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1.
Exp Neurol ; 370: 114548, 2023 12.
Article in English | MEDLINE | ID: mdl-37769794

ABSTRACT

Chordin-like 1 (Chrdl1) is an astrocyte-secreted protein that regulates synaptic maturation, and limits plasticity via GluA2-containing AMPA receptors (AMPARs). It was demonstrated that Chrdl1 expression is very heterogeneous throughout the brain, and it is enriched in astrocytes in cortical layers 2/3, with peak expression in the visual cortex at postnatal day 14. In response to ischemic stroke, Chrdl1 is upregulated during the acute and sub-acute phases in the peri-infarct region, potentially hindering recovery after stroke. Here, we used photothrombosis to model ischemic stroke in the motor cortex of adult male and female mice. In this study, we demonstrate that elimination of Chrdl1 in a global knock-out mouse reduces apoptotic cell death at early post-stroke stages and prevents ischemia-driven synaptic loss of AMPA receptors at later time points, all contributing to faster motor recovery. This suggests that synapse-regulating astrocyte-secreted proteins such as Chrdl1 have therapeutic potential to aid functional recovery after an ischemic injury.


Subject(s)
Ischemic Stroke , Stroke , Mice , Male , Female , Animals , Receptors, AMPA/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Eye Proteins/metabolism , Nerve Tissue Proteins/metabolism
2.
Methods Mol Biol ; 2616: 29-38, 2023.
Article in English | MEDLINE | ID: mdl-36715925

ABSTRACT

Photothrombosis is one of the techniques available to reproduce ischemic injuries in animal models. Most of the studies that use photothrombosis resort to this technique as it is highly reproducible and minimally invasive to target cortical brain regions, such as the motor or somatosensory areas. However, this technique can be modified and adapted to virtually target any brain region, including deeper tissue. Here, we describe some variations on the traditional protocol to use the photothrombotic technique to target the longitudinal hippocampal vein in the adult mouse and cause an ischemic injury in the hippocampus.


Subject(s)
Brain Ischemia , Stroke , Mice , Animals , Brain , Brain Ischemia/etiology , Hippocampus , Parietal Lobe , Infarction/complications , Disease Models, Animal , Stroke/etiology
3.
Sci Rep ; 12(1): 4176, 2022 03 09.
Article in English | MEDLINE | ID: mdl-35264691

ABSTRACT

Ischemic injury occurs when the brain is deprived of blood flow, preventing cells from receiving essential nutrients. The injury core is the brain region directly deprived and is surrounded by the peri-infarct area, the region with recovery potential. In the peri-infarct area neurons undergo acute loss of dendritic spines, which modifies synaptic plasticity and determines neuronal survival. Astrocytes can be protective or detrimental to the ischemic injury response depending on the specific stage, yet we lack clear understanding of the underlying mechanisms. Chordin-like 1 (Chrdl1) is an astrocyte-secreted protein that promotes synaptic maturation and limits experience-dependent plasticity in the mouse visual cortex. Given this plasticity-limiting function we asked if Chrdl1 regulates the response to ischemic injury, modelled using photothrombosis (PT). We find that Chrdl1 mRNA is upregulated in astrocytes in the peri-infarct area in both acute and sub-acute phases post-PT. To determine the impact of increased Chrdl1 on the response to PT we analyzed Chrdl1 knock-out mice. We find that absence of Chrdl1 prevents ischemia-induced spine loss in the peri-infarct area and reduces cell death in the core, without impacting gliosis. These findings highlight the important role of astrocyte-secreted proteins in regulating structural plasticity in response to brain ischemic injuries.


Subject(s)
Brain Injuries , Brain Ischemia , Animals , Astrocytes/metabolism , Brain Injuries/metabolism , Brain Ischemia/metabolism , Eye Proteins/metabolism , Glycoproteins , Infarction , Intercellular Signaling Peptides and Proteins , Ischemia/metabolism , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism
4.
Elife ; 102021 09 08.
Article in English | MEDLINE | ID: mdl-34494546

ABSTRACT

Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders.


Subject(s)
Astrocytes/metabolism , Glutamic Acid/metabolism , Neurodevelopmental Disorders/metabolism , Synapses/metabolism , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Neurodevelopmental Disorders/genetics , Neurons/metabolism , Presynaptic Terminals/metabolism , Rats , Rats, Sprague-Dawley , Synapses/genetics , Visual Cortex/growth & development , Visual Cortex/metabolism
5.
Neuron ; 100(5): 1116-1132.e13, 2018 12 05.
Article in English | MEDLINE | ID: mdl-30344043

ABSTRACT

In the developing brain, immature synapses contain calcium-permeable AMPA glutamate receptors (AMPARs) that are subsequently replaced with GluA2-containing calcium-impermeable AMPARs as synapses stabilize and mature. Here, we show that this essential switch in AMPARs and neuronal synapse maturation is regulated by astrocytes. Using biochemical fractionation of astrocyte-secreted proteins and mass spectrometry, we identified that astrocyte-secreted chordin-like 1 (Chrdl1) is necessary and sufficient to induce mature GluA2-containing synapses to form. This function of Chrdl1 is independent of its role as an antagonist of bone morphogenetic proteins (BMPs). Chrdl1 expression is restricted to cortical astrocytes in vivo, peaking at the time of the AMPAR switch. Chrdl1 knockout (KO) mice display reduced synaptic GluA2 AMPARs, altered kinetics of synaptic events, and enhanced remodeling in an in vivo plasticity assay. Studies have shown that humans with mutations in Chrdl1 display enhanced learning. Thus astrocytes, via the release of Chrdl1, promote GluA2-dependent synapse maturation and thereby limit synaptic plasticity.


Subject(s)
Astrocytes/metabolism , Eye Proteins/physiology , Nerve Tissue Proteins/physiology , Neuronal Plasticity , Receptors, AMPA/metabolism , Synapses/physiology , Animals , Cells, Cultured , Eye Proteins/metabolism , Female , Male , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/metabolism , Rats, Sprague-Dawley
6.
Adv Mater ; 30(12): e1706785, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29363828

ABSTRACT

Oriented composite nanofibers consisting of porous silicon nanoparticles (pSiNPs) embedded in a polycaprolactone or poly(lactide-co-glycolide) matrix are prepared by spray nebulization from chloroform solutions using an airbrush. The nanofibers can be oriented by an appropriate positioning of the airbrush nozzle, and they can direct growth of neurites from rat dorsal root ganglion neurons. When loaded with the model protein lysozyme, the pSiNPs allow the generation of nanofiber scaffolds that carry and deliver the protein under physiologic conditions (phosphate-buffered saline (PBS), at 37 °C) for up to 60 d, retaining 75% of the enzymatic activity over this time period. The mass loading of protein in the pSiNPs is 36%, and in the resulting polymer/pSiNP scaffolds it is 3.6%. The use of pSiNPs that display intrinsic photoluminescence (from the quantum-confined Si nanostructure) allows the polymer/pSiNP composites to be definitively identified and tracked by time-gated photoluminescence imaging. The remarkable ability of the pSiNPs to protect the protein payload from denaturation, both during processing and for the duration of the long-term aqueous release study, establishes a model for the generation of biodegradable nanofiber scaffolds that can load and deliver sensitive biologics.


Subject(s)
Nanofibers , Animals , Nanoparticles , Polymers , Porosity , Rats , Silicon , Tissue Engineering , Tissue Scaffolds
7.
J Physiol ; 595(6): 1903-1916, 2017 03 15.
Article in English | MEDLINE | ID: mdl-27381164

ABSTRACT

Astrocytes comprise half of the cells in the brain. Although astrocytes have traditionally been described as playing a supportive role for neurons, they have recently been recognized as active participants in the development and plasticity of dendritic spines and synapses. Astrocytes can eliminate dendritic spines, induce synapse formation, and regulate neurotransmission and plasticity. Dendritic spine and synapse impairments are features of many neurological disorders, including autism spectrum disorder, schizophrenia, and Alzheimer's disease. In this review we will present evidence from multiple neurological disorders demonstrating that changes in astrocyte-synapse interaction contribute to the pathologies. Genomic analysis has connected altered astrocytic gene expression with synaptic deficits in a number of neurological disorders. Alterations in astrocyte-secreted factors have been implicated in the neuronal morphology and synaptic changes seen in neurodevelopmental disorders, while alteration in astrocytic glutamate uptake is a core feature of multiple neurodegenerative disorders. This evidence clearly demonstrates that maintaining astrocyte-synapse interaction is crucial for normal central nervous system functioning. Obtaining a better understanding of the role of astrocytes at synapses in health and disease will provide a new avenue for future therapeutic targeting.


Subject(s)
Astrocytes/physiology , Nervous System Diseases/physiopathology , Neurodevelopmental Disorders/physiopathology , Synapses/physiology , Animals , Dendritic Spines/physiology , Humans
8.
J Cereb Blood Flow Metab ; 34(12): 1898-906, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25248834

ABSTRACT

Distinct neuronal populations show differential sensitivity to global ischemia, with hippocampal CA1 neurons showing greater vulnerability compared to cortical neurons. The mechanisms that underlie differential vulnerability are unclear, and we hypothesize that intrinsic differences in neuronal cell biology are involved. Dendritic spine morphology changes in response to ischemic insults in vivo, but cell type-specific differences and the molecular mechanisms leading to such morphologic changes are unexplored. To directly compare changes in spine size in response to oxygen/glucose deprivation (OGD) in cortical and hippocampal neurons, we used separate and equivalent cultures of each cell type. We show that cortical neurons exhibit significantly greater spine shrinkage compared to hippocampal neurons. Rac1 is a Rho-family GTPase that regulates the actin cytoskeleton and is involved in spine dynamics. We show that Rac1 and the Rac guanine nucleotide exchange factor (GEF) Tiam1 are differentially activated by OGD in hippocampal and cortical neurons. Hippocampal neurons express more Tiam1 than cortical neurons, and reducing Tiam1 expression in hippocampal neurons by shRNA enhances OGD-induced spine shrinkage. Tiam1 knockdown also reduces hippocampal neuronal vulnerability to OGD. This work defines fundamental differences in signalling pathways that regulate spine morphology in distinct neuronal populations that may have a role in the differential vulnerability to ischemia.


Subject(s)
Cerebral Cortex/metabolism , Dendritic Spines/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Hippocampus/metabolism , Ischemia/metabolism , Neoplasm Proteins/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Blood Glucose/metabolism , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Death/physiology , Cerebral Cortex/pathology , Dendritic Spines/pathology , Female , Guanine Nucleotide Exchange Factors/genetics , Hippocampus/pathology , Ischemia/pathology , Male , Neoplasm Proteins/genetics , Neurons/metabolism , Neurons/ultrastructure , Oxygen/metabolism , Pregnancy , Rats, Wistar , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , rac1 GTP-Binding Protein/genetics
9.
J Biol Chem ; 289(8): 4644-51, 2014 Feb 21.
Article in English | MEDLINE | ID: mdl-24403083

ABSTRACT

Brain ischemia occurs when the blood supply to the brain is interrupted, leading to oxygen and glucose deprivation (OGD). This triggers a cascade of events causing a synaptic accumulation of glutamate. Excessive activation of glutamate receptors results in excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts. The mechanisms that underlie this difference are unclear. Cultured hippocampal neurons respond to OGD with a rapid internalization of AMPA receptor (AMPAR) subunit GluA2, resulting in a switch from GluA2-containing Ca(2+)-impermeable receptors to GluA2-lacking Ca(2+)-permeable subtypes (CP-AMPARs). GluA2 internalization is a critical component of OGD-induced cell death in hippocampal neurons. It is unknown how AMPAR trafficking is affected in cortical neurons following OGD. Here, we show that cultured cortical neurons are resistant to an OGD insult that causes cell death in hippocampal neurons. GluA1 is inserted at the plasma membrane in both cortical and hippocampal neurons in response to OGD. In contrast, OGD causes a rapid endocytosis of GluA2 in hippocampal neurons, which is absent in cortical neurons. These data demonstrate that populations of neurons with different vulnerabilities to OGD recruit distinct cell biological mechanisms in response to insult, and that a crucial aspect of the mechanism leading to OGD-induced cell death is absent in cortical neurons. This strongly suggests that the absence of OGD-induced GluA2 trafficking contributes to the relatively low vulnerability of cortical neurons to ischemia.


Subject(s)
Glucose/deficiency , Hippocampus/pathology , Neurons/metabolism , Oxygen/pharmacology , Protein Subunits/metabolism , Receptors, AMPA/metabolism , Cell Hypoxia/drug effects , Cell Membrane/metabolism , Cells, Cultured , Endocytosis/drug effects , L-Lactate Dehydrogenase/metabolism , Neurons/drug effects , Neurons/pathology , Protein Transport/drug effects , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism
10.
Neuron ; 79(2): 293-307, 2013 Jul 24.
Article in English | MEDLINE | ID: mdl-23889934

ABSTRACT

Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.


Subject(s)
ADP-Ribosylation Factor 1/physiology , Actin-Related Protein 2-3 Complex/physiology , Actins/metabolism , Carrier Proteins/physiology , Neuronal Plasticity/physiology , Nuclear Proteins/physiology , Synapses/metabolism , Actin-Related Protein 2-3 Complex/antagonists & inhibitors , Actins/physiology , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Cytoskeletal Proteins , HEK293 Cells , Humans , Organ Culture Techniques , Polymerization , Rats , Rats, Wistar
11.
J Cell Sci ; 126(Pt 17): 3873-83, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23843614

ABSTRACT

Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.


Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Astrocytes/metabolism , Carrier Proteins/metabolism , Central Nervous System/metabolism , Nuclear Proteins/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Actin-Related Protein 2-3 Complex/genetics , Amides/pharmacology , Animals , Astrocytes/cytology , Astrocytes/drug effects , Carrier Proteins/genetics , Cells, Cultured , Colforsin/pharmacology , Enzyme Inhibitors/pharmacology , Fibroblasts , HEK293 Cells , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Mice , Myosin Type II/antagonists & inhibitors , Myosin Type II/metabolism , Nuclear Proteins/genetics , Pyridines/pharmacology , RNA Interference , RNA, Small Interfering , Rats , Thiazoles/pharmacology , Thiones/pharmacology , Uracil/analogs & derivatives , Uracil/pharmacology , Vasodilator Agents/pharmacology , Wiskott-Aldrich Syndrome Protein, Neuronal/genetics , rhoA GTP-Binding Protein/antagonists & inhibitors , rhoA GTP-Binding Protein/metabolism
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