Identification of 2,2-Dimethylbutanoic Acid (HST5040), a Clinical Development Candidate for the Treatment of Propionic Acidemia and Methylmalonic Acidemia.

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, caused by a deficiency in the enzymes P-CoA carboxylase and methylmalonyl-CoA (M-CoA) mutase, respectively. PA and MMA are classified as intoxication-type inborn errors of metabolism because the intramitochondrial accumulation of P-CoA, M-CoA, and other metabolites results in secondary inhibition of multiple pathways of intermediary metabolism, leading to organ dysfunction and failure. Herein, we describe the structure-activity relationships of a series of short-chain carboxylic acids which reduce disease-related metabolites in PA and MMA primary hepatocyte disease models. These studies culminated in the identification of 2,2-dimethylbutanoic acid (10, HST5040) as a clinical candidate for the treatment of PA and MMA. Additionally, we describe the in vitro and in vivo absorption, distribution, metabolism, and excretion profile of HST5040, data from preclinical studies, and the synthesis of the sodium salt of HST5040 for clinical trials.

A novel small molecule approach for the treatment of propionic and methylmalonic acidemias.

Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA) are inborn errors of metabolism affecting the catabolism of valine, isoleucine, methionine, threonine and odd-chain fatty acids. These are multi-organ disorders caused by the enzymatic deficiency of propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT), resulting in the accumulation of propionyl-coenzyme A (P-CoA) and methylmalonyl-CoA (M-CoA in MMA only). Primary metabolites of these CoA esters include 2-methylcitric acid (MCA), propionyl-carnitine (C3), and 3-hydroxypropionic acid, which are detectable in both PA and MMA, and methylmalonic acid, which is detectable in MMA patients only (Chapman et al., 2012). We deployed liver cell-based models that utilized PA and MMA patient-derived primary hepatocytes to validate a small molecule therapy for PA and MMA patients. The small molecule, HST5040, resulted in a dose-dependent reduction in the levels of P-CoA, M-CoA (in MMA) and the disease-relevant biomarkers C3, MCA, and methylmalonic acid (in MMA). A putative working model of how HST5040 reduces the P-CoA and its derived metabolites involves the conversion of HST5040 to HST5040-CoA driving the redistribution of free and conjugated CoA pools, resulting in the differential reduction of the aberrantly high P-CoA and M-CoA. The reduction of P-CoA and M-CoA, either by slowing production (due to increased demands on the free CoA (CoASH) pool) or enhancing clearance (to replenish the CoASH pool), results in a net decrease in the CoA-derived metabolites (C3, MCA and MMA (MMA only)). A Phase 2 study in PA and MMA patients will be initiated in the United States.

Specializations of intercellular junctions are associated with the presence and absence of hair cell regeneration in ears from six vertebrate classes.

Sensory hair cell losses lead to hearing and balance deficits that are permanent for mammals, but temporary for nonmammals because supporting cells in their ears give rise to replacement hair cells. In mice and humans, vestibular supporting cells grow exceptionally large circumferential F-actin belts and their junctions express E-cadherin in patterns that strongly correlate with postnatal declines in regeneration capacity. In contrast, chicken supporting cells retain thin F-actin belts throughout life and express little E-cadherin. To determine whether the junctions in chicken ears might be representative of other ears that also regenerate hair cells, we investigated inner ears from dogfish sharks, zebrafish, bullfrogs, Xenopus, turtles, and the lizard, Anolis. As in chickens, the supporting cells in adult zebrafish, Xenopus, and turtle ears retained thin circumferential F-actin belts and expressed little E-cadherin. Supporting cells in adult sharks and bullfrogs also retained thin belts, but were not tested for E-cadherin. Supporting cells in adult Anolis exhibited wide, but porous webs of F-actin and strong E-cadherin expression. Anolis supporting cells also showed some cell cycle reentry when cultured. The results reveal that the association between thin F-actin belts and low E-cadherin is shared by supporting cells in anamniotes, turtles, and birds, which all can regenerate hair cells. Divergent junctional specializations in supporting cells appear to have arisen independently in Anolis and mammals. The presence of webs of F-actin at the junctions in Anolis appears compatible with supporting cell proliferation, but the solid reinforcement of the F-actin belts in mammals is associated with its absence.

Variations in shape-sensitive restriction points mirror differences in the regeneration capacities of avian and mammalian ears.

When inner ear hair cells die, humans and other mammals experience permanent hearing and balance deficits, but non-mammalian vertebrates quickly recover these senses after epithelial supporting cells give rise to replacement hair cells. A postnatal decline in cellular plasticity appears to limit regeneration in mammalian balance organs, where declining proliferation responses are correlated with decreased spreading of supporting cells on artificial and native substrates. By culturing balance epithelia on substrates that differed in flexibility, we assessed spreading effects independent of age, showing a strong correlation between shape change and supporting cell proliferation. Then we made excision wounds in utricles cultured from young and old chickens and mice and compared quantified levels of spreading and proliferation. In utricles from young mice, and both young and old chickens, wounds re-epithelialized in <24 hours, while those in utricles from mature mice took three times longer. More cells changed shape in the fastest healing wounds, which accounted for some differences in the levels of proliferation, but inter-species and age-related differences in shape-sensitive restriction points, i.e., the cellular thresholds for shape changes that promote S-phase, were evident and may be particularly influential in the responses to hair cell losses in vivo.

The postnatal accumulation of junctional E-cadherin is inversely correlated with the capacity for supporting cells to convert directly into sensory hair cells in mammalian balance organs.

Mammals experience permanent impairments from hair cell (HC) losses, but birds and other non-mammals quickly recover hearing and balance senses after supporting cells (SCs) give rise to replacement HCs. Avian HC epithelia express little or no E-cadherin, and differences in the thickness of F-actin belts at SC junctions strongly correlate with different species' capacities for HC replacement, so we investigated junctional cadherins in human and murine ears. We found strong E-cadherin expression at SC-SC junctions that increases more than sixfold postnatally in mice. When we cultured utricles from young mice with γ-secretase inhibitors (GSIs), striolar SCs completely internalized their E-cadherin, without affecting N-cadherin. Hes and Hey expression also decreased and the SCs began to express Atoh1. After 48 h, those SCs expressed myosins VI and VIIA, and by 72 h, they developed hair bundles. However, some scattered striolar SCs retained E-cadherin and the SC phenotype. In extrastriolar regions, the vast majority of SCs also retained E-cadherin and failed to convert into HCs even after long GSI treatments. Microscopic measurements revealed that the junctions between extrastriolar SCs were more developed than those between striolar SCs. In GSI-treated utricles as old as P12, differentiated striolar SCs converted into HCs, but such responses declined with age and ceased by P16. Thus, temporal and spatial differences in postnatal SC-to-HC phenotype conversion capacity are linked to the structural attributes of E-cadherin containing SC junctions in mammals, which differ substantially from their counterparts in non-mammalian vertebrates that readily recover from hearing and balance deficits through hair cell regeneration.

Post-translational regulation of an Aplysia glutamate transporter during long-term facilitation.

Regulation of glutamate transporters accompanies plasticity of some glutamatergic synapses. The regulation of glutamate uptake at the Aplysia sensorimotor synapse during long-term facilitation (LTF) was investigated. Previously, increases in levels of ApGT1 (Aplysia glutamate transporter 1) in synaptic membranes were found to be related to long-term increases in glutamate uptake. In this study, we found that regulation of ApGT1 during LTF appears to occur post-translationally. Serotonin (5-HT) a transmitter that induces LTF did not increase synthesis of ApGT1. A pool of ApGT1 appears to exist in sensory neuron somata, which is transported to the terminals by axonal transport. Blocking the rough endoplasmic reticulum-Golgi-trans-Golgi network (TGN) pathway with Brefeldin A prevented the 5-HT-induced increase of ApGT1 in terminals. Also, 5-HT produced changes in post-translational modifications of ApGT1 as well as changes in the levels of an ApGT1-co-precipitating protein. These results suggest that regulation of trafficking of ApGT1 from the vesicular trafficking system (rough endoplasmic reticulum-Golgi-TGN) in the sensory neuron somata to the terminals by post-translational modifications and protein interactions appears to be the mechanism underlying the increase in ApGT1, and thus, glutamate uptake during memory formation.

Recent advances in hair cell regeneration research.

PURPOSE OF REVIEW: This review discusses recent progress in research that seeks to understand the regeneration of hair cells and highlights findings that may hold importance for the eventual development of regenerative therapies for hearing and balance impairments. RECENT FINDINGS: Signaling via the Notch receptor and the basic helix-loop-helix transcription factors has important roles in the development and regeneration of hair cells. The cytoskeletal properties and cell-matrix interactions of supporting cells in mice of different ages may hold part of the explanation for the age-related differences in their proliferative responses to damage and the differences between mammals and nonmammals in hair cell regeneration. Progress also has been made in deriving stem cells from inner ear tissues and other sources and in the evaluation of their potential uses as sources of new hair cells and as tools for biomedical research. SUMMARY: Much has been accomplished since the discovery of postembryonic hair cell production and hair cell regeneration in nonmammals decades ago. No therapies for hair cell regeneration are under clinical trials, but research is yielding potentially important discoveries that are likely to lead to the development of therapeutic methods for inducing hair cell regeneration in the mammalian inner ear.

Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds.

Debilitating hearing and balance deficits often arise through damage to the inner ear's hair cells. For humans and other mammals, such deficits are permanent, but nonmammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F-actin that bracket the apical junctions between supporting cells in the gravity-sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, whereas their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F-actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F-actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement and that the postnatal reinforcement of the F-actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration.

In vivo regulation of an Aplysia glutamate transporter, ApGT1, during long-term memory formation.

Regulation of glutamate transporters often accompanies glutamatergic synaptic plasticity. We investigated the mechanisms responsible for the increase in glutamate uptake associated with increased glutamate release at the Aplysia sensorimotor synapse during long-term sensitization (LTS) and long-term facilitation. An increase in the V(max) of transport, produced by LTS training, suggested that the increased glutamate uptake was due to an increase in the number of transporters in the membrane. We cloned a high-affinity, Na(+)-dependent glutamate transporter, ApGT1, from Aplysia central nervous system that is highly enriched in pleural sensory neurons, and in pleural-pedal synaptosome and cell/glial fractions. ApGT1, expressed in Xenopus oocytes, demonstrated a similar pharmacological profile to glutamate uptake in Aplysia synaptosome and cell/glial fractions (strong inhibition by threo-beta-benzyloxyaspartate and weak inhibition by dihydrokainate) suggesting that ApGT1 may be the primary glutamate transporter in pleural-pedal ganglia. Levels of ApGT1 and glutamate uptake were increased in synaptosomes 24 h after induction of LTS by electrical stimulation or serotonin. Regulation of ApGT1 during LTS appears to occur post-transcriptionally and results in an increased number of transporters in synaptic membranes. These results suggest that an increase in levels of ApGT1 is responsible, at least in part, for the long-term increase in glutamate uptake associated with long-term memory.

Different mechanisms exist for the plasticity of glutamate reuptake during early long-term potentiation (LTP) and late LTP.

Regulation of glutamate reuptake occurs along with several forms of synaptic plasticity. These associations led to the hypothesis that regulation of glutamate uptake is a general component of plasticity at glutamatergic synapses. We tested this hypothesis by determining whether glutamate uptake is regulated during both the early phases (E-LTP) and late phases (L-LTP) of long-term potentiation (LTP). We found that glutamate uptake was rapidly increased within minutes after induction of LTP and that the increase in glutamate uptake persisted for at least 3 h in CA1 of the hippocampus. NMDA receptor activation and Na+-dependent high-affinity glutamate transporters were responsible for the regulation of glutamate uptake during all phases of LTP. However, different mechanisms appear to be responsible for the increase in glutamate uptake during E-LTP and L-LTP. The increase in glutamate uptake observed during E-LTP did not require new protein synthesis, was mediated by PKC but not cAMP, and as previously shown was attributable to EAAC1 (excitatory amino acid carrier-1), a neuronal glutamate transporter. On the other hand, the increase in glutamate uptake during L-LTP required new protein synthesis and was mediated by the cAMP-PKA (protein kinase A) pathway, and it involved a different glutamate transporter, GLT1a (glutamate transporter subtype 1a). The switch in mechanisms regulating glutamate uptake between E-LTP and L-LTP paralleled the differences in the mechanisms responsible for the induction of E-LTP and L-LTP. Moreover, the differences in signaling pathways and transporters involved in regulating glutamate uptake during E-LTP and L-LTP indicate that different functions and/or sites may exist for the changes in glutamate uptake during E-LTP and L-LTP.

The circadian clock modulates core steps in long-term memory formation in Aplysia.

The circadian clock modulates the induction of long-term sensitization (LTS) in Aplysia such that long-term memory formation is significantly suppressed when animals are trained at night. We investigated whether the circadian clock modulated core molecular processes necessary for memory formation in vivo by analyzing circadian regulation of basal and LTS-induced levels of phosphorylated mitogen-activated protein kinase (P-MAPK) and Aplysia CCAAT/enhancer binding protein (ApC/EBP). No basal circadian regulation occurred for P-MAPK or total MAPK in pleural ganglia. In contrast, the circadian clock regulated basal levels of ApC/EBP protein with peak levels at night, antiphase to the rhythm in LTS. Importantly, LTS training during the (subjective) day produced greater increases in P-MAPK and ApC/EBP than training at night. Thus, circadian modulation of LTS occurs, at least in part, by suppressing changes in key proteins at night. Rescue of long-term memory formation at night required both facilitation of MAPK and transcription in conjunction with LTS training, confirming that the circadian clock at night actively suppresses MAPK activation and transcription involved in memory formation. The circadian clock appears to modulate LTS at multiple levels. 5-HT levels are increased more when animals receive LTS training during the (subjective) day compared with the night, suggesting circadian modulation of 5-HT release. Circadian modulation also occurred downstream of 5-HT release because animals treated with 5-HT to induce LTS exhibited significantly greater LTS when treated during the (subjective) day compared with the night. Together, our studies suggest that the circadian clock modulates LTS at multiple steps and locations during the formation of long-term memory.