Several of the best-studied sex differences in the mammalian brain are ascribed to the hormonal control of cell death. This conclusion is based primarily on correlations between pyknotic cell counts in development and counts of mature neurons in adulthood; the molecular mechanisms of hormone-regulated, sexually dimorphic cell death are unknown. We asked whether Bax, a member of the Bcl-2 family of proteins that is required for cell death in many developing neurons, might be essential for sex differences in neuron number. We compared Bax knockout mice and their WT siblings, focusing on two regions of the mouse forebrain that show opposite patterns of sexual differentiation: the principal nucleus of the bed nucleus of the stria terminalis, in which males have more neurons than do females, and the anteroventral periventricular nucleus (AVPV), where females have more neurons overall and many more dopaminergic neurons than do males. Testosterone, or its metabolites, is responsible for the sex differences in both nuclei. A null mutation of the Bax gene completely eliminated sex differences in overall cell number in both the principal nucleus of the bed nucleus of the stria terminalis and AVPV. Thus, Bax-dependent cell death is required for sexual differentiation of cell number, regardless of whether testosterone decreases or increases cell death. In contrast, the sex difference in AVPV dopaminergic cell number, as measured by tyrosine hydroxylase immunohistochemistry, was not affected by Bax gene deletion, demonstrating heterogeneity of mechanisms controlling cell number within a single nucleus.

ATP-binding cassette (ABC) drug efflux transporters in the CNS are predominantly localized to the luminal surface of endothelial cells in capillaries to impede CNS accumulation of xenobiotics. Inflammatory mediators and cellular stressors regulate their activity. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of upper and lower motor neurons characterized by extensive neuroinflammation. Here we tested the hypothesis that disease-driven changes in ABC transporter expression and function occur in ALS. Given the multitude of ABC transporters with their widespread substrate recognition, we began by examining expression levels of several ABC transporters. We found a selective increase in only two transporters; P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) both at mRNA and protein levels, in the SOD1-G93A mouse model of ALS, specifically in disease-affected CNS regions. Detailed analysis revealed a similar disease-driven increase in P-gp and BCRP levels in spinal cord microvessels, indicating that their altered expression occurs at the blood spinal cord barrier. Transport activity of P-gp and BCRP increased with disease progression in spinal cord and cerebral cortex capillaries. Finally, P-gp and BCRP protein expression also increased in spinal cords of ALS patients. Preclinical drug trials in the mouse model of ALS have failed to decisively slow or arrest disease progression; pharmacoresistance imparted by ABC transporters is one possible explanation for these failures. Our observations have large implications for ALS therapeutics in humans and suggest that the obstacle provided by these transporters to drug treatments must be overcome to develop effective ALS pharmacotherapies.

ObjectiveResearch identified promising therapeutics in cell models of Amyotrophic Lateral Sclerosis (ALS), but there is limited progress translating effective treatments to animal models and patients, and ALS remains a disease with no effective treatment. One explanation stems from an acquired pharmacoresistance driven by the drug efflux transporters P-glycoprotein (P-gp) and breast cancer-resistant protein (BCRP), which we have shown are selectively upregulated at the blood-brain and spinal cord barrier (BBB/BSCB) in ALS mice and patients. Pharmacoresistance is well appreciated in other brain diseases, but overlooked in ALS despite many failures in clinical trials.MethodsHere, we prove that a P-gp/BCRP-driven pharmacoresistance limits the bioavailability of ALS therapeutics using riluzole, the only FDA-approved drug for ALS and a substrate of P-gp and BCRP. ALS mice (SOD1-G93A) were treated with riluzole and elacridar, to block P-gp and BCRP, and monitored for survival as well as behavioral and physiological parameters.ResultsWe show that riluzole, which normally is not effective when given at onset of symptoms, is now effective in the ALS mice when administered in combination with the P-gp/BCRP inhibitor elacridar. Chronic elacridar treatment increases riluzole Central nervous system (CNS) penetration, improves behavioral measures, including muscle function, slowing down disease progression, and significantly extending survival.InterpretationOur approach improves riluzole efficacy with treatment beginning at symptom onset. Riluzole will not provide a cure, but enhancing its efficacy postsymptoms by addressing pharmacoresistance demonstrates a proof-of-principle concept to consider when developing new ALS therapeutic strategies. We highlight a novel improved therapeutic approach for ALS and demonstrate that pharmacoresistance can no longer be ignored in ALS.