Inhibiting drug efflux transporters improves efficacy of ALS therapeutics.

OBJECTIVE: Research 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. METHODS: Here, 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. RESULTS: We 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. INTERPRETATION: Our 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.

Selective increase of two ABC drug efflux transporters at the blood-spinal cord barrier suggests induced pharmacoresistance in ALS.

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.

The role of cell death in sexually dimorphic muscle development: male-specific muscles are retained in female bax/bak knockout mice.

The bulbocavernosus (BC) and levator ani (LA) muscles are present in males but absent or severely reduced in females, and the fate of these muscles controls the survival of motoneurons in the sexually dimorphic spinal nucleus of the bulbocavernosus. However, the mechanism underlying the sex difference in BC and LA development has been controversial. We examined the role of cell death in sexual differentiation of the bulbocavernosus BC/LA muscles in mice. Muscle development was mapped from embryonic day 16 (E16) to postnatal day 5 (P5). A sex difference (male>female) first arose on E17 (BC) or E18 (LA), and increased in magnitude postnatally. TUNEL labeling revealed dying cells in the BC and LA muscles of both sexes perinatally. However, females had a significantly higher density of TUNEL-positive cells than did males. A role for the proapoptotic factors, Bax and Bak, in BC/LA development was tested by examining mice lacking one or both of these proteins. In females lacking either Bax or Bak, the BC was absent and the LA rudimentary. Deletion of both bax and bak genes, however, rescued the BC, increased LA size approximately 20-fold relative to controls, and virtually eliminated TUNEL-positive cells in both muscles. We conclude that cell death plays an essential role in sexual differentiation of the BC/LA muscles. The presence of either Bax or Bak is sufficient for cell death in the BC/LA, whereas the absence of both prevents sexually dimorphic muscle cell death.

Development of sex differences in the principal nucleus of the bed nucleus of the stria terminalis of mice: role of Bax-dependent cell death.

Neuron number in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is greater in adult male mice than in females. Deletion of the proapoptotic gene, Bax, increases the number of BNSTp cells in adulthood and eliminates the sex difference in cell number. Here, we map the ontogeny of sex differences in nuclear volume and cell number in the BNSTp of neonatal mice, and evaluate the role of cell death in the development of these differences. We find that BNSTp volume and cell number do not differ between male and female wild-type mice on postnatal days P3, P5, or P7. Sex differences emerge after the first postnatal week and both measures are significantly greater in males than in females on P9 and P11. Cell death, assessed by TUNEL staining, was observed in the BNSTp of both sexes from P1-P8. Females had more TUNEL-positive cells than males from approximately P3-P6, with the maximum number of dying cells observed on P5/P6. To test whether the Bax gene is required for sexually dimorphic cell death in the BNSTp, TUNEL cells were counted on P6 in Bax -/- mice and their Bax +/+ siblings. Bax gene deletion nearly abolished TUNEL-positive cells in the BNSTp of both sexes. Together, these findings support the interpretation that the sex difference in BNSTp cell number seen in adulthood is due to Bax-dependent, sexually dimorphic cell death during the first week of life.

Effects of Bax gene deletion on muscle and motoneuron degeneration in a sexually dimorphic neuromuscular system.

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles in the perineum, bulbocavernosus (BC), and levator ani (LA) normally degenerate in female rodents. Death of the motoneurons and muscles can be prevented by androgen treatments around the time of birth. To identify the intracellular mechanisms underlying hormone-dependent survival of this neuromuscular system, we examined mice with a targeted disruption of the pro-death gene Bax. SNB motoneuron number was increased in female Bax-/- mice, whether measured using immunolabeling for a motoneuron-specific marker or retrograde labeling with the fluorescent tracer Fluoro-Gold. Based on retrograde tracing, the sex difference in SNB cell number is eliminated in Bax-/- mice. Thus, Bax is required for sexually dimorphic motoneuron death in the SNB, and motoneurons rescued by Bax deletion project their axons to the periphery. Mean soma size in the SNB of Bax-/- females is reduced, however, and there is a subpopulation of very small cells in the SNB of female knock-outs. In addition, the BC muscle was not identified in any female, regardless of Bax gene status. All females possessed a small LA muscle, and Bax deletion resulted in a tripling of LA fiber number in females. This increase was small, however, relative to the >50-fold sex difference in LA muscle fiber number. Thus, the sex difference in the perineal muscles is mostly unaffected by the absence of Bax protein, and SNB motoneuron number is dissociated from target muscle size in Bax-/- animals.

Deletion of Bax eliminates sex differences in the mouse forebrain.

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.