Exendin-4 Ameliorates Motor Neuron Degeneration in Cellular and Animal Models of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS

Ex-4 is neuroprotective in NSC19 cells expressing either WT SOD1 or G37R SOD1.

<p>(<b>A</b>) G37R SOD1 NSC19 cells are more vulnerable to oxidative stress (H₂O₂ (5 mM) for 24 h) than are WT SOD1 NSC19 cells. Cell viability assessed by MTS assay shows 22% cell survival in WT SOD1 cells <i>v.s.</i> 5% survival in G37R SOD1 cells (n = 6, * p<0.05). (<b>B</b>) Ex-4 reduced cell membrane permeability and protected cells from serum deprivation in both WT SOD1- and G37R SOD1-expressing NSC19 cells. Cells were cultured in no-serum media for 48 h in the presence and absence of 100 nM Ex-4. Cell viability was assessed by LDH assay. LDH levels were significantly reduced by Ex-4 treatment in both cells, under conditions of either Ex-4 alone or Ex-4 plus no-serum stress (C: vehicle-treated control; Ex-4: exendin-4; no serum: serum deprivation, n = 5, * p<0.05).</p

Behavioral effects of exendin-4 action in SOD1 (G93A) mice.

<p>(<b>A</b>) Comparison of average daily running distances of vehicle-treated and Ex-4-treated SOD1 (G93A) mice. During a presymptomatic stage (approximately 62–95 days of age), Ex-4 treatment significantly reduced average daily running distance of SOD1 (G93A) mice, resulting in 3.2–14.0 km/day (Ex-4 group) <i>v.s</i>. 7.8–21.0 km/day (vehicle group) (n = 10–15, * p<0.05, ** p<0.01, *** p<0.001). This Ex-4 induced decline brought the running in line with our prior studies of SOD1 WT non-ALS (B6SJL-Tg(SOD1)2Gur/J) mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032008#pone.0032008-Bruestle1" target="_blank">[31]</a>. At the age of 95 to 105 days, running activities quickly declined in both SOD1 (G93A) groups in a similar pattern, resulting in disease onset at the same time. (<b>B</b>) Neurological scores of SOD1 (G93A) mice. The neurological score between day 93–125 shows no difference between vehicle-treated and Ex-4-treated SOD1 (G93A) mice. (<b>C</b>) Kaplan-Meier plots of the survival of vehicle- and Ex-4 treated SOD1 (G93A) mice to reach a neurological score of 4 (paralysis of hind limbs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032008#pone.0032008-Martin1" target="_blank">[29]</a>). No difference was evident between the two groups (n = 10 per group, log-rank statistical analysis, p = 0.8).</p

Design of animal study and gluco-regulatory effects of Ex-4 in SOD1 (G93A) mice.

<p>(<b>A</b>) Scheme of animal study: SOD1 (G93A) mice were assigned to two groups (n = 15 mice/group), starting at the age of 6 weeks, saline or Ex-4 was delivered via subcutaneously implanted osmotic pumps in the control group and Ex-4 treatment group, respectively, throughout the study (treatment duration 12 weeks, hence mice were 18-weeks old at endstage) and pumps were changed every 3 weeks. (<b>B</b>) Baseline and weekly body weight of control (saline) and Ex-4-treated SOD1 (G93A) mice were largely similar throughout the study. Both mean baseline and weekly body weights were not different between the two groups of SOD1 (G93A) mice, except at the first measurement on day 2 post-treatment, when the body weight of Ex-4 treated mice transiently dropped compared to controls. The mean body weight of the Ex-4 group thereafter quickly caught up and remained comparable to controls throughout the study (n = 10–15, * p<0.05). (<b>C</b>) Baseline and weekly blood glucose levels of saline or Ex-4-treated SOD1 (G93A) mice. Morning fed blood glucose levels were measured weekly from tail tips. In general, blood glucose levels in Ex-4 treated mice were regulated between 120–140 mg/dL, whereas those for controls varied between 125–160 mg/dL. Mean blood glucose levels were largely comparable between the two groups, except during the first 2 weeks and last week of the study, when levels were significantly higher in the control group (n = 10–15, * p<0.05). (<b>D</b>) Intraperitoneal glucose tolerance test (IPGTT) at day 31 of treatment in SOD1 (G93A) mice. Assessment of mean blood glucose levels during a 2-hour IPGTT demonstrated significantly lower levels in Ex-4-treated mice at all time points (0, 10, 20, 30, 60, 90, 120 min) measured during IPGTT (n = 7, * p<0.05).</p

Ex-4 treatment modified cell type specific markers of spinal cord in SOD1 (G93A) mice towards levels present in control non-ALS WT mice.

<p>(<b>A</b> & <b>C</b>) Glial fibrillary acidic protein (GFAP) is a glial cell marker and is usually up regulated under neurodegenerative conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032008#pone.0032008-Middeldorp1" target="_blank">[86]</a>. Ex-4 treatment significantly reduced GFAP immunostaining in SOD1 (G93A) mice spinal cord. (<b>B</b> & <b>C</b>) Caspase-3 is an apoptotic marker. There are 3- to 4-fold less numerous activated caspase-3 neurons in Ex-4-treated G93A SOD1 mice lumbar spinal cord sections. (<b>D</b> & <b>F</b>) Choline acetyl transferase (ChAT) is a cholinergic neuron marker. Immunostaining with specific ChAT antibody shows significantly greater ChAT immunointensity in Ex-4-treated SOD1 (G93A) mice. (<b>E</b> & <b>F</b>) SMI-32 is a neurofilament marker that interacts with a nonphosphorylated epitope in neurofilament H of most mammalian species. There is greater SMI-32 immunointensity in SOD1 (G93A) mice (n = 5, ** p<0.01, *** p<0.001). For all investigated markers, Ex-4 treatment of SOD1 (G93A) mice substantially ameliorated the dramatic differences evident between vehicle SOD1 (G93A) and control non-ALS WT mice (C & F).</p

GLP-1R is expressed in NSC19 cells and mouse spinal cord.

<p>(<b>A</b>) A representative RT-PCR analysis showing expression of GLP-1R in stable transfected NSC19 cell lines (from left to right: NSC19 cells expressing wildtype SOD1, NSC19 cells expressing empty vector, NSC19 cells expressing G37R mutated SOD1). (<b>B</b>) A representative RT-PCR analysis showing expression of GLP-1R in wildtype C57Bl/6 mouse spinal cord (2 lanes represent 2 different mice). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as an internal control. Using specific primers, amplified mouse GLP-1R fragment is 250 bp and GAPDH is 452 bp.</p

Ex-4 treatment preserved lumbar spinal cord structure and neuron density.

<p>(<b>A</b>) Cresyl violet staining of lumbar spinal cord section of SOD1 (G93A) mice at both early (9 weeks of treatment at a pre-symptomatic stage) and end stages of disease. (<b>B</b>) Close-up of ventral horn of grey matter shows staining of motor neurons with large soma, scale bars on right inset is 25 µm. By comparison, (<b>C</b>) demonstrates similar lumbar spinal cord sections derived from control non-ALS WT mice, in which the grey matter tracts are clearly evident. (<b>D</b>) Quantification of motor neuron numbers in the ventral horn of grey matter. An approximately 3-fold greater neuron number was present in SOD1 (G93A) Ex-4-treated mice spinal cord sections than in vehicle-treated mice at both pre-symptomatic and end disease stages (9 and 12 week treatment times). Evaluation of motor neuron number in the lumbar ventral horn grey matter of age-matched control non-ALS WT mice were in the range of those present in SOD1 (G93A) mice treated with Ex-4. Selection of large pyramidal neurons was based on their characteristic nulceolus, angular and spindle-shaped <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032008#pone.0032008-Pendersen1" target="_blank">[85]</a>. A measuring bar of 25 µm is for reference only.</p

GLP-1R stimulation is neurotrophic and neuroprotective against oxidative stress and apoptosis in NSC19 cells.

<p>(<b>A</b>–<b>B</b>) Ex-4 is neurotrophic in NSC19 cells and pretreatment with Ex-4 protects cells from H₂O₂-induced cell death. Cells were pretreated with 100 nM Ex-4 for 4 h, and then exposed to 1.5 mM H₂O₂. Cell viability was assessed by MTS (<b>A</b>) after 24 h or LDH (<b>B</b>) after 48 h (C: vehicle-treated control; Ex-4: exendin-4 with and without H₂O₂, n≥6, * p<0.05). (<b>C</b>) Ex-4 pretreatment inhibits apoptosis in NSC19 cells. Exposure to 100 nM staurosporine significantly induced caspase-3 activity in NSC19 cells, pretreatment with 100 nM Ex-4 for 24 h reduced elevated caspase-3 activity in these cells (C: vehicle-treated control; Ex-4: exendin-4 with and without staurosporine, n = 3, * p<0.05). <b>(D</b>) Ex-4 elevates choline acetyltransferase (ChAT) activity in NSC 19 cells. NSC19 cells plated on 24-well plates were treated with 100 nM or 1 µM of Ex-4 as well as 10 µM forskolin for 72 h. ChAT activity was measured in cell lysates and normalized by protein content (n = 3, * p<0.05).</p