Astrocyte matricellular proteins that control excitatory synaptogenesis are regulated by inflammatory cytokines and correlate with paralysis severity during experimental autoimmune encephalomyelitis

Abstract

The matricellular proteins, secreted protein acidic and rich in cysteine (SPARC) and SPARC-like 1 (SPARCL1), are produced by astrocytes and control excitatory synaptogenesis in the central nervous system. While SPARCL1 directly promotes excitatory synapse formation in vitro and in the developing nervous system in vivo, SPARC specifically antagonizes the synaptogenic actions of SPARCL1. We hypothesized these proteins also help maintain existing excitatory synapses in adult hosts, and that local inflammation in the spinal cord alters their production in a way that dynamically modulates motor synapses and impacts the severity of paralysis during experimental autoimmune encephalomyelitis (EAE) in mice. Using a spontaneously remitting EAE model, paralysis severity correlated inversely with both expression of synaptic proteins and the number of synapses in direct contact with the perikarya of motor neurons in spinal gray matter. In both remitting and non-remitting EAE models, paralysis severity also correlated inversely with sparcl1:sparc transcript and SPARCL1:SPARC protein ratios directly in lumbar spinal cord tissue. In vitro, astrocyte production of both SPARCL1 and SPARC was regulated by T cell-derived cytokines, causing dynamic modulation of the SPARCL1:SPARC expression ratio. Taken together, these data support a model whereby proinflammatory cytokines inhibit SPARCL1 and/or augment SPARC expression by astrocytes in spinal gray matter that, in turn, cause either transient or sustained synaptic retraction from lumbar spinal motor neurons thereby regulating hind limb paralysis during EAE. Ongoing studies seek ways to alter this SPARCL1:SPARC expression ratio in favor of synapse reformation/maintenance and thus help to modulate neurologic deficits during times of inflammation. This could identify new astrocyte-targeted therapies for diseases such as multiple sclerosis.

Keywords: EAE; SPARC; SPARCL1; astrocytes; multiple sclerosis; synaptic plasticity.

Figure 1

Astrocytes are activated in both gray and white matter of the lumbar ventral spinal cord during relapsing EAE. (A) The clinical course of relapsing EAE induced by active PLP peptide immunization of SJL mice shows near complete resolution of paralysis over only a few days (n = 20). (B) Normalized spinal cord GFAP levels at defined stages of relapsing EAE shows evidence of astrocyte activation at disease onset (n = 5 samples per disease stage). (C) Representative immunohistochemical staining for GFAP expression in naïve SJL spinal cord shows expression in quiescent-appearing cells that predominate in white matter. Insert shows modest signal in ventral gray matter. (D) Representative GFAP staining of SJL spinal cord at peak EAE shows increased signal in both white and gray matter. Insert shows numerous activated GFAP+ astrocytes in ventral gray matter. ^*p < 0.05 compared to preclinical levels, Bar = 100 μm.

Figure 2

Expression of synaptic proteins in lumbar spinal gray matter is dynamically regulated during relapsing EAE. (A) Representative immunohistochemical staining for the presynaptic protein, synaptophysin, and the postsynaptic protein, MAP2, shows that both proteins localize to lumbar spinal gray matter at varying stages of relapsing EAE in SJL mice. Inserts show that synaptophysin labels gray matter neuropil while MAP2 labels neuronal cell bodies and proximal dendrites. (B) Normalized expression of both proteins shows reduced levels at peak disease, but notable recovery by the time of disease remission only a few days later (n = 5 samples per disease stage). Bar = 80 μm, ^*p < 0.05 compared to preclinical levels.

Figure 3

The number of synapses in direct contact with the proximal dendrites or cell bodies of lumbar ventral motor neurons changes over the course of relapsing EAE. (A) A representative electron micrographic image showing the proximal dendrite of a ventral motor neuron (green) with multiple postsynaptic densities (arrows) indicative of individual synapses (red), Bar = 500 nm. (B) Quantification of these synaptic contacts at different stages of relapsing EAE shows evidence of synaptic retraction from the perikarya of motor neurons (MN) at peak disease with full reestablishment of these structures around MN several days later in the setting of disease remission (n = 10 MN from each of 3 mice at each disease stage), ^*p < 0.05 naïve versus peak and peak versus remission. (C) A representative electron micrograph at peak disease shows many synapses (red, with arrows) displaced from a motor neuron cell body (green), Bar = 2 μm. (D) Another representative electron micrograph at higher magnification shows the physical separation of an axosomatic synapse (red) from the cell body of a ventral motor neuron (green), without any other cellular process interposed between the two structures, Bar = 500 nm.

Figure 4

Expression of both sparcl1 and sparc transcripts and SPARCL1 and SPARC proteins changes in lumbar spinal cord tissue over the course of relapsing EAE in SJL mice. (A) Relative sparcl1 mRNA expression fluxes over the course of relapsing EAE (n = 5 mice per disease stage). (B) Relative sparc mRNA expression also changes over the course of relapsing EAE (n = 5 mice per disease stage). (C) The calculated sparcl1 to sparc mRNA expression ratio shifts in favor of synapse inhibition at peak disease. (D) The same change in the SPARCL1 to SPARC protein concentration ratios is seen over the course of relapsing EAE (n = 5 mice per disease stage). ^*p < 0.05 compared to levels found in naïve spinal cord.

Figure 5

Expression of sparcl1 and sparc transcripts and SPARCL1 and SPARC proteins changes in lumbar spinal cord tissue over the course of non-relapsing EAE in C57BL/6 mice. (A,B) Representative immunofluorescence shows SPARCL1 expression co-localizes with GFAP staining in both white and gray matter of the spinal cord, Bar = 40 μm. (C) Relative sparcl1 mRNA expression fluxes over the course of non-relapsing EAE (n = 5 mice per disease stage). (D) Relative sparc mRNA expression does not change over the course of non-relapsing EAE (n = 5 mice per disease stage). (E) The calculated sparcl1 to sparc mRNA expression ratio shifts in favor of synapse inhibition at peak disease. (F) A similar change in the SPARCL1 to SPARC protein concentration ratios is seen over the course of non-relapsing EAE (n = 5 mice per disease stage). ^*p < 0.05 compared to levels found in naïve spinal cord.

Figure 6

T cell-derived cytokines regulate astrocyte production of SPARCL1 in a complex manner in vitro. (A) Astrocytes spontaneously secrete measurable amounts of SPARCL1 into culture supernatants (n = 4 experimental replicates per time point). (B) Interferon-gamma (IFN-γ) modestly increases astrocyte SPARCL1 release (n = 3 experimental replicates per time point), p = 0.003 comparing changes over time. (C) Interleukin (IL)-17 has no significant effect on astrocyte SPARCL1 production (n = 3 experimental replicates per time point). (D) IL-10 potently induces astrocyte SPARCL1 production (n = 3 experimental replicates per time point), p = 0.0003 comparing changes over time, p = 0.002 comparing fold change differences. (E) Granulocyte macrophage colony stimulating factor (GM-CSF) potently induces astrocyte SPARCL1 production (n = 3 experimental replicates per time point), p = 0.0024 comparing changes over time. (F) Tumor necrosis factor (TNF)-α suppresses astrocyte SPARCL1 production (n = 3 experimental replicates per time point), p = 0.0384 comparing fold change differences.

Figure 7

Some T cell-derived cytokines regulate astrocyte production of SPARC in vitro. (A) Astrocytes spontaneously secrete measurable amounts of SPARC into culture supernatants (n = 4 experimental replicates per time point). (B) TNF-α modestly induces astrocyte SPARC production (n = 3 experimental replicates per time point). (C) TNF-α suppresses the ratio of SPARCL1 to SPARC made by astrocytes over time, p < 0.0001 comparing ratio differences, p = 0.0002 comparing changes over time. (D) IL-10 augments the ratio of SPARCL1 to SPARC made by astrocytes over time, p < 0.0001 comparing ratio differences, p < 0.0001 comparing changes over time.