Toll-Like Receptor-4 Inhibitor TAK-242 Attenuates Motor Dysfunction and Spinal Cord Pathology in an Amyotrophic Lateral Sclerosis Mouse Model

Abstract

Neuroinflammation contributes to amyotrophic lateral sclerosis (ALS) progression. TLR4, a transmembrane protein that plays a central role in activation of the innate immune system, has been shown to induce microglial activation in ALS models. TLR4 is up-regulated in the spinal cords of hSOD1^G93A mice. We aimed to examine the effects of specific TLR4 inhibition on disease progression and survival in the hSOD1^G93A mouse model of ALS. Immunologic effect of TLR4 inhibition in vitro was measured by the effect of TAK-242 treatment on LPS-induced splenocytes proliferation. hSOD1^G93A transgenic mice were treated with TAK-242, a selective TLR4 inhibitor, or vehicle. Survival, body weight, and motor behavior were monitored. To evaluate in vivo immunologic modifications associated with TAK-242 treatment, we measured serum IL-1β in the plasma, as well as IL-1β and TNF-α mRNAs in the spinal cord in wild-type mice and in TAK-242-treated and vehicle-treated early symptomatic hSOD1^G93A mice. Immunohistochemical analysis of motor neurons, astrocytes, and microglial reactivity in the spinal cords were performed on symptomatic (100 days old) TAK-242-treated and vehicle-treated hSOD1^G93A mice. In vitro, splenocytes taken from 100 days old hSOD1^G93A mice showed significantly increased proliferation when exposed to LPS (p = 0.0002), a phenomenon that was reduced by TAK-242 (p = 0.0179). TAK-242 treatment did not attenuate body weight loss or significantly affect survival. However, TAK-242-treated hSOD1^G93A mice showed temporary clinical delay in disease progression evident in the ladder test and hindlimb reflex measurements. Plasma IL-1β levels were significantly reduced in TAK-242-treated compared to vehicle-treated hSOD1^G93A mice (p = 0.0023). TAK-242 treatment reduced spinal cord astrogliosis and microglial activation and significantly attenuated spinal cord motor neuron loss at early disease stage (p = 0.0259). Compared to wild-type animals, both IL-1β and TNF-α mRNAs were significantly upregulated in the spinal cords of hSOD1^G93A mice. Spinal cord analysis in TAK-242-treated hSOD1^G93A mice revealed significant attenuation of TNF-α mRNA (p = 0.0431), but no change in IL-1β mRNA. TLR4 inhibition delayed disease progression, attenuated spinal cord astroglial and microglial reaction, and reduced spinal motor neuron loss in the ALS hSOD1^G93A mouse model. However, this effect did not result in increased survival. To our knowledge, this is the first report on TAK-242 treatment in a neurodegenerative disease model. Further studies are warranted to assess TLR4 as a therapeutic target in ALS.

Keywords: ALS; SOD1 mice; TAK-242; TLR4; animal model; innate immune system; neuroinflammation.

Figure 1

LPS-induced splenocyte proliferation in vitro was significantly reduced by TAK-242 treatment. Splenocytes obtained from 100 days old hSOD^G93A mice were treated with TAK-242 or vehicle for three days. Proliferation was measured from the Alamar blue assay and quantified by fluorescence intensity. PBS: phosphate-buffered saline; LPS: lipopolysaccharide. Data are expressed as mean ± SEM, * p < 0.01, ** p < 0.05.

Figure 2

Behavioral motor performance analysis showed delay in disease progression in TAK-242- treated hSOD^G93A mice. (A) The ladder test. A score of 12 represents completely healthy mice and 0 correlates with disease end stage. Ladder testing showed statistically significant differences between hSOD^G93A mice treated with TAK-242 or vehicle. Data are expressed as mean ± SEM, * p < 0.05, ** p < 0.01; (B) A similar trend in disease progression was found in hind limb reflex score testing. Data are expressed as mean ± SEM; (C) Survival analysis by Kaplan–Meier curves of TAK-242 and vehicle-treated hSOD^G93A mice. All wild-type littermates survived and kept having a normal maximal score in all behavioral tests during the whole experiment period (not shown in the graphs).

Figure 3

Prolonged TAK-242 treatment attenuates serum IL1-β levels of early symptomatic hSOD^G93A mice. IL1-β levels were measured by ELISA testing in sera obtained at day 100 of life, after 50 days of treatment with TAK-242 or vehicle. Analysis showed an increase in IL1-β levels in vehicle-treated hSOD^G93A mice compared to WT mice, which was significantly attenuated by TAK-242 treatment. WT: wild type. * p < 0.01.

Figure 4

Preservation of spinal motor neurons in TAK-242-treated hSOD^G93A mice. Lumbar spinal cords sections (9 sections from each animal; 3 animals in each group) from early symptomatic hSOD^G93A mice (100 days old) were stained by H&E staining (A–C); Immunohistochemistry staining for the motor neuron marker ChAT is shown in (E–G); Motor neuron number was significantly reduced in hSOD^G93A mice treated with vehicle (B,F) compared to WT mice (A,E); Motor neuron loss was significantly reduced in hSOD^G93A mice treated with TAK-242 (C,G); Motor neurons quantification is shown in (D) and represents average number of motor neurons per one ventral horn in each group. An example of a motor neuron is marked by the arrow in A. WT: wild type; H&E: hematoxylin and eosin; ChAT: choline acetyltransferase; DAPI: 4,6-diamidino-2-phenylindole. Scale bars are 100 µm for (A–C) and 20 µm for (E–G). Data are expressed as mean ± SEM, * p < 0.05.

Figure 5

Prolonged TAK-242 treatment attenuates astroglial activation in hSOD^G93A mice spinal cords. Immunohistochemistry staining for the astrocyte markers GFAP (red; A–C) and S100β (green; E–G) at day 100 of life showed increased spinal astrogliosis in vehicle-treated hSOD^G93A mice (B,F) compared to WT littermates (A,E), that was prominently attenuated in TAK-242 treated hSOD^G93A mice (C (p = 0.0462); G (p = 0.0481)). Quantification of staining intensity is shown graphically in panels D (GFAP) and H (S100β). WT: wild type; GFAP: glial fibrillary acidic protein; DAPI: 4,6-diamidino-2-phenylindole. Scale bars for all panels = 20 µm.

Figure 6

Prolonged TAK-242 treatment attenuates microglial activation in hSOD^G93A mice spinal cords. Immunohistochemistry staining for the activated microglia marker IBA1 at day 100 of life showed increased spinal microglial activation in vehicle-treated hSOD^G93A mice (B) as compared to WT littermates (A). Microglial activation was prominently attenuated by prolonged TAK-242 treatment of hSOD^G93A mice (C; p = 0.0544). Quantification of staining intensity is shown graphically in panel (D). WT: wild type; IBA1: ionized calcium-binding adapter molecule-1; DAPI: 4,6-diamidino-2-phenylindole. Scale bars for all panels = 20 µm.

Figure 7

Prolonged TAK-242 treatment attenuated TNF-α expression but not IL1-β expression in the spinal cords of early symptomatic hSOD^G93A mice. TNF-α (A) and IL1-β (B) mRNA levels in the spinal cords of TAK-242-treated and vehicle-treated 100-days old WT and early symptomatic hSOD^G93A mice were quantified by real-time PCR as described in the methods section. WT: wild type; NS: non-significant. Data are expressed as mean ± SEM, * p < 0.05.