Selective and brain-permeable polo-like kinase-2 (Plk-2) inhibitors that reduce α-synuclein phosphorylation in rat brain.

Polo-like kinase-2 (Plk-2) has been implicated as the dominant kinase involved in the phosphorylation of α-synuclein in Lewy bodies, which are one of the hallmarks of Parkinson's disease neuropathology. Potent, selective, brain-penetrant inhibitors of Plk-2 were obtained from a structure-guided drug discovery approach driven by the first reported Plk-2-inhibitor complexes. The best of these compounds showed excellent isoform and kinome-wide selectivity, with physicochemical properties sufficient to interrogate the role of Plk-2 inhibition in vivo. One such compound significantly decreased phosphorylation of α-synuclein in rat brain upon oral administration and represents a useful probe for future studies of this therapeutic avenue toward the potential treatment of Parkinson's disease.

Pharmacological inhibition of polo like kinase 2 (PLK2) does not cause chromosomal damage or result in the formation of micronuclei.

Polo like kinase 2 (PLK2) phosphorylates α-synuclein and is considered a putative therapeutic target for Parkinson's disease. Several lines of evidence indicate that PLK2 is involved with proper centriole duplication and cell cycle regulation, inhibition of which could impact chromosomal integrity during mitosis. The objectives of the series of experiments presented herein were to assess whether specific inhibition of PLK2 is genotoxic and determine if PLK2 could be considered a tractable pharmacological target for Parkinson's disease. Several selective PLK2 inhibitors, ELN 582175 and ELN 582646, and their inactive enantiomers, ELN 582176 and ELN 582647, did not significantly increase the number of micronuclei in the in vitro micronucleus assay. ELN 582646 was administered to male Sprague Dawley rats in an exploratory 14-day study where flow cytometric analysis of peripheral blood identified a dose-dependent increase in the number of micronucleated reticulocytes. A follow-up investigative study demonstrated that ELN 582646 administered to PLK2 deficient and wildtype mice significantly increased the number of peripheral micronucleated reticulocytes in both genotypes, suggesting that ELN 582646-induced genotoxicity is not through the inhibition of PLK2. Furthermore, significant reduction of retinal phosphorylated α-synuclein levels was observed at three non-genotoxic doses, additional data to suggest that pharmacological inhibition of PLK2 is not the cause of the observed genotoxicity. These data, in aggregate, indicate that PLK2 inhibition is a tractable CNS pharmacological target that does not cause genotoxicity at doses and exposures that engage the target in the sensory retina.

Design and synthesis of highly selective, orally active Polo-like kinase-2 (Plk-2) inhibitors.

Polo-like kinase-2 (Plk-2) is a potential therapeutic target for Parkinson's disease and this Letter describes the SAR of a series of dihydropteridinone based Plk-2 inhibitors. By optimizing both the N-8 substituent and the biaryl region of the inhibitors we obtained single digit nanomolar compounds such as 37 with excellent selectivity for Plk-2 over Plk-1. When dosed orally in rats, compound 37 demonstrated a 41-45% reduction of pS129-α-synuclein levels in the cerebral cortex.

Development of an enzyme-linked immunosorbent assay (ELISA) to measure the level of tyrosine hydroxylase protein in brain tissue from Parkinson's disease models.

Tyrosine hydroxylase (TH) catalyses the rate-limiting step in the biosynthesis of catecholamines. TH expression is regulated in a tissue-specific manner during neuronal development and differentiation. Because of its key regulatory role in central and peripheral catecholamine synthesis, TH is associated with the pathogenesis of several neurological and psychiatric diseases, including Parkinson's disease, dystonia, schizophrenia, affective disorders, and cardiovascular diseases. Therefore, developing a quantitative method to monitor the changes in TH expression in disease models could facilitate the identification and characterisation of neuromodulatory and neuroprotective therapeutic agents. The present report describes the generation and characterisation of a new set of monoclonal TH antibodies and the development of a novel sandwich ELISA for the quantitative detection of the TH protein in rodent brain tissue. This ELISA exhibits excellent reproducibility and good linearity in the analysis of complex brain tissue lysates. The cross-validation of the TH ELISA using semi-quantitative TH Western blot methods and HPLC measurement of dopamine levels suggests that the new TH ELISA is sufficiently sensitive to detect small-to-moderate region-specific differences, developmental changes, and Parkinson's disease-related changes in TH expression in rodent brains. This new TH ELISA also offers greater flexibility than conventional HPLC-based dopamine assays because the optimal tissue lysis buffer used for the detection of TH in brain tissue is also compatible with the analysis of other proteins associated with Parkinson's disease, such as α-synuclein, suggesting that this TH ELISA could be used in a multiplexed format.

Effects of mGlu2 or mGlu3 receptor deletions on mGlu2/3 receptor agonist (LY354740)-induced brain c-Fos expression: specific roles for mGlu2 in the amygdala and subcortical nuclei, and mGlu3 in the hippocampus.

LY354740 is a potent and selective mGlu2/3 receptor agonist with activity in models of psychiatric disorders (anxiety, psychosis), and early clinical studies in anxiety patients. However, the specific receptor subtypes and brain regions which mediate mGlu2/3 receptor agonist pharmacology/efficacy are not well understood. Here we investigate the effects of deleting mGlu2 or mGlu3 receptors on basal and LY354740-regulated c-Fos expression in mouse brain using mGlu2 or mGlu3 knockout mice. Consistent with our earlier findings, LY354740 administration (20 mg/kg, i.p.) to wild-type mice increased c-Fos expression in specific limbic (central amygdala, bed nucleus of the stria terminalis, midline thalamic nuclei) and non-limbic (thalamic dorsolateral geniculate nucleus, superior colliculus, Edinger-Westphal) structures, while modestly suppressing hippocampal c-Fos expression. The LY354740-induced increases in c-Fos expression in all the above regions were abolished by mGlu2, but not mGlu3, receptor deletion. Interestingly, basal c-Fos expression was significantly increased in the hippocampus of mGlu3, but not mGlu2, receptor knockouts compared to wild-type mice. Moreover, this increase was not suppressed by LY354740, such that in the CA3 region LY354740 now increased c-Fos expression in the mGlu3 knockouts. These results demonstrate that the LY354740-induced increases of c-Fos expression in specific brain regions, including the central and extended amygdala are specifically linked to mGlu2 receptors, and LY354740 suppressions of neuronal activity in the hippocampus are linked to mGlu3 receptors.

Hypoxia-induced ischemic tolerance in neonatal rat brain involves enhanced ERK1/2 signaling.

Hypoxic preconditioning (HP) 24 h before hypoxic-ischemic (HI) injury confers significant neuroprotection in neonatal rat brain. Recent studies have shown that the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) intracellular signaling pathways play a role in the induction of tolerance to ischemic injury in heart and brain. To study the role of MAPK (ERK1/2, JNK, p38MAPK) and PI3K/Akt/GSK3beta signaling pathways in hypoxia-induced ischemic tolerance, we examined the brains of newborn rats at different time points after exposure to sublethal hypoxia (8% O(2) for 3 h). Immunoblot analysis showed that HP had no effect on the levels of phosphorylated Akt, GSK3beta, JNK and p38MAPK. In contrast, significantly increased levels of phosphorylated ERK1/2 were observed 0.5 h after HP. Double immunofluorescence staining showed that hypoxia-induced ERK1/2 phosphorylation was found mainly in microvessels throughout the brain and in astrocytes in white matter tracts. Inhibition of hypoxia-induced ERK1/2 pathway with intracerebral administration of U0126 significantly attenuated the neuroprotection afforded by HP against HI injury. These findings suggest that activation of ERK1/2 signaling may contribute to hypoxia-induced tolerance in neonatal rat brain in part by preserving vascular and white matter integrity after HI.

Systemic administration of the potent mGlu8 receptor agonist (S)-3,4-DCPG induces c-Fos in stress-related brain regions in wild-type, but not mGlu8 receptor knockout mice.

The effect of a novel and potent metabotropic glutamate 8 (mGlu8) receptor agonist, (S)-3,4-dicarboxyphenylglycine (DCPG), was studied in vivo in mouse brain. c-Fos expression was used as a marker of neuronal activity in specific brain regions 2 h after systemic (S)-3,4-DCPG (3-100 mg/kg, i.p.). The selectivity of (S)-3,4-DCPG on mGlu8 receptors was determined in mGlu8 receptor knockout mice. In wild-type mice, (S)-3,4-DCPG (100 mg/kg) significantly increased c-Fos expression in several stress-related brain regions: paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, lateral parabrachial nucleus and locus coeruleus. In the central nucleus of the amgydala, more than 92% of c-Fos positive neurons were identified as GABAergic inhibitory neurons after (S)-3,4-DCPG. Moreover, (S)-3,4-DCPG significantly induced c-Fos in the superficial gray layer of the superior colliculus, a central visual region. c-Fos expression was unchanged by (S)-3,4-DCPG in mGlu8 receptor knockout mice. Our results indicate that systemic (S)-3,4-DCPG alters neuronal excitability in specific brain regions via mGlu8 receptor. The prominent activation of stress areas suggests a role for mGlu8 receptors in the central integration of stress responses. Furthermore, our results indicate that systemic (S)-3,4-DCPG can be used as a tool to explore behavioral and cellular consequences of mGlu8 receptor activation.

Inducing photochemical cortical lesions in rat brain.

In the rat photochemical cortical lesion model described in this unit, an intravascular photochemical reaction induces endothelial damage resulting in platelet aggregation, thrombosis, thrombotic response (secretion of factors by the platelets) and permanent cerebral vascular occlusion. Because thrombosis is produced in pial vessels, the resulting cortical infarct is generally smaller and more reproducible than in the models involving occlusion of the middle cerebral artery. The surgical procedures involved are limited, making this model generally easier to perform and less invasive than most other models of permanent focal ischemia that involve mechanical occlusion of major cerebral arteries.