Roles of GABAB receptor subtypes in presynaptic auto- and heteroreceptor function regulating GABA and glutamate release.

Gamma-Aminobutyric acid B (GABA B) receptors are heterodimers composed of two subunits GABA B(1) and GABA B(2), the former existing in two isoforms GABA B(1a) and GABA B(1b). The contributions of individual receptor subunits and isoforms to GABA B auto- and heteroreceptor functions were investigated, using release experiments in cortical slice preparations from corresponding knockout mice. Presynaptic GABA B autoreceptors are located on GABAergic terminals and inhibit GABA release, whereas presynaptic GABA B heteroreceptors control the release of other neurotransmitters (e.g. glutamate). Neither baclofen nor the selective antagonist CGP55845 at maximally active concentrations affected [3H]GABA release in slices from GABA B(1)-/- mice. The amount of [3H]GABA released per pulse was unaffected by the stimulation frequency in slices from GABA B(1)-/- and GABA B(2)-/- demonstrating a loss of GABA B autoreceptor function in these knockout animals. The GABA B receptor agonist baclofen was ineffective in modulating glutamate release in cortical slices from GABA B(2)-/- mice, showing that heteroreceptor function was abolished as well. Next we investigated knockout mice for the two predominant GABA B(1) isoforms expressed in brain, GABA B(1a) and GABA B(1b). In cortical, hippocampal and striatal slices from both GABA B(1a)-/- and GABA B(1b)-/- mice, the frequency dependence of [3H]GABA released per pulse was maintained, suggesting that both isoforms participate or can substitute for each other in GABA B autoreceptor function. By contrast, the efficacy of baclofen to inhibit glutamate release was substantially reduced in GABA B(1a)-/-, but essentially unaltered in GABA B(1b)-/- mice. Our data suggest that functional GABA B heteroreceptors regulating glutamate release are predominantly, but not exclusively composed of GABA B(1a) and GABA B(2) subunits.

A small-molecule therapeutic lead for Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse.

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine expansion within huntingtin protein. The exact pathological mechanisms determining disease onset and progression remain unclear. However, aggregates of insoluble mutant huntingtin (mhtt), a hallmark of HD, are readily detected within neurons in HD brain. Although aggregated polyglutamines may not be inherently toxic, they constitute a biomarker for mutant huntingtin useful for developing therapeutics. We previously reported that the small molecule, C2-8, inhibits polyglutamine aggregation in cell culture and brain slices and rescues degeneration of photoreceptors in a Drosophila model of HD. In this study, we assessed the therapeutic potential of C2-8 in the R6/2 mouse model of HD, which has been used to provide proof-of-concept data in considering whether to advance therapies to human HD. We show that, at nontoxic doses, C2-8 penetrates the blood-brain barrier and is present in brain at a high concentration. C2-8-treated mice showed improved motor performance and reduced neuronal atrophy and had smaller huntingtin aggregates. There have been no prior drug-like, non-toxic, brain-penetrable aggregation inhibitors to arise from cell-based high-throughput screens for reducing huntingtin aggregation that is efficacious in preclinical in vivo models. C2-8 provides an essential tool to help elucidate mechanisms of neurodegeneration in HD and a therapeutic lead for further optimization and development.

Protective effects of NIM811 in transient focal cerebral ischemia suggest involvement of the mitochondrial permeability transition.

Cerebral ischemia followed by reperfusion activates numerous pathways that lead to cell death. One such pathway involves the release of large quantities of the excitatory amino acid glutamate into the synapse and activation of N-methyl-D-aspartate receptors. This causes an increase in mitochondrial calcium levels ([Ca(2+)](m)) and a production of reactive oxygen species (ROS), both of which may induce the mitochondrial permeability transition (MPT). As a consequence, there is eventual mitochondrial failure culminating in either apoptotic or necrotic cell death. Thus, agents that inhibit MPT might prove useful as therapeutic interventions in cerebral ischemia. In this study, we have investigated the neuroprotective efficacy of the novel compound NIM811. Similar in structure of its parent compound cyclosporin A, NIM811 is a potent inhibitor of the MPT. Unlike cyclosporin A, however, it is essentially void of immunosuppressive actions, allowing the role of MPT to be clarified in ischemia/reperfusion injury. The results of these studies demonstrate that NIM811 provides almost 40% protection in a model of transient focal cerebral ischemia. This was associated with a nearly 10% reduction in mitochondrial reactive species formation and 34% and 38% reduction of cytochrome c release in core and penumbra, respectively. Treatment with NIM811 also increased calcium retention capacity by approximately 20%. Interestingly, NIM811 failed to improve ischemia-induced impairment of bioenergetics. The neuroprotective effects of NIM811 were not due to drug-induced alterations in cerebral perfusion after ischemia. Activation of MPT appears to be an important process in ischemia/reperfusion injury and may be a therapeutic target.

Recent clinical failures in Parkinson's disease with apoptosis inhibitors underline the need for a paradigm shift in drug discovery for neurodegenerative diseases.

Understanding the mechanisms of neuronal death in concert with the identification of drugable molecular targets key to this process has held great promise for the development of novel chemical entities (NCEs) to halt neurodegenerative disease progression. Two key targets involved in the apoptotic process identified over the past decade include the mixed lineage kinase (MLK) family and glyceraldehyde phosphate dehydrogenase (GAPDH). Two NCEs, CEP-1347 and TCH346, directed against these respective targets have progressed to the clinic. For each, robust neuroprotective activity was demonstrated in multiple in vitro and in vivo models of neuronal cell death, but neither NCE proved effective Parkinson's disease (PD) patients. These recent clinical failures require a reassessment of both the relevance of apoptosis to neurodegenerative disease etiology and the available animal models used to prioritize NCEs for advancement to the clinic in this area.

Comparison of [18F]FDOPA, [18F]FMT and [18F]FECNT for imaging dopaminergic neurotransmission in mice.

INTRODUCTION: The clinically established positron emission tomography (PET) tracers 6-[(18)F]-fluoro-l-DOPA ([(18)F]FDOPA), 6-[(18)F]-fluoro-l-m-tyrosine ([(18)F]FMT) and 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-[(18)F]-fluoroethyl)-nortropane ([(18)F]FECNT) serve as markers of presynaptic integrity of dopaminergic nerve terminals in humans. This study describes our efforts to adopt the methodology of human Parkinson's disease (PD) PET studies to mice. METHODS: The PET imaging characteristics of [(18)F]FDOPA, [(18)F]FMT and [(18)F]FECNT were analyzed in healthy C57BL/6 mice using the dedicated small-animal PET tomograph quad-HIDAC. Furthermore, [(18)F]FECNT was tested in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. RESULTS: [(18)F]FDOPA and [(18)F]FMT failed to clearly visualize the mouse striatum, whereas PET experiments using [(18)F]FECNT proved that the employed methodology is capable of delineating the striatum in mice with exquisite resolution. Moreover, [(18)F]FECNT PET imaging of healthy and MPTP-lesioned mice demonstrated that the detection and quantification of striatal degeneration in lesioned mice can be accomplished. CONCLUSIONS: This study shows the feasibility of using [(18)F]FECNT PET to analyze noninvasively the striatal degeneration in the MPTP mouse model of PD. This methodology can be therefore considered as a viable complement to established in vivo microdialysis and postmortem techniques.

Hyperdopaminergia and altered locomotor activity in GABAB1-deficient mice.

GABAB1-/- mice, which are devoid of functional GABAB receptors, consistently exhibit marked hyperlocomotion when exposed to a novel environment. Telemetry recordings now revealed that, in a familiar environment, GABAB1-/- mice display an altered pattern of circadian activity but no hyperlocomotion. This indicates that hyperlocomotion is only triggered when GABAB1-/- mice are aroused by novelty. In microdialysis experiments, GABAB1-/- mice exhibited a 2-fold increased extracellular level of dopamine in the striatum. Following D-amphetamine administration, GABAB1-/- mice released less dopamine than wild-type mice, indicative of a reduced cytoplasmic dopamine pool. The hyperdopaminergic state of GABAB1-/- mice is accompanied by molecular changes, including reduced levels of tyrosine hydroxylase mRNA, D1 receptor binding-sites and Ser40 phosphorylation of tyrosine hydroxylase. Tyrosine hydroxylase activity, tissue dopamine content and dopamine metabolism do not appear to be measurably altered. Pharmacological and electrophysiological experiments support that the hyperdopaminergic state of GABAB1-/- mice is not severe enough to inactivate dopamine D2 receptors and to disrupt D2-mediated feedback inhibition of tyrosine hydroxylase activity. The data support that loss of GABAB activity results in a sustained moderate hyperdopaminergic state, which is phenotypically revealed by contextual hyperlocomotor activity. Importantly, the presence of an inhibitory GABA tone on the dopaminergic system mediated by GABAB receptors provides an opportunity for therapeutic intervention.

The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria.

Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.

Interrupting apoptosis in neurodegenerative disease: potential for effective therapy?

Current treatment options for neurodegenerative diseases are limited and mainly affect only the symptoms of disease. Because of the unknown and probably multiple causes of these diseases, they cannot be readily targeted. However, it has been established that apoptosis contributes to neuronal loss in most neurodegenerative diseases. A possible treatment option is to interrupt the signaling networks that link neuronal damage to apoptotic degradation in neurodegeneration. The viability of this option depends upon the extent to which apoptosis accounts for neuron loss, whether or not interruption of apoptosis signaling results in recovery of neurological function and whether or not there are significant downsides to targeting apoptosis. Several compounds acting at different sites in known apoptotic signaling networks are currently in development and a few are in clinical trial. If an apoptosis-targeted compound succeeds in slowing or halting neurological dysfunction in one or more neurodegenerative diseases, a new era in the treatment of neurodegenerative diseases will begin.

Specific gamma-hydroxybutyrate-binding sites but loss of pharmacological effects of gamma-hydroxybutyrate in GABA(B)(1)-deficient mice.

gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.

Cyclophilin D as a drug target.

The mitochondrial permeability transition (MPT) plays an important role in damage-induced cell death, and agents inhibiting the MPT may have a therapeutic potential for treating human conditions such as ischemia/reperfusion injury, trauma, and neurodegenerative diseases. The mitochondrial matrix protein, cyclophilin D (CYP D), a member of a family of highly homologous peptidylprolyl cis-trans isomerases (PPIases), plays a decisive role in MPT, being an integral constituent of the MPT pore. Other putative MPT pore proteins include the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC). In an alternative model, the MPT pore is formed by clusters of misfolded membrane proteins outlining aqueous channels that are regulated by CYP D and other chaperone-like proteins. Like cyclophilin A (CYP A) and other cyclophilin family members, CYP D is targeted by the immunosuppressant cyclosporin A (CsA). CsA is cytoprotective in many cellular and animal models, but protection may result from either inhibition of the MPT through an interaction with CYP D or inhibition of calcineurin-mediated dephosphorylation of BAD through an interaction with CYP A. The relevance of MPT inhibition by CsA for its cytoprotective effects is well documented in many cellular models. Mechanisms of action in vivo are more difficult to define, and accordingly the evidence is as yet less compelling in in vivo animal models of ischemia/reperfusion injury, trauma and neurodegenerative diseases. Notwithstanding, CYP D is a drug target of high interest. Structural considerations suggest feasibility of designing CYP D ligands without immunosuppressant properties. This is highly desirable, since they have the potential of being useful therapeutic agents in a variety of disease states. It might be a tougher challenge to obtain compounds specific for CYP D vs. other cyclophilins, and/or of small molecular weight, allowing brain penetration to make them suitable for treating neurodegenerative diseases.

Prospects for antiapoptotic drug therapy of neurodegenerative diseases.

The evidence for a role of apoptosis in the neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), and in the more acute conditions of cerebral ischemia, traumatic brain injury (TBI), and spinal cord injury (SCI) is reviewed with regard to potential intervention by means of small antiapoptotic molecules. In addition, the available animal models for these diseases are discussed with respect to their relevance for testing small antiapoptotic molecules in the context of what is known about the apoptotic pathways involved in the diseases and the models. The principal issues related to pharmacotherapy by apoptosis inhibition, i.e., functionality of rescued neurons and potential interference with physiologically occurring apoptosis, are pointed out. Finally, the properties of a number of small antiapoptotic molecules currently under clinical investigation are summarized. It is concluded that the evidence for a role of apoptosis at present is more convincing for PD and ALS than for AD. In PD, damage to dopaminergic neurons may occur through oxidative stress and/or mitochondrial impairment and culminate in activation of an apoptotic, presumably p53-dependent cascade; some neurons experiencing energy failure may not be able to complete apoptosis, end up in necrosis and give rise to inflammatory processes. These events are reasonably well reflected in some of the PD animal models, notably those involving 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone. In sporadic ALS, an involvement of pathways involving p53 and Bcl-2 family members appears possible if not likely, but is not established. The issue is important for the development of antiapoptotic compounds for the treatment of this disease because of differential involvement of p53 in different mutant superoxide dismutase (SOD) mice. Most debated is the role of apoptosis in AD; this implies that little is known about potentially involved pathways. Moreover, there is a lack of suitable animal models for compound evaluation. Apoptosis or related phenomena are likely involved in secondary cell death in cerebral ischemia, TBI, and SCI. Most of the pertinent information comes from animal experiments, which have provided some evidence for prevention of cell death by antiapoptotic treatments, but little for functional benefit. Much remains to be done in this area to explore the potential of antiapoptotic drugs. There is a small number of antiapoptotic compounds in clinical development. With some of them, evidence for maintenance of functionality of the rescued neurons has been obtained in some animal models, and the fact that they made it to phase II studies in patients suggests that interference with physiological apoptosis is not an obligatory problem. The prospect that small antiapoptotic molecules will have an impact on the therapy of neurodegenerative diseases, and perhaps also of ischemia and trauma, is therefore judged cautiously positively.

Inhibition of the mitochondrial permeability transition by the nonimmunosuppressive cyclosporin derivative NIM811.

Cyclosporin A (CsA) shows cytoprotective properties in many cellular and in vivo models that may depend on interference of the interaction of cyclophilin A with calcineurin or of cyclophilin D with the mitochondrial permeability transition (PT) pore. The nonimmunosuppressive cyclosporin derivative N-methyl-4-valine-cyclosporin (PKF220-384) inhibits the mitochondrial permeability transition (MPT) like CsA but without calcineurin inactivation. PKF220-384 has been used to discriminate between PT pore- and calcineurin mediated effects but is no longer available. Here, we evaluated the effects of another nonimmunosuppressive cyclosporin derivative, N-methyl-4-isoleucine-cyclosporin (NIM811) on the MPT. Using two newly developed microtiter plate assays, one measuring mitochondrial swelling from absorbance and the other measuring mitochondrial membrane potential from changes in safranin fluorescence, we show that NIM811 blocks the MPT induced by calcium and inorganic phosphate, alone or in combination with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the complex I inhibitor rotenone, and the prooxidant t-butylhydroperoxide. NIM811 was equipotent to CsA and half as potent as PKF220-384. Additionally, we show that NIM811 blocks cell killing and prevents in situ mitochondrial inner membrane permeabilization and depolarization during tumor necrosis factor-alpha-induced apoptosis to cultured rat hepatocytes. NIM811 inhibition of apoptosis was equipotent with CsA except at higher concentrations: CsA lost efficacy but NIM 811 did not. We conclude that NIM811 is a useful alternative to PKF220-384 to investigate the role of the mitochondrial permeability transition in apoptotic and necrotic cell death.