Zoe A. Hughes

BACE1 (β-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes

BACE1 is a key enzyme in the generation of Abeta, the major component of senile plaques in the brains of Alzheimers disease patients. We have generated transgenic mice expressing human BACE1 with the Cam Kinase II promoter driving neuronal-specific expression. The transgene contains the full-length coding sequence of human BACE1 preceding an internal ribosome entry site element followed by a LacZ reporter gene. These animals exhibit a bold, exploratory behavior and show elevated 5-hydroxytryptamine turnover. We have also generated a knockout mouse in which LacZ replaces the first exon of murine BACE1. Interestingly these animals show a contrasting behavior, being timid and less exploratory. Despite these clear differences both mouse lines are viable and fertile with no changes in morbidity. These results suggest an unexpected role for BACE1 in neurotransmission, perhaps through changes in amyloid precursor protein processing and Abeta levels.

LPA1 receptor-deficient mice have phenotypic changes observed in psychiatric disease

Several psychiatric diseases, including schizophrenia, are thought to have a developmental aetiology, but to date no clear link has been made between psychiatric disease and a specific developmental process. LPA(1) is a G(i)-coupled seven transmembrane receptor with high affinity for lysophosphatidic acid. Although LPA(1) is expressed in several peripheral tissues, in the nervous system it shows relatively restricted temporal expression to neuroepithelia during CNS development and to myelinating glia in the adult. We report the detailed neurological and behavioural analysis of mice homozygous for a targeted deletion at the lpa(1) locus. Our observations reveal a marked deficit in prepulse inhibition, widespread changes in the levels and turnover of the neurotransmitter 5-HT, a brain region-specific alteration in levels of amino acids, and a craniofacial dysmorphism in these mice. We suggest that the loss of LPA(1) receptor generates defects resembling those found in psychiatric disease.

High-performance liquid chromatography/tandem mass spectrometry assay for the rapid high sensitivity measurement of basal acetylcholine from microdialysates

A high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the analysis of acetylcholine (ACh) in brain dialysates. This separation of ACh is based on cation exchange chromatography with elution buffer consisting of a mixture of ammonium acetate, ammonium formate and acetonitrile. Using isocratic separation conditions, ACh was resolved within a minute and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for ACh was found to be 1 fmol on column with a S/N ratio of 3:1. The assay has been used routinely for the measurement of ACh in brain dialysates from awake freely moving rats. Furthermore, separation conditions were modified to allow simultaneous measurement of ACh and the acetylcholine esterase inhibitor, neostigmine.

Neurochemical Changes in LPA1 Receptor Deficient Mice – A Putative Model of Schizophrenia

LPA1 is a Gi-coupled seven transmembrane receptor with high affinity for the ligand lysophosphatidic acid. We have investigated the effect of targeted deletion at the lpa1 locus on evoked release of amino acids from hippocampal slices, using in vitro superfusion techniques, and evoked 5-HT efflux from the dorsal raphe nucleus, using invitro fast cyclic voltammetry. Superfusion of hippocampal slices revealed that basal levels of tyrosine, aspartate and glutamate release were significantly increased while K+-evoked release of glutamate and GABA were significantly decreased in lpa1(−/−) mice. Fast cyclic voltammetry measurements in the dorsal raphe nucleus demonstrated significant decreases in electrically evoked 5-HT efflux in lpa1(−/−) mice. In summary, these data demonstrate that the lpa1 mutation produces a number of changes in neurotransmitters that have been associated with a schizophrenic-like pathology.

Estrogen Receptor Neurobiology and its Potential for Translation into Broad Spectrum Therapeutics for CNS Disorders

Estrogens are hormones that modulate a diverse array of effects during development and adulthood. The effects of estrogen are mediated by two estrogen receptor (ER) isotypes, ERalpha and ERbeta, which classically function as transcription factors to modulate specific target gene expression and in addition regulate a growing list of intracellular signaling cascades. These receptors share protein sequence homology and protein-motif organization but have distinct differences in their tissue distribution and binding affinities for their ligands. In the nervous system estrogen has been implicated to play a role in a number of processes which regulate synaptic plasticity including synaptogenesis and neurogenesis. The role for estrogen in a range of neurological and neuropsychiatric diseases is also becoming very apparent. Estrogen is able to regulate processes and behaviours relevant for both Alzheimers disease and schizophrenia and to modulate neuroendocrine and inflammatory processes important in neuroinflammation, anxiety and depressive disorders as well as chronic pain. We will consider the rationale for estrogen-based therapies for diseases of the nervous system. In particular we will highlight the molecular mechanisms and signal transduction pathways most likely underlying the effects of estrogen in the CNS.

5-Piperazinyl-3-sulfonylindazoles as Potent and Selective 5-Hydroxytryptamine-6 Antagonists

As part of our efforts to develop agents for CNS diseases, we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands for cognitive enhancement. Herein we report the identification of a novel series of 5-piperazinyl-3-sulfonylindazoles as potent and selective 5-HT(6) antagonists. The synthesis, SAR, and pharmacokinetic and pharmacological activities of some of the compounds including 3-(naphthalen-1-ylsulfonyl)-5-(piperazin-1-yl)-1H-indazole (WAY-255315 or SAM-315) will be described.

Correlating Efficacy in Rodent Cognition Models with in Vivo 5-Hydroxytryptamine1A Receptor Occupancy by a Novel Antagonist, (R)-N-(2-Methyl-(4-indolyl-1-piperazinyl)ethyl)-N-(2-pyridinyl)-cyclohexane Carboxamide (WAY-101405)

5-Hydroxytryptamine (5-HT)1A receptors play an important role in multiple cognitive processes, and compelling evidence suggests that 5-HT1A antagonists can reverse cognitive impairment. We have examined the therapeutic potential of a potent (Ki = 1.1 nM), selective (>100-fold), orally bioavailable, silent 5-HT1A receptor antagonist (KB = 1.3 nM) (R)-N-(2-methyl-(4-indolyl-1-piperazinyl)-ethyl)-N-(2-pyridinyl)-cyclohexane carboxamide (WAY-101405). Oral administration of WAY-101405 was shown to be effective in multiple rodent models of learning and memory. In a novel object recognition paradigm, 1 mg/kg enhanced retention (memory) for previously learned information, and it was able to reverse the memory deficits induced by scopolamine. WAY-101405 (1 mg/kg) was also able to reverse scopolamine-induced deficits in a rat contextual fear conditioning model. In the Morris water maze, WAY-101405 (3 mg/kg) significantly improved learning in a paradigm of increasing task difficulty. In vivo microdialysis studies in the dorsal hippocampus of freely moving adult rats demonstrated that acute administration of WAY-101405 (10 mg/kg) increased extracellular acetylcholine levels. The selective radioligand [3H]WAY-100635, administered i.v., was used for in vivo receptor occupancy studies, where WAY-101405 occupied 5-HT1A receptors in the rat cortex, with an ED50 value of 0.1 mg/kg p.o. Taken together, these studies demonstrate that WAY-101405 is a potent and selective, brain penetrant, orally bioavailable 5-HT1A receptor “silent” antagonist that is effective in preclinical memory paradigms at doses where approximately 90% of the postsynaptic 5-HT1A receptors are occupied. These results further support the rationale for use of this compound class in the treatment of cognitive dysfunction associated with psychiatric and neurological conditions.

Prefrontal Cortical Kappa Opioid Receptors Attenuate Responses to Amygdala Inputs

Kappa opioid receptors (KORs) have been implicated in anxiety and stress, conditions that involve activation of projections from the basolateral amygdala (BLA) to the medial prefrontal cortex (mPFC). Although KORs have been studied in several brain regions, their role on mPFC physiology and on BLA projections to the mPFC remains unclear. Here, we explored whether KORs modify synaptic inputs from the BLA to the mPFC using in vivo electrophysiological recordings with electrical and optogenetic stimulation. Systemic administration of the KOR agonist U69,593 inhibited BLA-evoked synaptic responses in the mPFC without altering hippocampus-evoked responses. Intra-mPFC U69,593 inhibited electrical and optogenetic BLA-evoked synaptic responses, an effect blocked by the KOR antagonist nor-BNI. Bilateral intra-mPFC injection of the KOR antagonist nor-BNI increased center time in the open field test, suggesting an anxiolytic effect. The data demonstrate that mPFC KORs negatively regulate glutamatergic synaptic transmission in the BLA–mPFC pathway and anxiety-like behavior. These findings provide a framework whereby KOR signaling during stress and anxiety can regulate the flow of emotional state information from the BLA to the mPFC.

WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one]: A Novel Dopamine D2 Receptor Partial Agonist/Serotonin Reuptake Inhibitor with Preclinical Antipsychotic-Like and Antidepressant-Like Activity

The preclinical characterization of WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one] is described. In vitro binding and functional studies revealed highest affinity to the D2 receptor (D2L Ki, 4.0 nM) and serotonin transporter (Ki, 7.1 nM), potent D2 partial agonist activity (EC50, 0.38 nM; Emax, 30%), and complete block of the serotonin transporter (IC50, 56.4 nM). Consistent with this in vitro profile, WS-50030 (10 mg/kg/day, 21 days) significantly increased extracellular 5-HT in the rat medial prefrontal cortex, short-term WS-50030 treatment blocked apomorphine-induced climbing (ID50, 0.51 mg/kg) in a dose range that produced minimal catalepsy in mice and induced low levels of contralateral rotation in rats with unilateral substantia nigra 6-hydroxydopamine lesions (10 mg/kg i.p.), a behavioral profile similar to that of the D2 partial agonist aripiprazole. In a rat model predictive of antipsychotic-like activity, WS-50030 and aripiprazole reduced conditioned avoidance responding by 42 and 55% at 10 mg/kg, respectively. Despite aripiprazoles reported lack of effect on serotonin transporters, long-term treatment with aripiprazole or WS-50030 reversed olfactory bulbectomy-induced hyperactivity at doses that did not reduce activity in sham-operated rats, indicating antidepressant-like activity for both compounds. Despite possessing serotonin reuptake inhibitory activity in addition to D2 receptor partial agonism, WS-50030 displays activity in preclinical models predictive of antipsychotic- and antidepressant efficacy similar to aripiprazole, suggesting potential efficacy of WS-50030 versus positive and negative symptoms of schizophrenia, comorbid mood symptoms, bipolar disorder, major depressive disorder, and treatment-resistant depression. Furthermore, WS-50030 provides a tool to further explore how combining these mechanisms might differentiate from other antipsychotics or antidepressants.

The synthesis and biological evaluation of quinolyl-piperazinyl piperidines as potent serotonin 5-HT1A antagonists.

As part of an effort to identify 5-HT(1A) antagonists that did not possess typical arylalkylamine or keto/amido-alkyl aryl piperazine scaffolds, prototype compound 10a was identified from earlier work in a combined 5-HT(1A) antagonist/SSRI program. This quinolyl-piperazinyl piperidine analogue displayed potent, selective 5-HT(1A) antagonism but suffered from poor oxidative metabolic stability, resulting in low exposure following oral administration. SAR studies, driven primarily by in vitro liver microsomal stability assessment, identified compound 10b, which displayed improved oral bioavailability and lower intrinsic clearance. Further changes to the scaffold (e.g., 10r) resulted in a loss in potency. Compound 10b displayed cognitive enhancing effects in a number of animal models of learning and memory, enhanced the antidepressant-like effects of the SSRI fluoxetine, and reversed the sexual dysfunction induced by chronic fluoxetine treatment.