Novel LRRK2 GTP-binding inhibitors reduced degeneration in Parkinson's disease cell and mouse models.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.

Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine.

We investigated the subcellular distribution of dopamine D(1), D(2) and D(5) receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D(1) receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D(2) receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D(5) receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D(5) receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D(1) receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D(5) and D(2) receptor subtypes were not significantly altered by cocaine treatment. These data imply that D(5) receptors are predominantly cytoplasmic, D(2) receptors are diffusely distributed within the cell, whereas D(1) receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation.

Characterization of the transport, metabolism, and pharmacokinetics of the dopamine D3 receptor-selective fluorenyl- and 2-pyridylphenyl amides developed for treatment of psychostimulant abuse.

The recent discovery of novel high-affinity and selective dopamine D3 receptor (DA D3R) antagonists and partial agonists has provided tools with which to further elucidate the role DA D3R plays in substance abuse. The present study was conducted to evaluate the transport, metabolism, pharmacokinetics, and brain uptake of the DA D3R-selective fluorenyl amides, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide] fumarate) and JJC 4-077 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-9H-fluorene-2-carboxamide hydrochloride], and the 2-pyridylphenyl amides, CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridine-2-yl)benzamide hydrochloride] and PG 01037 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-(pyridine-2-yl)benzamide hydrochloride], all of which have been studied in animal models of psychostimulant abuse. Additional screening with a panel of human and rat Supersomes was performed for NGB 2904 and PG 01037. Drug-stimulated ATPase activation assays and bidirectional transport and efflux assays were used to test for substrate specificity of NGB 2904 and PG 01037 for human and rat efflux transporters. All compounds exhibited moderate elimination half-lives, ranging from 1.49 to 3.27 h, and large volumes of distribution (5.95-14.19 l/kg). The brain-to-plasma ratios ranged from 2.93 to 11.81 and were higher than those previously reported for cocaine. Brain exposure levels of NGB 2904 and PG 01037 were significantly reduced after intraperitoneal administration compared with intravenous administration. The metabolism of these compounds was mediated primarily by CYP3A subfamilies. PG 01037 was a P-glycoprotein-transported substrate. Higher doses of these compounds are often required for in vivo action, suggesting decreased bioavailability via extravascular administration that may be attributed to high drug efflux and hepatic metabolism. These studies provide important preclinical information for optimization of next-generation D3R selective agents for the treatment of drug addiction.

Estradiol and progesterone-mediated regulation of P-gp in P-gp overexpressing cells (NCI-ADR-RES) and placental cells (JAR).

The effect of progesterone and estrogen treatment on the expression and function of P-glycoprotein (P-gp) was evaluated in JAR cells and a P-gp overexpressing cell line, NCI-ADR-RES. Western blot analysis and real-time Q-PCR were used to evaluate P-gp protein and MDR1 mRNA expression respectively in the cells following incubation with progesterone (P4) and/or beta-estradiol (E2). Cellular uptake studies of the P-gp substrates, saquinavir and paclitaxel, were performed to evaluate function. Treatment with either E2 or P4 resulted in a significant increase in P-gp protein levels in the NCI-ADR-RES cells at concentrations of or greater than 100 nM or 10 nM, respectively. JAR cells also had increased levels of P-gp with 100 nM of P4 but were much more sensitive to E2 showing increased P-gp at a concentration of 1 nM. Furthermore, E2 or P4 treatment resulted in a significant decrease in cellular uptake of the P-gp substrates tested in these cells lines. Based on mRNA quantitation, a transient increase (2-fold) in MDR1 levels was observed at 8 h postincubation with either E2 or P4, while MDR1 levels remained high in the JAR cells treated with E2 for 72 h postincubation. The addition of actinomycin D, a transcription inhibitor negated the increase in P-gp by P4 and E2. P4 and E2 increase P-gp expression and function in NCI-ADR-RES and JAR cells with the ERalpha-expressing cells (JAR) much more sensitive to E2. Furthermore, transcriptional regulation by E2 and P4 likely contributes to the modulation of P-gp levels.

Sex differences in the expression of calcitonin gene-related peptide receptor components in the spinal trigeminal nucleus.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) plays an important role in migraine pathophysiology. CGRP acts primarily by activating a receptor composed of 3 proteins: calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP). We tested the hypothesis that sex differences exist in protein levels of two key components of this CGRP receptor: CLR and RCP. METHODS: We used specific antibodies to assess baseline protein levels of CLR and RCP in the spinal trigeminal nucleus caudalis (SpVc) and upper cervical spinal cord of both male and female rats. We also tested if manipulations that knock-down the expression of RCP in SpVc, using locally-mediated gene transfer of short hairpin RNA (shRNA), ameliorate pain in an animal model of intracranial migraine-like pain induced by chemical noxious stimulation of the meninges. To assess pain, we used tests of ongoing pain (rat face grimace test and freezing behavior) and tests of facial mechanical hypersensitivity and allodynia. RESULTS: There was no difference in CLR levels between male and female animals (p > 0.11) in SpVc and the upper cervical cord. However, female animals exhibited greater baseline levels of RCP (up to 3-fold higher) compared to males (p < 0.002). The knock-down of RCP expression in SpVc attenuated mechanical facial allodynia induced by chemical noxious stimulation of the meninges, but had little effect on ongoing pain behaviors in female and male animals. CONCLUSIONS: RCP is an integral component of the CGRP receptor and may play a key role in mediating CGRP induced central sensitization after noxious stimulation of the meninges. RCP expression in the SpVc and upper cervical cord is sexually dimorphic, with higher levels of expression in females. This dimorphism may be related to the increased incidence of migraines in females-a hypothesis that should be tested in the future.

Loss of dopamine D1 receptors and diminished D1/5 receptor-mediated ERK phosphorylation in the periaqueductal gray after spinal cord lesion.

Neuropathic pain resulting from spinal cord injury is often accompanied by maladaptive plasticity of the central nervous system, including the opioid receptor-rich periaqueductal gray (PAG). Evidence suggests that sensory signaling via the PAG is robustly modulated by dopamine D1- and D2-like receptors, but the effect of damage to the spinal cord on D1 and D2 receptor protein expression and function in the PAG has not been examined. Here we show that 21days after a T10 or C6 spinothalamic tract lesion, both mice and rats display a remarkable decline in the expression of D1 receptors in the PAG, revealed by western blot analysis. These changes were associated with a significant reduction in hindpaw withdrawal thresholds in lesioned animals compared to sham-operated controls. We investigated the consequences of diminished D1 receptor levels by quantifying D1-like receptor-mediated phosphorylation of ERK1,2 and CREB, events that have been observed in numerous brain structures. In naïve animals, western blot analysis revealed that ERK1,2, but not CREB phosphorylation was significantly increased in the PAG by the D1-like agonist SKF 81297. Using immunohistochemistry, we found that SKF 81297 increased ERK1,2 phosphorylation in the PAG of sham animals. However, in lesioned animals, basal pERK1,2 levels were elevated and did not significantly increase after exposure to SKF 81297. Our findings provide support for the hypothesis that molecular adaptations resulting in a decrease in D1 receptor expression and signaling in the PAG are a consequence of SCL.

Metabotropic glutamate receptors and dopamine receptors cooperate to enhance extracellular signal-regulated kinase phosphorylation in striatal neurons.

Striatal medium spiny neurons are an important site of convergence for signaling mediated by the neurotransmitters dopamine and glutamate. We report that in striatal neurons in primary culture, signaling through group I metabotropic glutamate receptors (mGluRs) 1/5 and the D1 class of dopamine receptors (DRs) 1/5 converges to increase phosphorylation of the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2). Induction of mitogen-activated protein kinase kinase-dependent signaling cascades by either mGluR1/5 or DR1/5 gave rise to increases in phosphorylation of ERK2. Coactivation of mGluR1/5 and DR1/5 with (S)-3,5-dihydroxyphenylglycine and (+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride enhanced the phosphorylation of ERK2. This interaction between mGluR1/5 and DR1/5 required protein kinase C (PKC), because the PKC inhibitors calphostin C, bisindolylmaleimide I, and Gö6976 blocked DR1/5-enhanced phosphorylation of ERK2. Use of the phosphatase inhibitors calyculin and okadaic acid indicated that inhibition of protein phosphatases 1 and 2A dramatically enhanced ERK2 phosphorylation by mGluR1/5. Coactivation of mGluR1/5 and DR1/5 also enhanced cAMP-response element binding protein (CREB) phosphorylation (compared with each receptor agonist alone) but did not enhance CREB-mediated transcriptional activity. Thus, signal transduction pathways activated by DR1/5 and mGluR5 interact to modify downstream events in striatal neurons while retaining numerous regulatory checkpoints.

Motor cortex stimulation suppresses cortical responses to noxious hindpaw stimulation after spinal cord lesion in rats.

BACKGROUND: Motor cortex stimulation (MCS) is a potentially effective treatment for chronic neuropathic pain. The neural mechanisms underlying the reduction of hyperalgesia and allodynia after MCS are not completely understood. OBJECTIVE: To investigate the neural mechanisms responsible for analgesic effects after MCS. We test the hypothesis that MCS attenuates evoked blood oxygen-level dependent signals in cortical areas involved in nociceptive processing in an animal model of chronic neuropathic pain. METHODS: We used adult female Sprague-Dawley rats (n = 10) that received unilateral electrolytic lesions of the right spinal cord at the level of C6 (SCL animals). In these animals, we performed magnetic resonance imaging (fMRI) experiments to study the analgesic effects of MCS. On the day of fMRI experiment, 14 days after spinal cord lesion, the animals were anesthetized and epidural bipolar platinum electrodes were placed above the left primary motor cortex. Two 10-min sessions of fMRI were performed before and after a session of MCS (50 µA, 50 Hz, 300 µs, for 30 min). During each fMRI session, the right hindpaw was electrically stimulated (noxious stimulation: 5 mA, 5 Hz, 3 ms) using a block design of 20 s stimulation off and 20 s stimulation on. A general linear model-based statistical parametric analysis was used to analyze whole brain activation maps. Region of interest (ROI) analysis and paired t-test were used to compare changes in activation before and after MCS in these ROI. RESULTS: MCS suppressed evoked blood oxygen dependent signals significantly (Family-wise error corrected P < 0.05) and bilaterally in 2 areas heavily implicated in nociceptive processing. These areas consisted of the primary somatosensory cortex and the prefrontal cortex. CONCLUSIONS: These findings suggest that, in animals with SCL, MCS attenuates hypersensitivity by suppressing activity in the primary somatosensory cortex and prefrontal cortex.