AAV2/9-mediated overexpression of MIF inhibits SOD1 misfolding, delays disease onset, and extends survival in mouse models of ALS

Abstract

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the loss of upper and lower motor neurons. Transgenic mice that overexpress mutant SOD1 develop paralysis and accumulate misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit mutant SOD1 misfolding and binding to intracellular membranes. In addition, complete elimination of endogenous MIF accelerated disease onset and late disease progression, as well as shortened the lifespan of mutant SOD1 mice with higher amounts of misfolded SOD1 detected within the spinal cord. Based on these findings, we used adeno-associated viral (AAV) vectors to overexpress MIF in the spinal cord of mutant SOD1^G93A and loxSOD1^G37R mice. Our data show that MIF mRNA and protein levels were increased in the spinal cords of AAV2/9-MIF-injected mice. Furthermore, mutant SOD1^G93A and loxSOD1^G37R mice injected with AAV2/9-MIF demonstrated a significant delay in disease onset and prolonged survival compared with their AAV2/9-GFP-injected or noninjected littermates. Moreover, these mice accumulated reduced amounts of misfolded SOD1 in their spinal cords, with no observed effect on glial overactivation as a result of MIF up-regulation. Our findings indicate that MIF plays a significant role in SOD1 folding and misfolding mechanisms and strengthen the hypothesis that MIF acts as a chaperone for misfolded SOD1 in vivo and may have further implications regarding the therapeutic potential role of up-regulation of MIF in modulating the specific accumulation of misfolded SOD1.

Keywords: AAV; ALS; MIF; misfolded SOD1; mutant SOD1.

Fig. 1.

AAV2/9 vector shows efficient transduction of MIF protein in vitro and in vivo. (A) AAV2/9-MIF and AAV2/9-GFP vector design is shown. (B) Virion particles (AAV2/9-MIF and AAV2/9-GFP) were evaluated for infectivity by immunoblot in primary motor neurons using anti-MIF and anti-EGFP antibodies. Anti-p75 was used as a motor neuron marker and anti-actin as a loading control. (C) Confocal imaging of immunocytochemistry analysis of hippocampal neurons infected with AAV2/9-GFP (green) and AAV2/9-MIF (red). DAPI in blue was used for nuclear staining. (D) Confocal imaging of immunohistochemistry analysis of lumbar spinal cord sections of 1-mo-old mice injected at P1 with AAV2/9-GFP (green). DAPI in blue was used for nuclear staining. (Scale bar: 50 μm.) (E) Two microliters of AAV2/9-MIF virions were intraspinally injected into P1 mice that were killed 1 or 2 mo postinjection to determine the efficiency and long-lasting effects of AAV2/9-MIF expression. Immunoblot analysis was performed using anti-MIF antibody and ponceau staining is shown as a loading control. (Scale bar: 200 μm.) (F and G) RNA isolated from the spinal cords of AAV2/9-GFP (red) or AAV2/9-MIF (blue) injected SOD1^G93A (F) or loxSOD1^G37R (G) mice was used to analyze MIF levels by quantitative reverse-transcriptase PCR. RNA collected from AAV2/9-MIF–injected animals had an increase of 6 and 2.5 folds in MIF RNA levels in SOD1^G93A and loxSOD1^G37R, respectively, at the end stage. The bar graph represents mean ± SEM. P value was determined by Student’s t test. ***P < 0.001, *P < 0.05.

Fig. 2.

Overexpression of MIF in the spinal cord delays disease onset and extends the survival of SOD1^G93A mice. SOD1^G93A mice received a single intraspinal injection of AAV2/9-MIF at P1 (n = 15, blue) or AAV2/9-GFP (n = 9, red). Treated mice were monitored up to end stage. AAV2/9-MIF injection into P1 SOD1^G93A mice significantly delayed median disease onset (A and C) compared with AAV2/9-GFP–injected mice (AAV2/9-GFP: 112 d; AAV2/9-MIF: 125 d; P < 0.05) and extended median disease progression (AAV2/9-GFP: 36 d; AAV2/9-MIF: 53 d; P < 0.01) (D) and median survival (B and E) (AAV2/9-GFP: 148 d; AAV2/9-MIF: 178 d; P < 0.01). Mean age of disease onset was determined as the time when mice reached peak body weight. Disease progression was defined as the time from onset to end stage. Disease end stage was determined as the time when the mouse could not right itself within 20 s when placed on its side. At each time point, P value was determined by t test. Error bars denote SEM. **P < 0.01; *P < 0.05.

Fig. 3.

Overexpression of MIF in the spinal cord suppresses the accumulation of misfolded SOD1 in the SOD1^G93A mouse model of ALS. Representative micrographs of lumbar spinal cord sections from AAV2/9-GFP–treated SOD1^G93A mice (A–D) and AAV2/9-MIF–treated SOD1^G93A mice (E–H), processed for immunofluorescence by using the B8H10 antibody, which detects misfolded SOD1 (A and E), anti-MIF (B and F), and anti-NeuN antibody, which detects neuronal cells (C and G). Overexpression of MIF inhibits the accumulation of misfolded SOD1 species as detected by B8H10 antibody. (Scale bar: 25 μm.) (I and J) Quantification of the relative fluorescence intensity of the B8H10 (I) or anti-MIF (J) staining of lumbar spinal cord from SOD1^G93A mice injected with AAV2/9-GFP (red) or AAV2/9-MIF (blue). About 30 to 35 different areas from different mice of each treatment were analyzed. The bar graph represents mean ± SEM. P value was determine by Student’s t test. **P < 0.01; *P < 0.05.

Fig. 4.

Overexpression of MIF in the spinal cord does not affect the overactivation of astrocytes and microglia in SOD1^G93A mice. Representative micrographs of lumbar spinal cord sections from AAV2/9-GFP–treated SOD1^G93A mice (A–C and G–I) and AAV2/9-MIF–treated SOD1^G93A mice (D–F and J–L), processed for immunofluorescence by using the anti-MIF antibody (A, D, G, and J) anti-Iba1 antibody, which detects activated microglia (B and E) and anti-GFAP, which detects activated astrocytes (H and K). No significant difference was observed in microglia (A–F) or astrocyte (G–L) activation following the overexpression of MIF in the spinal cord. (Scale bar: 25 μm.)

Fig. 5.

Overexpression of MIF in the spinal cord delays disease onset and extends the survival of loxSOD1^G37R mice. loxSOD1^G37R mice received a single intraspinal injection of AAV2/9-MIF at P1 (n = 8, blue). Treated mice were monitored up to end stage and compared with AAV2/9-GFP–injected (n = 5, green) or noninjected mice (n = 20, red). AAV2/9-MIF injection into P1 SOD1^G37R mice significantly delayed median disease onset (A and C) compared with AAV2/9-GFP–injected or to noninjected mice (AAV2/9-GFP: 220 d; noninjected: 235 d; AAV2/9-MIF: 253 d; P < 0.05) and extended median survival (B and D) (AAV2/9-GFP: 384 d; noninjected: 389 d; AAV2/9-MIF: 411 d; P < 0.01). Mean age of disease onset was determined as the time when mice reached peak body weight. Disease end stage was determined as the time when the mouse could not right itself within 20 s when placed on its side. At each time point, P value was determined by t test. Error bars denote SEM. **P < 0.01; *P < 0.05; NS, no significance.