Neuroprotective efficacy of aminopropyl carbazoles in a mouse model of amyotrophic lateral sclerosis

Abstract

We previously reported the discovery of P7C3, an aminopropyl carbazole having proneurogenic and neuroprotective properties in newborn neural precursor cells of the hippocampal dentate gyrus. We have further found that chemicals having efficacy in this in vivo screening assay also protect dopaminergic neurons of the substantia nigra following exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a mouse model of Parkinson disease. Here, we provide evidence that an active analog of P7C3, known as P7C3A20, protects ventral horn spinal cord motor neurons from cell death in the G93A-SOD1 mutant mouse model of amyotrophic lateral sclerosis (ALS). P7C3A20 is efficacious in this model when administered at disease onset, and protection from cell death correlates with preservation of motor function in assays of walking gait and in the accelerating rotarod test. The prototypical member of this series, P7C3, delays disease progression in G93A-SOD1 mice when administration is initiated substantially earlier than the expected time of symptom onset. Dimebon, an antihistaminergic drug with significantly weaker proneurogenic and neuroprotective efficacy than P7C3, confers no protection in this ALS model. We propose that the chemical scaffold represented by P7C3 and P7C3A20 may provide a basis for the discovery and optimization of pharmacologic agents for the treatment of ALS.

Fig. 1.

P7C3A20 and P7C3 block motor neuron cell death in the spinal cord when administered at the time of disease onset to G93A-SOD1 mutant mice. Treatment of G93A-SOD1 mutant mice with 20 mg⋅kg⁻¹⋅d⁻¹ of P7C3A20, P7C3, or Dimebon, or the appropriate vehicle, was initiated on day 80. Five mice from each group were killed on days 90, 100, 110, and 120. The number of spinal cord motor neurons per cubic millimeter of lumbar spinal cord was determined through immunohistochemical staining for ChAT and quantification with National Institutes of Health Image J software. All images were analyzed blind to treatment group. Spinal cord motor neurons in G93A-SOD1 mutant mice died over time as expected. Spinal cord motor neuron cell death was blocked by administration of P7C3A20. P7C3 was intermediately protective, whereas Dimebon had no neuroprotective efficacy. (Upper) Representative staining of ChAT of one ventral horn from each of the five mice in each treatment group at 110 d is shown above the graph.

Fig. 2.

P7C3A20 preserves performance in the accelerating rotarod test when administered at the time of disease onset to G93A-SOD1 mutant mice. Treatment of G93A-SOD1 mutant mice with 20 mg⋅kg⁻¹⋅d⁻¹ of P7C3A20, P7C3, or Dimebon, or the appropriate vehicle, was initiated on day 80, with 20 mice per group. All compounds were administered at 20 mg⋅kg⁻¹⋅d⁻¹ i.p. in divided doses. Each compound-treated mouse had a sex-matched sibling that received vehicle. Only sibling pairs were analyzed at each time point. By week 16, there were 13 compound–vehicle pairs remaining in each group. All vehicle-treated mice showed the expected decline in retention time on the accelerating rotarod over time, and P7C3 and Dimebon groups showed no difference in retention time compared with their vehicle groups. Mice treated with P7C3A20 showed significantly higher retention time on the rotarod at weeks 13, 14, 15, and 16. All testing and analysis were performed blind to treatment group.

Fig. 3.

P7C3A20 preserves walking gait when administered at the time of disease onset to G93A-SOD1 mutant mice. (A) The schematic diagram shows parameters used to measure gait. Front stride and back stride were collected as a straight line from back paw print to the following paw print. Back-to-front distance was collected as a straight line from back paw print to the corresponding front paw print. Twenty measurements (10 on each side) for each parameter were measured per mouse, and 20 mice per group were evaluated at 90- and 118-d time points. All measurements were conducted blind to treatment group, and Student’s t test was used for statistical comparison of a treatment group to its matched vehicle group. (B) At 90 d, there were no differences between any groups in back stride, front stride, and back-to-front distance. By day 118, all vehicle groups, and P7C3- and Dimebon-treated mice, showed a significant difference in these measures, reflecting disease progression. Back stride and front stride were preserved to near-normal levels in P7C3A20-treated mice on day 118. By day 132, P7C3- and Dimebon-treated mice were too sick to participate in the task. At this time point, P7C3A20-treated mice continued to show normalized back stride and front stride levels.

Fig. 4.

Plasma, brain, and spinal cord levels of P7C3A20, P7C3, and Dimebon. Five mice for each compound group were treated for 21 d with 20 mg⋅kg⁻¹⋅d⁻¹ of the compound, starting on day 85. Blood, brain, and spinal cord were harvested 6 h after the last injection and compound levels were measured by LC/MS/MS. Concentrations are presented as mean ± SEM.