Immunomodulatory therapy with glatiramer acetate reduces endoplasmic reticulum stress and mitochondrial dysfunction in experimental autoimmune encephalomyelitis

Abstract

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are found in lesions of multiple sclerosis (MS) and animal models of MS such as experimental autoimmune encephalomyelitis (EAE), and may contribute to the neuronal loss that underlies permanent impairment. We investigated whether glatiramer acetate (GA) can reduce these changes in the spinal cords of chronic EAE mice by using routine histology, immunostaining, and electron microscopy. EAE spinal cord tissue exhibited increased inflammation, demyelination, mitochondrial dysfunction, ER stress, downregulation of NAD+ dependent pathways, and increased neuronal death. GA reversed these pathological changes, suggesting that immunomodulating therapy can indirectly induce neuroprotective effects in the CNS by mediating ER stress.

Figure 1

Glatiramer Acetate treatment improves clinical score, reduces demyelination, and suppresses inflammation in EAE mice. (1A) Graph of mean EAE scores for both groups of mice over a 30-day period. (1A1) The clinical severity of EAE disease was reduced by GA treatment. (1A2) Mean score for all animals in each group during the disease phase (days 10–30). The mean score was significantly lower in the GA treated EAE (EAE + GA) mice (P < 0.0001) compared to untreated EAE mice. N = 20/group, t-test. (B) Demyelination in EAE was reduced by GA treatment. Demyelination was assessed on LFB-stained (1B1–1B3) and MBP stained (1B5–1B7) sections of spinal cord white matter. Original magnification was × 400. B4 and B8 are graphs of the number of quadrants with demyelination expressed as a percentage of the total number of quadrants examined (n = 4), with statistics based on the t-test. B9–B11 shows transmission electron microscographs (TEM) at 6500 × magnification in normal mouse (1B9) untreated EAE mice (1B10) and EAE with GA treatment (1B11). These images are representative of at least 3 grids from each of five mice in each group. (1C) Inflammation in EAE was reduced by GA treatment. Inflammation was assessed in spinal cord white matter by (1C1–1C4) cells staining positively for antibodies to IL-17 and (1C5–1C8) cells staining positively for IFNγ. Original magnification was × 400. Bar Graphs show cell counts and statistical comparisons based on one-way ANOVA. Hematoxylin and eosin (H&E) stained sections show infiltration of mononuclear cells in white matter of EAE (1C10) and EAE + GA (1C11) mice. However, the number of inflammatory pockets and inflammatory cells is fewer in EAE + GA compared to EAE. Normal (1C9) mice show no inflammatory infiltrates. (1C12) The number of positive quadrants with inflammation was scored and expressed as a percentage of the total number of quadrants (H&E; n = 4).

Figure 2

Endoplasmic reticulum stress seen in EAE was reduced by GA treatment. Endoplasmic reticulum stress was assessed with antibodies to CHOP (A1–A4) and PERK (B1–B4). Panels 1–3 show representative staining patterns in each treatment group for each antibody. Original magnification: X400. Panel 4 in each case shows a graph of the mean number of positive cells per field for each treatment group (CHOP: n = 4; PERK: n = 4) with intergroup comparisons based on a one-way ANOVA. Transmission electron microscopic examination (C1–C3) showed vesiculated endoplasmic reticulum, irregularly arranged and disrupted endoplasmic reticulum (arrows) in untreated EAE (C2) while regular and parallel organized endoplasmic reticulum in normal (C1) and GA treated EAE mice (C3).

Figure 3

Glatiramer Acetate treatment improves mitochondrial function, fission/fusion, and biogenesis in EAE mice. (A,B) EAE caused loss of mitochondrial integrity that was reduced by GA treatment. Mitochondrial integrity was assessed by staining for PINK-1 (A1–A4) and staining for PARKIN (B1–B4). Panels 1–3 show representative staining patterns in each treatment group for each antibody. Original magnification: X400. Panel 4 in each case shows a graph of the mean number of positive cells per field for each treatment group (n = 4) with intergroup comparisons based on a one-way ANOVA. (C, D) Changes in mitochondrial biogenesis and morphology in EAE are reduced by GA treatment. Mitochondrial biogenesis was assessed with antibody to PGC1-α, a regulator of mitochondrial biogenesis (C1–C4) Panels 1–3 show representative staining patterns in each treatment group for each antibody. Original magnification: X400. Panel 4 shows a graph of the mean number of positive cells per field for each treatment group (n = 3; n = 4) with intergroup comparisons based on a one-way ANOVA. Mitochondrial morphology was assessed by transmission electron microscopy (TEM) at 6500 × magnification. Morphological changes were observed in mitochondria in EAE (D2) that were reduced by treatment (D3) compared to the mitochondria in normal mice (D1). In EAE many of the mitochondria showed mitochondrial membrane disruption with disruption or loss of cristae and changes in mitochondrial size and shape with increased fission. The results shown are typical of at least 3 grids for each mouse (n = 5). (E–G) EAE caused changes in mitochondrial dynamics that were reduced by GA treatment. Mitochondrial fusion was assessed by staining with antibody to Mitofusin–2 (MFN-2) (E1–E4) and mitochondrial fission was assessed with antibody to FIS-1 (F1–F4) and DNM1-L (G1–G4). Panels 1 to 3 show representative staining patters in each treatment group with each antibody. Original magnification was × 400. Panel 4 in each case shows a graph of the mean number of positive cells per field for each treatment group (MFN-2, n = 4; FIS-1, n = 3; DNM1-L, n = 3) with intergroup comparisons based on a one-way ANOVA. (E5) Western Blot anasupplemental filelysis of mitochondrial fraction of spinal cord shows an increase in MFN-2 in the EAE + GA mice compared to EAE and Normal. (G5) Western Blot analysis of mitochondrial fraction of spinal cord shows a decrease in DNM1-L in the EAE + GA mice compared to EAE and Normal. Full length membranes of the blots for MNF-2 and DNM1-L are provided as a . Full length membranes of the blots for B-actin are unable to be provided as the team only saved the files of the cropped images.

Figure 4

Changes in the mitochondria associated membrane (MAM) associated with EAE were reduced by GA treatment. Antibodies to PACS2 (A1–A4) and VDAC-1 (B1–B4) were studied as markers of the MAM. (A1–A3 and B1–B3) show the antibody staining in each treatment group with each antibody at an original magnification of × 400. (A4 and B4) show quantitation of staining in each treatment group with comparisons by one-way ANOVA (n = 5).

Figure 5

EAE associated reductions in NAD-dependent pathways were reduced by GA treatment. NAD+ dependent pathways were assessed with antibodies to NAMPT (A1–A4), Sirt-1 (B1–B4), and Sirt-3 (C1–C4). For each antibody, panels 1–3 show typical staining in each treatment group at an original magnification of × 400 and panel 4 is a graph of the quantification of positive cells in the spinal cord with comparisons made using one-way ANOVA (n = 4).

Figure 6

EAE related increases in apoptosis were reduced by GA treatment. Apoptosis was assessed in spinal cord tissue with in situ TUNEL staining (A1–A4) and with antibodies to FOXO-1 (B1–B4), BAX (C1–C4), and Cytochrome C (D1–D4). Panels 1–3 show typical staining patterns for each treatment group at an original magnification of × 400 while panels 4 provide quantitation of numbers of positive cells in each group (n = 4) with comparisons using one-way ANOVA.

Figure 7

Proposed neuroprotective mechanism underlying glatiramer acetate treatment in EAE. GA induces a shift towards the Th2 response in the CNS. This reduced inflammatory environment contributes to controlling the synergistic ER stress response and mitochondrial dysfunction. By doing so, apoptotic activity in the CNS is downregulated, and in turn, disease progression is slowed. CNS AG: CNS antigen; MHC: major histocompatibility complex; TCR: T cell receptor.