The oral antidiabetic pioglitazone protects from neurodegeneration and amyotrophic lateral sclerosis-like symptoms in superoxide dismutase-G93A transgenic mice

Abstract

Amyotrophic lateral sclerosis (ALS) represents a fatal neurodegenerative disorder characterized by progressive death of the upper and lower motor neurons. Because accompanying inflammation may interact with and promote neurodegeneration, anti-inflammatory treatment strategies are being evaluated. Because peroxisome proliferator-activated receptor gamma (PPARgamma) agonists act as potent anti-inflammatory drugs, we tested whether superoxide dismutase (SOD1)-G93A transgenic mice, a mouse model of ALS, benefit from oral treatment with the PPARgamma agonist pioglitazone (Pio). Pio-treated transgenic mice revealed improved muscle strength and body weight, exhibited a delayed disease onset, and survived significantly longer than nontreated SOD1-G93A mice. Quantification of motor neurons of the spinal cord at day 90 revealed complete neuroprotection by Pio, whereas nontreated SOD1-G93A mice had lost 30% of motor neurons. This was paralleled by preservation of the median fiber diameter of the quadriceps muscle, indicating not only morphological but also functional protection of motor neurons by Pio. Activated microglia were significantly reduced at sites of neurodegeneration in Pio-treated SOD1-G93A mice, as were the protein levels of cyclooxygenase 2 and inducible nitric oxide synthase. Interestingly, mRNA levels of the suppressor of cytokine signaling 1 and 3 genes were increased by Pio, whereas both the mRNA and protein levels of endogenous mouse SOD1 and of transgenic human SOD1 remained unaffected.

Figure 1.

Effects of pioglitazone treatment on survival, body weight, and motor functions in SOD1-G93A mice. a, Probability of survival in nontreated (SOD1; n = 10) compared with Pio-treated (SOD1-Pio; n = 7) mice. Data are expressed as mean ± SEM. b, Time course of changes in body weight of nontreated (wt; n = 8) and Pio-treated (wt-Pio; n = 13) wild-type mice compared with their respective SOD1 and SOD1-Pio mice. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 compared with SOD1-Pio. c, Probability of onset of motor dysfunctions in nontreated (SOD1) compared with Pio-treated (SOD1-Pio) mice. d, Time course of motor performance in the paw grip endurance test. ^***p < 0.001 compared with SOD1 mice.

Figure 2.

Effect of pioglitazone treatment on motor neuron degeneration and muscle fiber morphology. a, CGRP-positive motor neuron numbers in the lumbar spinal cord (n = 5 for wild-type group; n = 6 for SOD1 groups) and median fiber diameters in the quadriceps muscle (n = 3 for wt; n = 4 for wt-Pio; n = 4 for SOD1; n = 5 for SOD1-Pio) at day 90 of life. ^*p < 0.05 and ^**p < 0.01 compared with Pio-treated SOD1 mice. Error bars represent SEM (CGRP⁺ cells) or SD (median fiber diameter). b, Evaluation of the spinal cord motor neuron area by CGRP immunohistochemistry and muscle fiber morphology by histological HE stain.

Figure 3.

Effect of pioglitazone treatment on the activation of astroglia and inflammation markers. a, Immunofluorescence colabeling of CD11b (green) and CGRP (red) in the spinal cord motor neuron area at day 90 of life (top). Examples for the classification of microglia activation states by bright-field (blue/black reaction products) and green fluorescent CD11b immunohistochemistry (bottom left) are shown. Quantification of microglia at day 90 of life (bottom right; n = 4 for wt and wt-Pio; n = 5 for SOD1 and SOD1-Pio) and of COX-2 and iNOS Western blots (n = 4 for all groups) from spinal cord samples derived from 90-d-old mice (b) are shown. ^**p < 0.01 and ^***p < 0.001 compared with the respective nontreated group. c, Quantification of SOCS-1 and SOCS-3 mRNA levels in the spinal cord at day 90 of life (n = 5 for all groups). Representative gels of two animals per group are shown. ^***p < 0.001 compared with nontreated SOD1 mice. Error bars represent SEM

Figure 4.

Effect of pioglitazone treatment on the expression levels of endogenous and transgenic SOD1. a-d, Quantification of mouse SOD1 (a) and human SOD1 (b) mRNA expression levels, and of mouse SOD1 (c) and human SOD1 (d) protein levels in the spinal cord at day 90 of life (n = 4 for all groups). Note the variations in mouse SOD1 expression levels between the groups in a and c. All data are represented as mean ± SEM.