Brain-penetrant cyanoindane and cyanotetralin inhibitors of G2019S-LRRK2 kinase activity.

The G2019S variant of LRRK2, which causes an increase in kinase activity, is associated with the occurrence of Parkinson's disease (PD). Potent, mutation-selective, and brain penetrant inhibitors of LRRK2 can suppress the biological effects specific to G2019S-LRRK2 that cause pathogenicity. We report the discovery of a series of cyanoindane and cyanotetralin kinase inhibitors culminating in compound 34 that demonstrated selective inhibition of phosphorylation of LRRK2 in the mouse brain. These novel inhibitors may further enable the precision medicine path for future PD therapeutics.

Crowdsourced estimation of cognitive decline and resilience in Alzheimer's disease.

Identifying accurate biomarkers of cognitive decline is essential for advancing early diagnosis and prevention therapies in Alzheimer's disease. The Alzheimer's disease DREAM Challenge was designed as a computational crowdsourced project to benchmark the current state-of-the-art in predicting cognitive outcomes in Alzheimer's disease based on high dimensional, publicly available genetic and structural imaging data. This meta-analysis failed to identify a meaningful predictor developed from either data modality, suggesting that alternate approaches should be considered for prediction of cognitive performance.

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum.

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.

Degree Adjusted Large-Scale Network Analysis Reveals Novel Putative Metabolic Disease Genes.

A large percentage of the global population is currently afflicted by metabolic diseases (MD), and the incidence is likely to double in the next decades. MD associated co-morbidities such as non-alcoholic fatty liver disease (NAFLD) and cardiomyopathy contribute significantly to impaired health. MD are complex, polygenic, with many genes involved in its aetiology. A popular approach to investigate genetic contributions to disease aetiology is biological network analysis. However, data dependence introduces a bias (noise, false positives, over-publication) in the outcome. While several approaches have been proposed to overcome these biases, many of them have constraints, including data integration issues, dependence on arbitrary parameters, database dependent outcomes, and computational complexity. Network topology is also a critical factor affecting the outcomes. Here, we propose a simple, parameter-free method, that takes into account database dependence and network topology, to identify central genes in the MD network. Among them, we infer novel candidates that have not yet been annotated as MD genes and show their relevance by highlighting their differential expression in public datasets and carefully examining the literature. The method contributes to uncovering connections in the MD mechanisms and highlights several candidates for in-depth study of their contribution to MD and its co-morbidities.

Design, synthesis and SAR of a novel series of benzimidazoles as potent NPY Y5 antagonists.

A novel class of benzimidazole NPY Y5 receptor antagonists was prepared exploiting a privileged spirocarbamate moiety. The structure-activity relationship of this series and efforts to achieve a profile suitable for further development and an appropriate pharmacokinetic profile in rat are described. Optimisation led to the identification of the brain penetrant, orally bioavailable Y5 antagonist 9b which significantly inhibited the food intake induced by a Y5 selective agonist with a minimal effective dose of 30mg/kg po.

Synthesis and structure-activity relationship of N-(3-azabicyclo[3.1.0]hex-6-ylmethyl)-5-(2-pyridinyl)-1,3-thiazol-2-amines derivatives as NPY Y5 antagonists.

A novel class of small molecule NPY Y5 antagonists based around an azabicyclo[3.1.0]hexane scaffold was identified through modification of a screening hit. Structure-activity relationships and efforts undertaken to achieve a favourable pharmacokinetic profile in rat are described.

Discovery and structure-activity relationship of a novel spirocarbamate series of NPY Y5 antagonists.

A novel series of trans-8-aminomethyl-1-oxa-3-azaspiro[4.5]decan-2-one derivatives was identified with potent NPY Y5 antagonist activity. Optimization of the original lead furnished compounds 23p and 23u, which combine sub-nanomolar Y5 activity with metabolic stability, oral bioavailability, brain penetration and strong preclinical profile for development. Both compounds significantly inhibited the food intake induced by a Y5 selective agonist with minimal effective doses of 3mg/kg po.

The identification of a series of novel, soluble non-peptidic neuropeptide Y Y2 receptor antagonists.

The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.

Gene expression signature of antidepressant treatment response/non-response in Flinders Sensitive Line rats subjected to maternal separation.

Neurobiological underpinnings of treatment-resistant depression, a debilitating condition associated with significant functional impairment, have not been elucidated. Consequently, the aim of this study was to use animal models of response and resistance to antidepressant treatment, in an attempt to identify differences in associated transcriptional responses. Flinders Sensitive Line rats were subjected to maternal separation (MS) and chronically treated with Escitalopram or Nortriptyline. Antidepressants reduced immobility time in the forced swim test in non-MS rats, while lack of antidepressant behavioural response was observed in MS animals. We developed a novel bioinformatic algorithm that enabled identification of transcriptional signatures in hippocampus and pre-frontal cortex that discriminate vehicle- and antidepressant-treated subjects in both MS and non-MS rats. Functional annotation analysis showed that in antidepressant-responder rats the most enriched pathways included IQGAPs activation, toll-like receptor trafficking, energy metabolism, and regulation of endopeptidase activity. The analysis of interacting proteins implicated synaptic vesicles and neurotransmitter release, ubiquitin regulation, cytoskeleton organisation and carbohydrate metabolism. In contrast, in treatment-resistant MS rats, main expression changes were revealed in ribosomal proteins, inflammatory responses, transcriptional/epigenetic regulation, and small GTPases. Susceptibility signature shared Rtn1, Zdhhc5, Igsf6, and Sim1 genes with the latest depression GWAS meta-analysis, while antidepressant resistance signature shared Ctnnd1, Rbms3, Atp1a3, and Pla2r1 genes. In conclusion, this study demonstrated that distinct transcriptional signatures are associated with behavioural response or non-response to antidepressant treatment. The identification of genes involved in antidepressant response will increase the comprehension of the neurobiological underpinnings of treatment-resistant depression, thus contributing to identification of novel therapeutic targets.

Selective Inhibitors of G2019S-LRRK2 Kinase Activity.

Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.

Evaluation of expression and function of the H+/myo-inositol transporter HMIT.

BACKGROUND: The phosphoinositide (PIns) signalling pathway regulates a series of neuronal processes, such as neurotransmitter release, that are thought to be altered in mood disorders. Furthermore, mood-stabilising drugs have been shown to inhibit key enzymes that regulate PIns production and alter neuronal growth cone morphology in an inositol-reversible manner. Here, we describe analyses of expression and function of the recently identified H+/myo-inositol transporter (HMIT) investigated as a potential regulator of PIns signalling. RESULTS: We show that HMIT is primarily a neuronal transporter widely expressed in the rat and human brain, with particularly high levels in the hippocampus and cortex, as shown by immunohistochemistry. The transporter is localised at the Golgi apparatus in primary cultured neurones. No HMIT-mediated electrophysiological responses were detected in rat brain neurones or slices; in addition, inositol transport and homeostasis were unaffected in HMIT targeted null-mutant mice. CONCLUSION: Together, these data do not support a role for HMIT as a neuronal plasma membrane inositol transporter, as previously proposed. However, we observed that HMIT can transport inositol triphosphate, indicating unanticipated intracellular functions for this transporter that may be relevant to mood control.

The identification and optimisation of novel and selective diamide neuropeptide Y Y2 receptor antagonists.

A novel small molecule NPY Y2 antagonist (3) identified from high throughput screening is described. A subsequent SAR study and optimisation programme based around this molecule is also described, leading to the identification of potent and soluble pyridyl analogue 36.

Cross-disease analysis of Alzheimer's disease and type-2 Diabetes highlights the role of autophagy in the pathophysiology of two highly comorbid diseases.

Evidence is accumulating that the main chronic diseases of aging Alzheimer's disease (AD) and type-2 diabetes mellitus (T2DM) share common pathophysiological mechanisms. This study aimed at applying systems biology approaches to increase the knowledge of the shared molecular pathways underpinnings of AD and T2DM. We analysed transcriptomic data of post-mortem AD and T2DM human brains to obtain disease signatures of AD and T2DM and combined them with protein-protein interaction information to construct two disease-specific networks. The overlapping AD/T2DM network proteins were then used to extract the most representative Gene Ontology biological process terms. The expression of genes identified as relevant was studied in two AD models, 3xTg-AD and ApoE3/ApoE4 targeted replacement mice. The present transcriptomic data analysis revealed a principal role for autophagy in the molecular basis of both AD and T2DM. Our experimental validation in mouse AD models confirmed the role of autophagy-related genes. Among modulated genes, Cyclin-Dependent Kinase Inhibitor 1B, Autophagy Related 16-Like 2, and insulin were highlighted. In conclusion, the present investigation revealed autophagy as the central dys-regulated pathway in highly co-morbid diseases such as AD and T2DM allowing the identification of specific genes potentially involved in disease pathophysiology which could become novel targets for therapeutic intervention.

Chronic stress-induced alterations in amygdala responsiveness and behavior--modulation by trait anxiety and corticotropin-releasing factor systems.

The basolateral nucleus of the amygdala (BLA) plays a key role in emotional arousal and anxiety, and expresses high levels of corticotropin-releasing factor receptor (CRFR)1. In rat brain slices, we have recently shown that afferent activation of the BLA is increased following application of exogenous corticotropin-releasing factor (CRF). Here we examined the impact of chronic unpredictable stress (CUS) on this effect of CRF and whether blockade of CRFR1 could prevent stress-induced changes in the electrophysiological response, the animal's behavior and in cell proliferation in the hippocampus. The behavior of the rats was monitored via a series of tests that formed part of the CUS. Electrophysiological measures of the BLA response to CRF, cell proliferation in the dentate gyrus and the expression of CRF and CRFR1 mRNA in amygdaloid nuclei were determined ex vivo after completion of the CUS. CRF-induced potentiation of afferent activation of the BLA was reduced in rats exposed to CUS, an effect that was inhibited by chronic antagonism of CRFR1. Furthermore, the reduction in BLA response to CRF was correlated with the anxiety trait of the animals, determined prior to initiation of the CUS. These results implicate CRFR1 in chronic stress-induced alterations in amygdala function and behavior. Furthermore, they show that CRFR1 antagonists can prevent changes induced by chronic stress, in particular in those animals that are highly anxious.

Acute stress differentially affects corticotropin-releasing hormone mRNA expression in the central amygdala of the "depressed" flinders sensitive line and the control flinders resistant line rats.

Preclinical and clinical evidence suggests that neuropeptides play a role in the pathophysiology of mood disorders. In the present study, we investigated the involvement of the peptides corticotropin-releasing hormone (CRH), neuropeptide Y (NPY) and nociceptin/orphanin FQ (N/OFQ) and of their receptors in the regulation of emotional behaviours. In situ hybridization experiments were performed in order to evaluate the mRNA expression levels of these neuropeptidergic systems in limbic and limbic-related brain regions of the Flinders Sensitive Line (FSL) rats, a putative genetic animal model of depression. The FSL and their controls, the Flinders Resistant Line (FRL) rats, were subjected to one hour acute restraint and the effects of the stress exposure, including possible strain specific changes on these neuropeptidergic systems, were studied. In basal conditions, no significant differences between FSL and FRL rats in the CRH mRNA expression were found, however an upregulation of the CRH mRNA hybridization signal was detected in the central amygdala of the stressed FRL, compared to the non stressed FRL rats, but not in the FSL, suggesting a hypoactive mechanism of response to stressful stimuli in the "depressed" FSL rats. Baseline levels of NPY and N/OFQ mRNA were lower in the FSL rats compared to the FRL in the dentate gyrus of hippocampus and in the medial amygdala, respectively. However, the exposure to stress induced a significant upregulation of the N/OFQ mRNA levels in the paraventricular thalamic nucleus, while in the same nucleus the N/OFQ receptor mRNA expression was higher in the FSL rats. In conclusion, selective alterations of the NPY and N/OFQ mRNA in limbic and limbic-related regions of the FSL rats, a putative animal model of depression, provide further support for the involvement of these neuropeptides in depressive disorders. Moreover, the lack of CRH activation following stress in the "depressed" FSL rats suggests a form of allostatic load, that could alter their interpretation of environmental stimuli and influence their behavioural response to stressful situations.

Combined use of protein biomarkers and network analysis unveils deregulated regulatory circuits in Duchenne muscular dystrophy.

Although the genetic basis of Duchenne muscular dystrophy has been known for almost thirty years, the cellular and molecular mechanisms characterizing the disease are not completely understood and an efficacious treatment remains to be developed. In this study we analyzed proteomics data obtained with the SomaLogic technology from blood serum of a cohort of patients and matched healthy subjects. We developed a workflow based on biomarker identification and network-based pathway analysis that allowed us to describe different deregulated pathways. In addition to muscle-related functions, we identified other biological processes such as apoptosis, signaling in the immune system and neurotrophin signaling as significantly modulated in patients compared with controls. Moreover, our network-based analysis identified the involvement of FoxO transcription factors as putative regulators of different pathways. On the whole, this study provided a global view of the molecular processes involved in Duchenne muscular dystrophy that are decipherable from serum proteome.

Cross-species evidence from human and rat brain transcriptome for growth factor signaling pathway dysregulation in major depression.

An enhanced understanding of the pathophysiology of depression would facilitate the discovery of new efficacious medications. To this end, we examined hippocampal transcriptional changes in rat models of disease and in humans to identify common disease signatures by using a new algorithm for signature-based clustering of expression profiles. The tool identified a transcriptomic signature comprising 70 probesets able to discriminate depression models from controls in both Flinders Sensitive Line and Learned Helplessness animals. To identify disease-relevant pathways, we constructed an expanded protein network based on signature gene products and performed functional annotation analysis. We applied the same workflow to transcriptomic profiles of depressed patients. Remarkably, a 171-probesets transcriptional signature which discriminated depressed from healthy subjects was identified. Rat and human signatures shared the SCARA5 gene, while the respective networks derived from protein-based significant interactions with signature genes contained 25 overlapping genes. The comparison between the most enriched pathways in the rat and human signature networks identified a highly significant overlap (p-value: 3.85 × 10-6) of 67 terms including ErbB, neurotrophin, FGF, IGF, and VEGF signaling, immune responses and insulin and leptin signaling. In conclusion, this study allowed the identification of a hippocampal transcriptional signature of resilient or susceptible responses in rat MDD models which overlapped with gene expression alterations observed in depressed patients. These findings are consistent with a loss of hippocampal neural plasticity mediated by altered levels of growth factors and increased inflammatory responses causing metabolic impairments as crucial factors in the pathophysiology of MDD.

Electroconvulsive stimuli selectively affect behavior and neuropeptide Y (NPY) and NPY Y(1) receptor gene expressions in hippocampus and hypothalamus of Flinders Sensitive Line rat model of depression.

Previously we reported that basal neuropeptide Y (NPY)-like immunoreactivity-(LI) in hippocampus of the "depressed" Flinders Sensitive Line (FSL) rats was lower compared to the control Flinders Resistant Line (FRL) and that electroconvulsive stimuli (ECS) raise NPY-LI in discrete brain regions. Here we studied NPY mRNA expression, NPY Y(1) receptor (Y(1)) mRNA expression and binding sites, and behavior under basal conditions (Sham) and after repeated ECS. Baseline NPY and Y(1) mRNAs in the CA1-2 regions and dentate gyrus were lower while the Y(1) binding was higher in the FSL. ECS had larger effects on both NPY and behavior in the FSL rats. ECS increased NPY mRNA in the CA1-2, dentate gyrus and hypothalamus in FSL, but only in the dentate gyrus in FRL. ECS also increased Y(1) mRNA in the CA1-2, dentate gyrus and the parietal cortex in both strains, while in the hypothalamus the increase was observed only in the FSL rats. Consistently with Y(1) mRNA increase, Y(1) binding was downregulated in the corresponding regions. ECS decreased FSL immobility in the Porsolt swim test. These findings suggest that NPY is involved in depressive disorder and that antidepressant effects of ECS may in part be mediated through NPY.

Nutrition for the ageing brain: Towards evidence for an optimal diet.

As people age they become increasingly susceptible to chronic and extremely debilitating brain diseases. The precise cause of the neuronal degeneration underlying these disorders, and indeed normal brain ageing remains however elusive. Considering the limits of existing preventive methods, there is a desire to develop effective and safe strategies. Growing preclinical and clinical research in healthy individuals or at the early stage of cognitive decline has demonstrated the beneficial impact of nutrition on cognitive functions. The present review is the most recent in a series produced by the Nutrition and Mental Performance Task Force under the auspice of the International Life Sciences Institute Europe (ILSI Europe). The latest scientific advances specific to how dietary nutrients and non-nutrient may affect cognitive ageing are presented. Furthermore, several key points related to mechanisms contributing to brain ageing, pathological conditions affecting brain function, and brain biomarkers are also discussed. Overall, findings are inconsistent and fragmented and more research is warranted to determine the underlying mechanisms and to establish dose-response relationships for optimal brain maintenance in different population subgroups. Such approaches are likely to provide the necessary evidence to develop research portfolios that will inform about new dietary recommendations on how to prevent cognitive decline.

Anxiolytic-like effect of the selective neuropeptide Y Y2 receptor antagonist BIIE0246 in the elevated plus-maze.

The involvement of Neuropeptide Y (NPY) in the pathophysiology of mood disorders has been suggested by clinical and preclinical evidence. NPY Y1 and Y2 receptors have been proposed to mediate the NPY modulation of stress responses and anxiety related behaviors. To further investigate the role of Y2 receptors in anxiety we studied the effect of BIIE0246, a selective Y2 receptor antagonist, in the elevated plus-maze test. Rats treated with 1.0 nmol BIIE0246 showed an increase in the time spent on the open arm of the maze. In addition, to study the effects of the Y2 antagonism on NPY protein level, NPY-like immunoreactivity was measured in different brain regions following treatment with BIIE0246, but no statistically significant effects were observed. These results suggest that BIIE0246 has an anxiolytic-like profile in the elevated plus-maze.