Ablation of proliferating cells in the CNS exacerbates motor neuron disease caused by mutant superoxide dismutase

Abstract

Proliferation of glia and immune cells is a common pathological feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here, to investigate the role of proliferating cells in motor neuron disease, SOD1(G93A) transgenic mice were treated intracerebroventicularly (i.c.v.) with the anti-mitotic drug cytosine arabinoside (Ara-C). I.c.v. delivery of Ara-C accelerated disease progression in SOD1(G93A) mouse model of ALS. Ara-C treatment caused substantial decreases in the number of microglia, NG2+ progenitors, Olig2+ cells and CD3+ T cells in the lumbar spinal cord of symptomatic SOD1(G93A) transgenic mice. Exacerbation of disease was also associated with significant alterations in the expression inflammatory molecules IL-1β, IL-6, TGF-β and the growth factor IGF-1.

Figure 1. Ara-C treatment caused a decrease in microglia, NG2+ progenitors, oligodendrocytes, astrocytes and T cells in the spinal cord of SOD1^G93A mice.

Immunofluorescence for cell markers Iba1 (A), CD68 (B), NG2 (C), GFAP (D), CD3 (E) and Olig2 (F) in the lumbar spinal cord of mutant SOD1 transgenic mice treated with vehicle or Ara-C. (G) Quantification of Iba positive cells showed a 1.5 folds reduction of cells in Ara-C treated mice compared to controls (**p = 0.0086). (H) Quantification of CD68 marker showed a 1.5 folds reduction of cells in Ara-C treated mice compared to controls (**p = 0.0099). (I) Quantification of NG2+ marker showed a 1.7 folds reduction of cells in Ara-C treated mice compared to controls (p = 0.0713) (J) Quantification of GFAP marked cells showed a slightly reduced number of astrocytes (1.2 folds) in Ara-C treated mice compared to controls, although this result was not significant (p = 0.3430). (K) Quantification of Olig2 positive cells showed a 2.0 folds reduction of cells in Ara-C treated mice compared to controls (*p = 0.0236) (L) Quantification of CD3+ cells showed 3.8 folds reduction of cells in Ara-C treated mice compared to controls (*p = 0.0122). All mice were analyzed at 115 days. All values are means ± SEM. Scale bars: 100 µm.

Figure 2. Ablation of proliferating cells in CNS decreases lifespan of SOD1^G93A mice.

Kaplan-Meier survival curve shows that transgenic mice Sod1^G93A (n = 7) treated with Ara-C between 75 and 115 days had a mean survival of 134 days while untreated SOD1^G93A (n = 9) had a mean survival of 141 days. Log-rank test shows that this difference is significant (p = 0.0081).

Figure 3. Motoneuron degeneration of SOD1^G93A mice was not affected by Ara-C treatment.

(A) Axons in the ventral root of transgenic mice treated or with Ara-C or vehicle. Quantification of the total number of axons showed no difference between both groups (p = 0.1790). (B) Nissl stain for neuronal cells. Quantification of motoneurons showed no difference between both groups (p = 0.9937). (C) Immunofluorecence for innervation of muscle fibres in the gastrocnemius muscle. Number of innervated, partially innervated and denervated fibre in Ara-C treated mice did not differ from vehicle-treated controls (Denervated: p = 0.8326, partially innervated: p = 0.9281, innervated: p = 0.5504). (A–B) Mice were analyzed at 115 days and (C) end-stage (±140 days). All values are means ± SEM. Scale bars: (A) 100 µm and (B–C) 50 µm.

Figure 4. Modulation of the inflammatory response in Ara-C treated SOD1^G93A mice.

Quantitative RT-PCR results (values are normalized to GADPH and relative to wild-type control treated with vehicle; no significant difference was found between wild-type controls treated with vehicle or M-CSF for any marker). Significant differences were found between Ara-C and vehicle treated SOD1^G93A in levels of mRNA for TGF-b1 (*p = 0.0148), IL-1b (**p = 0.0072), IL-6 (**p = 0.0014) and IGF-1 (*p = 0.0108). Note that all mRNA levels were significantly higher in vehicle treated transgenic mice compared to WT, treated or not (p<0.0025), except for IL-4 (p = 0.7582). All values are mean ± SEM; n(WT) = 9, n(Tg-Vehicle) = 6, n(Tg-Ara-C) = 15.