Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson's disease (PD). To support clinical development of LRRK2 inhibitors as ...disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC
's equivalent to IC
's on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.
Background The hemizygous 22q11.2 microdeletion is a common copy number variant in humans. The deletion confers high risk for neurodevelopmental disorders, including autism and schizophrenia. Up to ...41% of deletion carriers experience psychotic symptoms. Methods We present a new mouse model (Df(h22q11)/+) of the deletion syndrome (22q11.2DS) and report on, to our knowledge, the most comprehensive study undertaken to date in 22q11.2DS models. The study was conducted in male mice. Results We found elevated postpubertal N -methyl- d -aspartate (NMDA) receptor antagonist–induced hyperlocomotion, age-independent prepulse inhibition (PPI) deficits and increased acoustic startle response (ASR). The PPI deficit and increased ASR were resistant to antipsychotic treatment. The PPI deficit was not a consequence of impaired hearing measured by auditory brain stem responses. The Df(h22q11)/+ mice also displayed increased amplitude of loudness-dependent auditory evoked potentials. Prefrontal cortex and dorsal striatal elevations of the dopamine metabolite DOPAC and increased dorsal striatal expression of the AMPA receptor subunit GluR1 was found. The Df(h22q11)/+ mice did not deviate from wild-type mice in a wide range of other behavioural and biochemical assays. Limitations The 22q11.2 microdeletion has incomplete penetrance in humans, and the severity of disease depends on the complete genetic makeup in concert with environmental factors. In order to obtain more marked phenotypes reflecting the severe conditions related to 22q11.2DS it is suggested to expose the Df(h22q11)/+ mice to environmental stressors that may unmask latent psychopathology. Conclusion The Df(h22q11)/+ model will be a valuable tool for increasing our understanding of the etiology of schizophrenia and other psychiatric disorders associated with the 22q11DS.
Background Genome-wide scans have uncovered rare copy number variants conferring high risk of psychiatric disorders. The 15q13.3 microdeletion is associated with a considerably increased risk of ...idiopathic generalized epilepsy, intellectual disability, and schizophrenia. Methods A 15q13.3 microdeletion mouse model ( Dfh15q13/+ ) was generated by hemizygous deletion of the orthologous region and characterized with focus on schizophrenia- and epilepsy-relevant parameters. Results Df(h15q13)/+ mice showed marked changes in neuronal excitability in acute seizure assays, with increased propensity to develop myoclonic and absence-like seizures but decreased propensity for clonic and tonic seizures. Furthermore, they had impaired long-term spatial reference memory and a decreased theta frequency in hippocampus and prefrontal cortex. Electroencephalogram characterization revealed auditory processing deficits similar to those observed in schizophrenia. Gamma band power was increased during active state, but evoked gamma power following auditory stimulus (40 Hz) was dramatically reduced, mirroring observations in patients with schizophrenia. In addition, Df(h15q13)/+ mice showed schizophrenia-like decreases in amplitudes of auditory evoked potentials. Although displaying a grossly normal behavior, Df(h15q13)/+ mice are more aggressive following exposure to mild stressors, similar to what is described in human deletion carriers. Furthermore, Df(h15q13)/+ mice have increased body weight, and a similar increase in body weight was subsequently found in a sample of human subjects with 15q13.3 deletion. Conclusions The Df(h15q13)/+ mouse shows similarities to several alterations related to the 15q13.3 microdeletion syndrome, epilepsy, and schizophrenia, offering a novel tool for addressing the underlying biology of these diseases.
Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint ...kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo2,3-bpyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC50 values for 22 in mouse brain and kidney being 1.3 and 5 nM, respectively.
1q21.1 hemizygous microdeletion is a copy number variant leading to eightfold increased risk of schizophrenia. In order to investigate biological alterations induced by this microdeletion, we ...generated a novel mouse model (Df(h1q21)/+) and characterized it in a broad test battery focusing on schizophrenia-related assays. Df(h1q21)/+ mice displayed increased hyperactivity in response to amphetamine challenge and increased sensitivity to the disruptive effects of amphetamine and phencyclidine hydrochloride (PCP) on prepulse inhibition. Probing of the direct dopamine (DA) pathway using the DA D1 receptor agonist SKF-81297 revealed no differences in induced locomotor activity compared to wild-type mice, but Df(h1q21)/+ mice showed increased sensitivity to the DA D2 receptor agonist quinpirole and the D1/D2 agonist apomorphine. Electrophysiological characterization of DA neuron firing in the ventral tegmental area revealed more spontaneously active DA neurons and increased firing variability in Df(h1q21)/+ mice, and decreased feedback reduction of DA neuron firing in response to amphetamine. In a range of other assays, Df(h1q21)/+ mice showed no difference from wild-type mice: gross brain morphology and basic functions such as reflexes, ASR, thermal pain sensitivity, and motor performance were unaltered. Similarly, anxiety related measures, baseline prepulse inhibition, and seizure threshold were unaltered. In addition to the central nervous system-related phenotypes, Df(h1q21)/+ mice exhibited reduced head-to tail length, which is reminiscent of the short stature reported in humans with 1q21.1 deletion. With aspects of both construct and face validity, the Df(h1q21)/+ model may be used to gain insight into schizophrenia-relevant alterations in dopaminergic transmission.
Our understanding of Parkinson's disease (PD) has been revolutionized by the discovery of disease-causing genetic mutations. The most common of these is the G2019S mutation in the LRRK2 kinase gene, ...which leads to increased kinase activity. However, the link between increased kinase activity and PD is unclear. Previously, we showed that dopaminergic expression of the human LRRK2-G2019S transgene in flies led to an activity-dependent loss of vision in older animals and we hypothesized that this may have been preceded by a failure to regulate neuronal activity correctly in younger animals. To test this hypothesis, we used a sensitive measure of visual function based on frequency-tagged steady-state visually evoked potentials. Spectral analysis allowed us to identify signals from multiple levels of the fly visual system and wild-type visual response curves were qualitatively similar to those from human cortex. Dopaminergic expression of hLRRK2-G2019S increased contrast sensitivity throughout the retinal network. To test whether this was due to increased kinase activity, we fed Drosophila with kinase inhibitors targeted at LRRK2. Contrast sensitivity in both day 1 and day 14 flies was normalized by a novel LRRK2 kinase inhibitor 'BMPPB-32'. Biochemical and cellular assays suggested that BMPPB-32 would be a more specific kinase inhibitor than LRRK2-IN-1. We confirmed this in vivo, finding that dLRRK(-) null flies show large off-target effects with LRRK2-IN-1 but not BMPPB-32. Our data link the increased Kinase activity of the G2019S-LRRK2 mutation to neuronal dysfunction and demonstrate the power of the Drosophila visual system in assaying the neurological effects of genetic diseases and therapies.
The amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was ...analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined.
Cyclooxygenase-2 (Cox-2), Protocadherin-8 (Pcdh8) and TGF-beta-inducible early response gene-1 (TIEG1) were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-alpha, RGS2, Egr2/krox-20 and beta-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs) as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD) duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration.
The present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus.
Drugs acting at dopamine D2-like receptors play a pivotal role in the treatment of both schizophrenia and Parkinson's disease. Recent studies have demonstrated a role for G-protein independent D2 ...receptor signaling pathways acting through β-arrestin. In this study we describe the establishment of a Bioluminescence Resonance Energy Transfer (BRET) assay for measuring dopamine induced recruitment of human β-arrestin2 to the human dopamine D2 receptor. Dopamine, as well as the dopamine receptor agonists pramipexole and quinpirole, acted as full agonists in the assay as reflected by their ability to elicit marked concentration dependent increases in the BRET signal signifying β-arrestin2 recruitment to the D2 receptor. As expected from their effect on G-protein coupling and cAMP levels mediated through the D2 receptor RNPA, pergolide, apomorphine, ropinirole, bromocriptine, 3PPP, terguride, aripiprazole, SNPA all acted as partial agonists with decreasing efficacy in the BRET assay. In contrast, a wide selection of typical and atypical anti-psychotics was incapable of stimulating β-arrestin2 recruitment to the D2 receptor. Moreover, we observed that haloperidol, sertindole, olanzapine, clozapine and ziprasidone all fully inhibited the dopamine induced β-arrestin2 recruitment to D2 receptor (short variant) in a concentration dependent manner. We conclude that most anti-psychotics are incapable of stimulating β-arrestin2 recruitment to the dopamine D2 receptor, in accordance with their antagonistic properties at the level of G-protein coupling.
The purpose of this study was to investigate the influence of an energy drink (ED) on cycling performance and immune-related variables. Eleven trained male cyclists (33.4 ± 8.9 years; 81 ± 7.6 kg; ...maximal VO2, 52 ± 3.4 ml·kg(-1)·min(-1)) consumed 500 ml of (a) ED (2.0 g taurine, 1.2 g glucuronolactone, 160 mg caffeine, 56 g carbohydrate CHO, and B vitamins), (b) cola matched for caffeine and CHO (CC), or (c) flavored placebo (PL: sparking water and flavoring) 50 minutes before racing in a randomized, crossover design. Performance was measured as time to complete (TTC) a 25-mile simulated road race. Blood was collected at baseline, 30 minutes after drink consumption, during exercise at miles 5 (M5), 15 (M15), and immediately (POEX) and 30 minutes (30minPO) after exercise. TTC was not different (p > 0.05) among trials (ED, 68.6 ± 2.7; CC, 68.9 ± 3.8; PL, 69.6 ± 3.8 minutes). Consumption of CC and ED elicited a mild hypoglycemia elicited a mild hypoglycemia during cycling. POEX interleukin-6 (IL-6) was greatest after ED, whereas CC IL-6 was greater than PL (10.2 ± 1.6, 6.7 ± 0.6, and 4.8 ± 0.7 pg·ml(-1), respectively; p < 0.001). Cycling increased leukocyte number in all conditions with ED leukocyte number greater than that of PL at M15 (9.8 ± 0.6, 8.5 ± 0.3 × 10(6) cells·mL(-1)). Energy drink induced an earlier recruitment of monocytes to the blood stream than CC. Mean fat oxidation was greater in PL compared with CC (0.43 ± 0.06 and 0.28 ± 0.04 g·min(-1); p = 0.033) but did not differ between ED (0.32 ± 0.06) and PL. Lactate was higher in ED compared with CC and PL at M5 and M15 (p = 0.003), but there was no significant influence of either ED or CC on performance. Carbohydrate and caffeine consumption before endurance cycling significantly increased the IL-6 release and leukocytosis, and the additional ingredients in ED seem to have further augmented these responses.
The glucocorticoid receptor (GR) and myocyte enhancer factor 2 (MEF2) are transcription factors involved in neuronal plasticity. c-JUN, a target gene of GR and MEF2, plays a role in regulating both ...synaptic strength and synapse number. The aim of this study was to investigate the nature of this dual regulation of c-JUN by GR and MEF2 in a neuronal context. First, we showed that GR mediates the dexamethasone-induced suppression of c-JUN mRNA expression. Next, we observed that GR activation resulted in an increase in phosphorylation of MEF2, a post-translational modification known to change MEF2 from a transcriptional enhancer to a repressor. In addition, we observed an enhanced binding of MEF2 to genomic sites directly upstream of the c-JUN gene upon GR activation. Finally, in primary hippocampal neuronal cultures, knockdown of MEF2 not only reduced c-JUN expression levels but abolished GR regulation of c-JUN expression. This suggests that MEF2 is necessary for GR regulation of c-JUN. In conclusion, for the first time, we show that activated GR requires MEF2 to regulate c-JUN. At the same time, GR influences MEF2 activity and DNA binding. These results give novel insight into the molecular interplay of GR and MEF2 in the control of genes important for neuronal plasticity.
PDF
