for mediating the behavioral abnormalities. Our results also provide the first evidence that SAPAP isoforms may be localized to synapses according to circuit-selective principles.National Institute of Mental Health (U.S.) (Grant MH081201

Thrombospondin receptor α2δ-1 promotes synaptogenesis and spinogenesis via postsynaptic Rac1

Astrocytes control excitatory synaptogenesis by secreting thrombospondins (TSPs), which function via their neuronal receptor, the calcium channel subunit α2δ-1. α2δ-1 is a drug target for epilepsy and neuropathic pain; thus the TSP–α2δ-1 interaction is implicated in both synaptic development and disease pathogenesis. However, the mechanism by which this interaction promotes synaptogenesis and the requirement for α2δ-1 for connectivity of the developing mammalian brain are unknown. In this study, we show that global or cell-specific loss of α2δ-1 yields profound deficits in excitatory synapse numbers, ultrastructure, and activity and severely stunts spinogenesis in the mouse cortex. Postsynaptic but not presynaptic α2δ-1 is required and sufficient for TSP-induced synaptogenesis in vitro and spine formation in vivo, but an α2δ-1 mutant linked to autism cannot rescue these synaptogenesis defects. Finally, we reveal that TSP–α2δ-1 interactions control synaptogenesis postsynaptically via Rac1, suggesting potential molecular mechanisms that underlie both synaptic development and pathology

Astrocytes refine cortical connectivity at dendritic spines

During cortical synaptic development, thalamic axons must establish synaptic connections despite the presence of the more abundant intracortical projections. How thalamocortical synapses are formed and maintained in this competitive environment is unknown. Here, we show that astrocyte-secreted protein hevin is required for normal thalamocortical synaptic connectivity in the mouse cortex. Absence of hevin results in a profound, long-lasting reduction in thalamocortical synapses accompanied by a transient increase in intracortical excitatory connections. Three-dimensional reconstructions of cortical neurons from serial section electron microscopy (ssEM) revealed that, during early postnatal development, dendritic spines often receive multiple excitatory inputs. Immuno-EM and confocal analyses revealed that majority of the spines with multiple excitatory contacts (SMECs) receive simultaneous thalamic and cortical inputs. Proportion of SMECs diminishes as the brain develops, but SMECs remain abundant in Hevin-null mice. These findings reveal that, through secretion of hevin, astrocytes control an important developmental synaptic refinement process at dendritic spines. DOI: http://dx.doi.org/10.7554/eLife.04047.00

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

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

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