Laura Caberlotto

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.

Exogenous neuropeptide Y (NPY) reduces experimental anxiety in a wide range of animal models. The generation of an NPY-transgenic rat has provided a unique model to examine the role of endogenous NPY in control of stress and anxiety-related behaviors using paradigms previously used by pharmacological studies. Locomotor activity and baseline behavior on the elevated plus maze were normal in transgenic subjects. Two robust phenotypic traits were observed. (i) Transgenic subjects showed a markedly attenuated sensitivity to behavioral consequences of stress, in that they were insensitive to the normal anxiogenic-like effect of restraint stress on the elevated plus maze and displayed absent fear suppression of behavior in a punished drinking test. (ii) A selective impairment of spatial memory acquisition was found in the Morris water maze. Control experiments suggest these traits to be independent. These phenotypic traits were accompanied by an overexpression of prepro-NPY mRNA and NPY peptide and decreased NPY-Y1 binding within the hippocampus, a brain structure implicated both in memory processing and stress responses. Data obtained using this unique model support and extend a previously postulated anti-stress action of NPY and provide novel evidence for a role of NPY in learning and memory.

Abstract 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.

Substance P exerts its various biochemical effects mainly via interactions through neurokinin-1 receptors (NK1). Recently, the NK1 receptor has attracted considerable interest for its possible role in a variety of psychiatric disorders including depression and anxiety. However, little is known regarding the anatomical distribution of NK1 in the human central nervous system (CNS). Riboprobe in situ hybridization, quantitative PCR and in vitro autoradiography were performed. Highest NK1 mRNA levels were localized in the locus coeruleus and ventral striatum, while moderate hybridization signals were observed in the cerebral cortex (most abundant in the visual cortex), hippocampus and different amygdaloid nuclei. Very low levels of the NK1 mRNA were detected in the cerebellum and thalamus. In view of the existence of a long and short isoform of the NK1 receptor, it was of interest to assess whether there was a differential distribution of the two splice variants in the human CNS and peripheral tissues. A quantitative TaqMan PCR analysis showed that the long NK1 isoform was the most prevalent throughout the human brain, while in peripheral tissues the truncated form was the most represented. 3H-Substance P autoradiography revealed a good correlation between receptor binding sites and NK1 mRNA expression throughout the brain, with the highest levels of binding in the locus coeruleus. These results provide the anatomical evidence that the NK1 receptors have a strong association with neuronal systems relevant to mood regulation and stress in the human brain, but do not suggest a region-specific role of the two isoforms in the CNS.

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.

To investigate the possible link between neuropeptide Y (NPY) and depression, we analyzed NPY and its receptors in different limbic-related regions in the Flinder sensitive line (FSL), a genetic animal model of depression. In situ hybridization histochemistry was used to measure mRNA expression levels of NPY and NPY receptors, Y1 and Y2, in the FSL as compared to the control Flinder resistant Line rats (FRL). In the FSL rats, NPY mRNA expression levels were significantly decreased in the nucleus accumbens and CA regions, but increased in the arcuate nucleus and anterior cingulate cortex. Y1 receptor mRNA expression was decreased in different cortical regions (retrosplenial, anterior cingulate, and occipital) and in the hippocampal dentate gyrus. Y2 mRNA expression levels did not differ between FSL and FRL animals. The effect of the antidepressant drug fluoxetine (a serotonin reuptake inhibitor) in the two rat strains was also studied. There was an increase of the NPY mRNA hybridization signal in the arcuate nucleus of both strains following the antidepressant treatment (10 μmol/kg; daily for 14 days). However, in other brain regions, fluoxetine administration caused a differential effect on the induction of NPY-related genes in the two rat strains: in the CA region and dentate gyrus NPY mRNA expression was increased in the FSL, but decreased in the FRL. In contrast, Y1 mRNA levels tended to be decreased by fluoxetine in the nucleus accumbens of the FSL rats, but increased in the FRL. These findings suggest an involvement of the Y1, but not the Y2, receptor subtype in depressive disorder. Overall, the results appear to sustain the importance of the FSL rats as an animal model of depression in view of the impairment of NPY genes and the ability of fluoxetine treatment to normalize NPY-related gene expression selectively in this strain.

Previously, we observed specific alterations of neuropeptide Y (NPY) and Y1 receptor mRNA expression in discrete regions of the Flinders Sensitive Line rats (FSL), an animal model of depression. In order to clarify the correlation between mRNA expression and protein content, radioimmunoassay and receptor autoradiography were currently performed. In the FSL rats, NPY-like immunoreactivity (NPY-LI) was decreased in the hippocampal CA region, while Y1 binding sites were increased; NPY-LI was increased in the arcuate nucleus. Fluoxetine treatment elevated NPY-LI in the arcuate and anterior cingulate cortex and increased Y1 binding sites in the medial amygdala and occipital cortex in both strains. No differences were found regarding the Y2 binding sites. The results demonstrate a good correlation between NPY peptide and mRNA expression, and sustain the possible involvement of NPY and Y1 receptors in 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 Y1 receptor (Y1) mRNA expression and binding sites, and behavior under basal conditions (Sham) and after repeated ECS. Baseline NPY and Y1 mRNAs in the CA1–2 regions and dentate gyrus were lower while the Y1 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 Y1 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 Y1 mRNA increase, Y1 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.

Abstract 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.

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.

Background: Central neuropeptide Y (NPY) is known to control feeding and stress responses. Recently, it has been suggested that NPY also has a role in regulation of alcohol consumption. Methods: NPY and NPY receptor expression in genetically selected alcohol‐preferring (AA), alcohol‐nonpreferring (ANA), and Wistar rats were investigated. Expression was assessed using in situ hybridization histochemistry with riboprobes specific for preproNPY, Y1, and Y2 receptors. Effects of central NPY administration on ethanol self‐administration were also examined in AA, ANA, and Wistar rats by using oral operant self‐administration. Results: NPY mRNA expression was higher in ANA than in both AA and Wistar rats in the hippocampal CA region and dentate gyrus, whereas AA and Wistar did not differ from each other. No differences in NPY expression were found in the other regions analyzed: cingulate cortex, medial nucleus of the amygdala, arcuate, and paraventricular nuclei of the hypothalamus. Y1 receptor mRNA expression did not differ between the three lines. Y2 expression was higher in the dentate gyrus of both AA and ANA rats than in Wistar subjects. In the medial amygdala, Y2 mRNA was reduced in the AA line, compared to both ANA and Wistar rats. NPY injected intracerebroventricularly (1.5–3.0 nmol) did not affect operant ethanol self‐administration in any of the three lines examined. Conclusion: The NPY system seems to differ in several respects between rat lines with different levels of alcohol preference. Differences observed within the hippocampus could be related to behavioral traits other than alcohol intake but it is also possible that elevated hippocampal expression of NPY in the ANA rats contributes to the low alcohol intake of this line. Aberrant NPY expression and/function within the amygdala complex could contribute to alcohol preference and constitute an anatomic substrate of the effects of NPY expression on alcohol intake observed previously in genetically modified animals.

In the present study, we compared neuropeptide Y mRNA expression levels in the prefrontal cortex (Brodmann area 9 and 46) of subjects diagnosed with major depression, bipolar disorder and schizophrenia with those in normal controls without a psychiatric history. No correlation was found regarding neuropeptide Y mRNA expression and postmortem interval, age, gender, hemisphere side, suicide as cause of death, or the history of use of substances such as alcohol, marihuana and cocaine/amphetamine. The only significant alteration found was related to the clinical diagnosis; neuropeptide Y mRNA expression was reduced in the group of bipolar subjects as compared to the controls. Overall, the present results confirm an involvement of neuropeptide Y in affective disorders, and show for the first time a specific association between NPY and bipolar disorder.

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.

Selective NPY-Y5 antagonists are known to reduce NPY-evoked increase of food intake under free feeding conditions and drug-reinforced operant responding in rodents suggesting that NPY-Y5 receptors can regulate reinforcers, potentially by modulating the hypothalamic-limbic reward system. However, evidence published to date has revealed a limited expression of NPY-Y5 in the limbic areas. Thus, the first aim of the present study was to investigate the distribution of NPY-Y5 receptor binding sites in rat mesocorticolimbic projection areas such as the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and lateral hypothalamus (LH). Since mesocorticolimbic release of monoamines has been typically associated to the rewarding and motivational significance of reinforcers, we then compared the ability of NPY and an NPY-Y5 selective agonist, [cPP1–7,NPY19–23,Ala31,Aib32,Gln34]hPP, to evoke changes in extracellular monoamines from these brain regions using in vivo microdialysis techniques. Intracerebral doses of each compound were selected on the basis of those previously demonstrated to trigger food intake in a separate set of animals. We found that NPY-Y5 receptors were widely distributed in both the NAc and mPFC but not in the LH nuclei. Central administration of either NPY (4.5 nmol/rat) or the NPY-Y5 agonist (0.6 nmol/rat) induced a significant increase of dopamine (DA) output of up to 150% of basal values in the NAc. In addition, NPY induced a stepped increase of norepinephrine (NE) outflow in the NAc area. Also extracellular levels of NE levels were increased by both treatments in the mPFC (150% vs basal concentration). Hypothalamic monoamine levels were unaffected by both treatments. Extracellular serotonin (5-HT) levels were also unchanged in all regions. Given the NPY-Y5 agonist paralleled the in vivo ability of NPY to increase DA, these data suggest that the release of NPY may modulate behaviours associated to accumbal DA release such reward and reinforcement by, at least in part, acting on mesocorticolimbic NPY-Y5 receptors.

Alzheimer's disease is the most common cause of dementia worldwide, affecting the elderly population. It is characterized by the hallmark pathology of amyloid-β deposition, neurofibrillary tangle formation, and extensive neuronal degeneration in the brain. Wealth of data related to Alzheimer's disease has been generated to date, nevertheless, the molecular mechanism underlying the etiology and pathophysiology of the disease is still unknown. Here we described a method for the combined analysis of multiple types of genome-wide data aimed at revealing convergent evidence interest that would not be captured by a standard molecular approach. Lists of Alzheimer-related genes (seed genes) were obtained from different sets of data on gene expression, SNPs, and molecular targets of drugs. Network analysis was applied for identifying the regions of the human protein-protein interaction network showing a significant enrichment in seed genes, and ultimately, in genes associated to Alzheimer's disease, due to the cumulative effect of different combinations of the starting data sets. The functional properties of these enriched modules were characterized, effectively considering the role of both Alzheimer-related seed genes and genes that closely interact with them. This approach allowed us to present evidence in favor of one of the competing theories about AD underlying processes, specifically evidence supporting a predominant role of metabolism-associated biological process terms, including autophagy, insulin and fatty acid metabolic processes in Alzheimer, with a focus on AMP-activated protein kinase. This central regulator of cellular energy homeostasis regulates a series of brain functions altered in Alzheimer's disease and could link genetic perturbation with neuronal transmission and energy regulation, representing a potential candidate to be targeted by therapy.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system. Its effects on, for example, cognition, memory and motor functions are thought to be mediated mainly via its interactions with the NPY Y1 and Y2 receptor subtypes. We had previously described the neuroanatomical organization of the Y1 and Y2 mRNA expression in humans. However, in view of the lack of information regarding the overall detailed distribution of NPY mRNA expression in the human brain, a complete picture of the anatomical organization of the NPY-related genes was still missing. Thus, in the present study, the regional distribution of NPY mRNA-expressing cells was analyzed in the post-mortem human brain. In addition, double labeling in situ hybridization was performed to characterize the NPY neuronal populations in relation to the Y1 and/or Y2 receptor mRNA localization in the human cerebral cortex, striatum, and amygdala. NPY mRNA was found to be abundant in layers II and VI of the neocortex, polymorphic layer of the dentate gyrus, basal ganglia, and amygdala. Double labeling in situ hybridization showed the co-expression of NPY mRNA with the Y2, but not with the Y1, mRNA in the human cerebral cortex, hippocampus, amygdala, striatum, and nucleus accumbens, and the existence of co-expression of the Y1 and Y2 mRNAs in the cerebral cortex and amygdala. Overall, these results suggest a role for the Y2, but not Y1, as an autoreceptor in the NPY neuronal populations of the human brain.

Double immunolabelling immunohistochemistry in the arcuate nucleus of the rat demonstrates that neuropeptide Y (NPY) Y1 receptor like immunoreactivity of β-endorphin is strongly present in a subpopulation β-endorphin immunoreactive nerve cell bodies, while the small NPY immunoreactive nerve cell bodies lie in an arcuate area rich in NPY immunoreactive nerve terminals forming an uniform plexus. It is postulated that NPY Y1 receptors in β-endorphin neurons may mediate some actions of NPY on motivational processes and pain control as well as on hypophyseal hormone secretion, involving at least in part a regulation of the tubero-infundibular dopamine (DA) neurons.

It has been hypothesized that the neuropeptide Y (NPY) system is involved in the pathogenesis of mood disorder. In this study, Y1 and Y2 receptor mRNA expression levels were analyzed in the dorsolateral prefrontal cortex of subjects affected with major depression, bipolar disorder, or schizophrenia and compared to normal controls. No significant alterations in Y1 or Y2 mRNA expression levels were observed between the groups. However, the Y2 mRNA expression was elevated in layer IV in subjects with suicide as a cause of death. For the Y1 mRNA expression, there was a negative correlation with increasing subject age in the prefrontal cortex. Analysis of covariance revealed a significant elevation of the Y1 mRNA expression levels in individuals with a current history of marijuana use but no other drug. In summary, the current results suggest distinct alterations of the prefrontal Y1 and Y2 neuronal populations in aging and suicide.

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.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.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.

Abstract Many neurobiological functions have been ascribed to the NPY Y1 receptor subtype, but autoradiographic analysis has failed to detect Y1 binding sites in most human brain areas, in contrast to the rat. We examined the regional distribution of Y1 mRNA‐containing cells in the post‐mortem human brain to clarify if there is a major species difference in terms of the existence of Y1 receptors in the human telencephalon, in particular the striatum and cortex. In situ hybridization experiments revealed widespread distribution of Y1 mRNA signals in all layers of most limbic and neocortical regions, predominantly in layer IV (most cortical regions) and layer VI. The striatum showed moderate Y1 receptor mRNA expression levels with intensely expressing cells localized to the nucleus accumbens. The highest Y1 receptor mRNA expression was apparent within the dentate gyrus, and the lowest in the subiculum, parahippocampal gyrus, cerebellum, and thalamus. In vitro autoradiography using [ 125 I]Leu 31 Pro 34 ‐PYY and [ 125 I]PYY with NPY (13–36) or Leu 31 Pro 34 NPY; confirmed the presence of low Y1–like binding in the human brain despite abundant Y1 mRNA expression. However, using a rat model of the human autopsy process, it was apparent that the inability to reveal high Y1– versus Y2–like receptors in the human brain was related in part to marked reductions of Y1–like, but not Y2–like, receptors within a 4 h post‐mortem delay. Altogether, the results indicate that the Y1 receptor gene is abundant in the human brain and this receptor may have important roles in cognitive, limbic and motor function.

The neuropeptide Y (NPY) system has been largely studied in relation to affective disorders, in particular for its role in the mechanisms regulating the pathophysiology of anxiety and depression and in the stress-related behaviours. Although NPY has been previously investigated in a variety of animal models of mood disorders, the receptor subtype mainly involved in the modulation of the stress response has not been identified. In the present study, the chronic psychosocial stress based on the resident-intruder protocol-an ethologically relevant paradigm known to induce behavioural and endocrine modifications which mimic depression-like symptoms-was used. Two different species were investigated: rat and tree shrew (Tupaia belangeri); the latter is regarded as an intermediate between insectivores and primates and it was chosen in this study for its pronounced territoriality. In these animals, the regulation of NPY and of Y(1), Y(2) and Y(5) receptors mRNA expression was evaluated after chronic stress and chronic antidepressant treatment by in situ hybridization in selected brain regions known to be involved in the pathophysiology of mood disorders. The animals were exposed to psychosocial stress for 35 days and concomitant daily fluoxetine treatment (10 mg/kg for rats and 15 mg/kg for tree shrews) after the first week of stress. The results confirmed a major role for hippocampal and hypothalamic NPY system in the pathophysiology of mood disorders. Although there were no evident differences between rat and tree shrew in the NPY system distribution, an opposite effect of chronic psychosocial stress was observed in the two species. Moreover, chronic antidepressant treatment was able to counteract the effects of stress and restored basal expression levels, suggesting the utility of these paradigms as preclinical models of stress-induced depression. Overall, although evident species differences were found in response to chronic psychosocial stress, the present study suggests a role for NPY receptors in the stress response and in the action of antidepressant drugs, providing further support for an involvement of this neuropeptidergic system in the pathophysiology of depression and anxiety.

Neuropeptide Y (NPY) has been extensively studied in relation to anxiety and depression but of the seven NPY receptors known to date, it is not yet clear which one is mainly involved in mediating its effects in emotional behavior. Mice lacking the NPY-Y2 receptors were previously shown to be less anxious due to their improved ability to cope with stressful situations. In the present study, the behavioral phenotype including the response to challenges was analyzed in NPY-Y2 knockout (KO) mice backcrossed in to congenic C57BL/6 background. In the elevated plus-maze (EPM) and the forced swim test (FST), the anxiolytic-like or antidepressant-like phenotype of the NPY-Y2 KO mice could not be confirmed, although this study differs from the previous one only with regard to the genetic background of the mice. In addition, no differences in response to acute stress or to the antidepressant desipramine in the FST were detected between wild type (WT) and NPY-Y2 KO animals. These results suggest that the genetic background of the animals appears to have a strong influence on the behavioral phenotype of NPY-Y2 KO mice. Additionally, to further characterize the animals by their biochemical response to a challenge, the neurochemical changes induced by the anxiogenic compound yohimbine were measured in the medial prefrontal cortex (mPFC) of NPY-Y2 KO and compared to WT mice. Dopamine (DA) levels were significantly increased by yohimbine in the WT but unaffected in the KO mice, suggesting that NPY-Y2 receptor exerts a direct control over both the tonic and phasic release of DA and that, although the anxiety-like behavior of these NPY-Y2 KO mice is unaltered, there are clear modifications of DA dynamics. However, yohimbine led to a significant increase in noradrenaline (NA) concentration and a slight reduction in serotonin concentration that were identical for both phenotypes.

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.

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.

Neurodegenerative dementia comprises chronic and progressive illnesses with major clinical features represented by progressive and permanent loss of cognitive and mental performance, including impairment of memory and brain functions. Many different forms of neurodegenerative dementia exist, but they are all characterized by death of specific subpopulation of neurons and accumulation of proteins in the brain. We incorporated data from OMIM and primary molecular targets of drugs in the different phases of the drug discovery process to try to reveal possible hidden mechanism in neurodegenerative dementia. In the present study, a systems biology approach was used to investigate the molecular connections among seemingly distinct complex diseases with the shared clinical symptoms of dementia that could suggest related disease mechanisms.Network analysis was applied to characterize an interaction network of disease proteins and drug targets, revealing a major role of metabolism and, predominantly, of autophagy process in dementia and, particularly, in tauopathies. Different phases of the autophagy molecular pathway appear to be implicated in the individual disease pathophysiology and specific drug targets associated to autophagy modulation could be considered for pharmacological intervention. In particular, in view of their centrality and of the direct association to autophagy proteins in the network, PP2A subunits could be suggested as a suitable molecular target for the development of novel drugs.The present systems biology investigation identifies the autophagy pathway as a central dis-regulated process in neurodegenerative dementia with a prevalent involvement in diseases characterized by tau inclusion and indicates the disease-specific molecules in the pathway that could be considered for therapy.

Despite significant advances in the study of the molecular mechanisms altered in the development and progression of neurodegenerative diseases (NDs), the etiology is still enigmatic and the distinctions between diseases are not always entirely clear. We present an efficient computational method based on protein-protein interaction network (PPI) to model the functional network of NDs. The aim of this work is fourfold: (i) reconstruction of a PPI network relating to the NDs, (ii) construction of an association network between diseases based on proximity in the disease PPI network, (iii) quantification of disease associations, and (iv) inference of potential molecular mechanism involved in the diseases. The functional links of diseases not only showed overlap with the traditional classification in clinical settings, but also offered new insight into connections between diseases with limited clinical overlap. To gain an expanded view of the molecular mechanisms involved in NDs, both direct and indirect connector proteins were investigated. The method uncovered molecular relationships that are in common apparently distinct diseases and provided important insight into the molecular networks implicated in disease pathogenesis. In particular, the current analysis highlighted the Toll-like receptor signaling pathway as a potential candidate pathway to be targeted by therapy in neurodegeneration.

Among the genetic factors known to increase the risk of late onset Alzheimer's diseases (AD), the presence of the apolipoproteine e4 (APOE4) allele has been recognized as the one with the strongest effect. However, despite decades of research, the pathogenic role of APOE4 in Alzheimer's disease has not been clearly elucidated yet. In order to investigate the pathogenic action of APOE4, we applied a systems biology approach to the analysis of transcriptomic and genomic data of APOE44 vs. APOE33 allele carriers affected by Alzheimer's disease. Network analysis combined with a novel technique for biomarker computation allowed the identification of an alteration in aging-associated processes such as inflammation, oxidative stress and metabolic pathways, indicating that APOE4 possibly accelerates pathological processes physiologically induced by aging. Subsequent integration with genomic data indicates that the Notch pathway could be the nodal molecular mechanism altered in APOE44 allele carriers with Alzheimer's disease. Interestingly, PSEN1 and APP, genes whose mutation are known to be linked to early onset Alzheimer's disease, are closely linked to this pathway. In conclusion, APOE4 role on inflammation and oxidation through the Notch signaling pathway could be crucial in elucidating the risk factors of Alzheimer's disease.

The neuropeptide Y Y2 receptor is one of six receptor subtypes mediating the multiform physiological actions of neuropeptide Y. The Y2 receptor has been demonstrated to be the most predominant receptor subtype in the human brain and appears to be involved in many neuropeptide Y actions, such as the regulation of locomotor activity, cardiovascular functions, memory processing, circadian rhythms and release of other neurotransmitters. We have recently demonstrated the widespread and abundant distribution of neuropeptide Y Y1 receptor messenger RNA in the human cerebral cortex (different laminar patterns within distinct cortical regions), hippocampal dentate gyrus and striatum. To assess a possible differential distribution of Y1 and Y2 receptor messenger RNAs, the regional expression of neuropeptide Y Y2 messenger RNA-containing cells in the human brain was analysed, in particular within the cerebral cortex and striatum. In situ hybridization experiments revealed the localization of the Y2 messenger RNA signal throughout all cortical regions, with the highest intensity per cell apparent in lamina IV, with the exception of the striate cortex, which showed an intense labelling primarily in layer VI. The striatum expressed low to undetectable levels of the Y2 receptor messenger RNA. The dentate gyrus and the CA2 region presented the highest hybridization signals, while a very weak Y2 messenger RNA expression was found in the CA1 region and subiculum. Positive Y2 messenger RNA hybridization signals were also detected in the lateral geniculate nucleus, amygdala, substantia nigra, hypothalamus, cerebellum and choroid plexus. These results demonstrate the widespread distribution of neuropeptide Y Y2 receptor messenger RNA in the human brain, with a pattern of expression distinct from the Y1 subtype, suggesting that these two receptor subtypes may mediate different neuropeptide Y functions in the human brain, mainly through actions on different neuronal systems.

Chronic treatment with cannabinoid agonists leads to tolerance. One possible mechanism for this is receptor internalization, but tolerance has also been reported with compounds that only cause internalization to a low degree. Furthermore, cannabinoid antagonist administration precipitates a characteristic withdrawal syndrome in tolerant subjects, accompanied by neuronal activation and enhanced release of corticotropin-releasing hormone (CRH) in the central amygdala. The underlying molecular mechanisms are unknown. We examined the role of cannabinoid tolerance and withdrawal for the expression of the cannabinoid 1 (CB1) receptor and of CRH in rats. Tolerance was first established functionally. An acute dose (100 μg/kg) of the CB1 agonist HU-210 suppressed locomotor activity, and had an anxiogenic-like effect on the elevated plus-maze. Both effects were absent following daily treatment with the same agonist or a lower (40 μg/kg) dose for 14 days. Next, withdrawal was reliably precipitated by a single dose (3 mg/kg) of the CB1 antagonist SR141716A in rats treated subchronically with 14-day HU-210. Using in situ hybridization, a robust suppression of CB1 mRNA expression was found in the caudate-putamen, indicating a downregulation of CB1 expression levels as one mechanism for tolerance to the locomotor suppressant effects of HU-210. The CRH transcript was upregulated in the central amygdala in precipitated withdrawal compared to nonwithdrawn tolerant subjects, suggesting that increased gene expression contributes to the previously reported CRH release in withdrawal. Most importantly, this increase occurred from a suppressed level in tolerant subjects, and behavioral signs of withdrawal, presumably mediated by CRH, were seen at the CRH expression that had only returned to normal nontolerant levels. This suggests the possibility of an allostatic shift, as previously proposed on theoretical grounds. The expression of CRH-R1, CRH-R2α, NPY, and its Y1 receptor mRNA was analyzed in search of neural substrates for the allostatic shift observed, but did not seem to contribute to the dysregulated state.

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.

Worldwide population is aging, and a large part of the growing burden associated with age-related conditions can be prevented or delayed by promoting healthy lifestyle and normalizing metabolic risk factors. However, a better understanding of the pleiotropic effects of available nutritional interventions and their influence on the multiple processes affected by aging is needed to select and implement the most promising actions. New methods of analysis are required to tackle the complexity of the interplay between nutritional interventions and aging, and to make sense of a growing amount of -omics data being produced for this purpose. In this paper, we review how various systems biology-inspired methods of analysis can be applied to the study of the molecular basis of nutritional interventions promoting healthy aging, notably caloric restriction and polyphenol supplementation. We specifically focus on the role that different versions of network analysis, molecular signature identification and multi-omics data integration are playing in elucidating the complex mechanisms underlying nutrition, and provide some examples on how to extend the application of these methods using available microarray data.

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.

The next generation of biomarkers and companion diagnostics will require the development of technologies capable of conjugating the advances in high-throughput techniques in biology with computational methods. Systems biology is poised to contribute through an integrated view, capturing the complexity of the system, both in terms of a collection of interacting molecular components and also in terms of multiple intersecting views. Following this system-centered view, novel approaches have been developed for the identification of signatures of both disease processes and drug modes of action with the promising perspectives of better diagnosis of disease and of the discovery of more efficacious and safe drugs. The application of systems biology to the development of companion diagnostics is very recent and to date a few pioneering steps have been made in this direction. In this review, we describe the ongoing studies and the potential developments in this area of research.

Neuropeptide Y is the most abundant peptide in the mammalian central nervous system and exhibits a variety of potent neurobiological functions. In the present study, double immunolabelling histochemistry was performed, using previously characterized antibodies against neuropeptide Y and the neuropeptide Y Y1 receptor subtype, to clarify the cellular distribution of Y1 receptors in the rat brain in relation to the neuropeptide Y-immunoreactive systems. Based on fluorescence and confocal laser microscopy analysis, morphological evidence is presented that the perikaryal and dendritic Y1 receptor-like immunoreactivity demonstrated in discrete regions of the tel-, diencephalon and of the lower brain stem, shown to be cytoplasmic and membrane associated, in many brain regions is not co-distributed with the neuropeptide Y-immunoreactive terminal network. These findings may partly be explained by the existence of volume transmission in Y1 receptor-mediated neuropeptide Y transmission involving short to long distance diffusion and/or convection of neuropeptide Y from its site of release to the neuronal target cells, containing the high-affinity Y1 receptors. Furthermore, neuropeptide Y and Y1 receptor-like immunoreactivities were in no case co-localized in the same nerve cell, suggesting that, in the rat brain, the Y1 receptor subtype may not be a neuropeptide Y autoreceptor.

Abstract Increasing evidence suggests that substance P (SP) neurokinin‐1 (NK1) receptors are involved in stress and emotional responses, representing a potential target for the treatment of anxiety and depression in humans. Given the important role of the amygdaloid complex in the regulation of emotional behavior, we examined the mRNA levels of preprotachykinin A [PPT‐A, a precursor of both SP and neurokinin A (NKA)] and 3 H‐SP binding sites in the amygdala of patients affected by bipolar disorder, major depression or schizophrenia as compared with matched control individuals. By means of in situ hybridization, a significant reduction of PPT‐A mRNA expression levels was detected in the three diagnostic groups, mainly in the basal, lateral and accessory basal amygdaloid nuclei, but not in the temporal cortical area proximal to the amygdala. Receptor autoradiography performed on adjacent sections showed no change in 3 H‐SP binding sites as compared with controls. No significant correlation was found between levels of PPT‐A mRNA expression or binding sites and subject age, gender, hemisphere side, cause of death or history of substance misuse (marijuana, alcohol, cocaine/amphetamine). An inverse relationship between PPT‐A mRNA expression levels and lifetime antipsychotic treatment (Fluphenazine) in the schizophrenic and bipolar disorder groups was found. Post‐mortem delay was also negatively correlated with NK1 binding sites. The results confirm an involvement of the tachykinins in psychiatric disorders, suggesting there is a generalized impairment of the SP system in the amygdala in mood disorders and schizophrenia rather than this being a disease‐related phenomenon.

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.

Ethanol preference and behavioral disinhibition in AA (alcohol accepting) animals is a behavioral constellation similar to that seen in human type II alcoholism, for which considerable genetic loading has been shown. In search of novel neural substrates for this phenotype, we compared gene expression in the cerebral cortex of the AA rat with two groups of control animals, the ANA (alcohol non-accepting) line and heterogeneous Wistar animals, by differential display RT-PCR. We identified two transcripts, ribosomal protein L18a mRNA and diacyglycerol kinase iota mRNA, which are differentially expressed between AA and ANA rats. Ribosomal protein L18A mRNA is evenly expressed throughout the brain, but strongly reduced in cortex of AA rats vs controls. Diacylglycerol kinase iota is exclusively found in the brain, and expressed in a distinct regional pattern. Its cortical expression is about 25% higher in AA than ANA rats. Differential display RT-PCR seems to provide a feasible strategy to identify previously unknown genes whose differential expression correlates with behavioral phenotypes related to dependence.

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.

Abstract Dementia is a neurodegenerative condition of the brain in which there is a progressive and permanent loss of cognitive and mental performance. Despite the fact that the number of people with dementia worldwide is steadily increasing and regardless of the advances in the molecular characterization of the disease, current medical treatments for dementia are purely symptomatic and hardly effective. We present a novel multi-relational association mining method that integrates the huge amount of scientific data accumulated in recent years to predict potential novel targets for innovative therapeutic treatment of dementia. Owing to the ability of processing large volumes of heterogeneous data, our method achieves a high performance and predicts numerous drug targets including several serine threonine kinase and a G-protein coupled receptor. The predicted drug targets are mainly functionally related to metabolism, cell surface receptor signaling pathways, immune response, apoptosis and long-term memory. Among the highly represented kinase family and among the G-protein coupled receptors, DLG4 (PSD-95) and the bradikynin receptor 2 are highlighted also for their proposed role in memory and cognition, as described in previous studies. These novel putative targets hold promises for the development of novel therapeutic approaches for the treatment of dementia.

Leptin decreases food intake through actions in the hypothalamus, partly through interactions with neuropeptide Y (NPY). However, NPY also produces behavioral antistress effects mediated inter alia through the amygdala. If leptin generally suppresses NPY function, the utility of leptin-mimics for treatment of obesity might be limited. Here, we therefore compared the effects of intracerebroventricular leptin on hypothalamic and amygdala NPY expression, as well as the respective related behaviors, i.e., feeding and experimental anxiety. Rats were injected intracerebroventricularly with leptin once daily for 6 days. Leptin-treated subjects consumed significantly less chow and had reduced body weight at the end of the treatment period compared to saline-treated controls. This was accompanied by a significant suppression of hypothalamic NPY expression. In contrast, the expression of NPY within the amygdala was unaffected by leptin. In parallel, in an established animal model of anxiety, the elevated plus-maze, no effect of leptin on anxiety-related behaviors was observed. In conclusion, leptin selectively affects the hypothalamic NPY system and its functional outflow, i.e., feeding and endocrine stress responses. Despite modifying endocrine responses, leptin treatment does not affect behavioral measures of experimental anxiety.

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.

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.

The pathophysiological basis of major depression is incompletely understood. Recently, numerous proteomic studies have been performed in rodent models of depression to investigate the molecular underpinnings of depressive-like behaviours with an unbiased approach. The objective of the study is to integrate the results of these proteomic studies in depression models to shed light on the most relevant molecular pathways involved in the disease.Network analysis is performed integrating preexisting proteomic data from rodent models of depression. The IntAct mouse and the HRPD are used as reference protein-protein interaction databases. The functionality analyses of the networks are then performed by testing overrepresented GO biological process terms and pathways.Functional enrichment analyses of the networks revealed an association with molecular processes related to depression in humans, such as those involved in the immune response. Pathways impacted by clinically effective antidepressants are modulated, including glutamatergic signaling and neurotrophic responses. Moreover, dysregulations of proteins regulating energy metabolism and circadian rhythms are implicated. The comparison with protein pathways modulated in depressive patients revealed significant overlapping.This systems biology study supports the notion that animal models can contribute to the research into the biology and therapeutics of depression.

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 30 mg/kg po.

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 3 mg/kg po.

To properly understand the function of genes of neurological interest, in vivo manipulation in the adult is essential, particularly when the target gene is involved in brain development. Moreover, since the physiological effects of target protein may be region-specific, targeting a distinct brain region could be required to dissect these effects in specific brain locations. Infection of somatic tissues of transgenic mice bearing loxP-flanked gene sequences with a viral vector expressing Cre recombinase provides a means of allowing flexible spatio-temporal control of target gene expression. Viral vector-mediated Cre expression could be used to mediate localized gene modulation in a specific brain region. In the present study this technology was applied to the glycine transporter type-1 (GlyT1) protein which is responsible for the uptake of synaptic glycine in the forebrain and has been implicated as a therapeutic target for the treatment of schizophrenia. Since GlyT1 is widely expressed in glial cells, we employed an adenoviral-based vector (Ad5) to deliver Cre protein, due to the preferentially transduction of glial cells by adenoviral vectors in rodent brain. We show significant reduced GlyT1 binding specifically in the thalamic area of conditional GlyT1 (GlyT1c) transgenic mice injected with Ad5-Cre virus, as measured by GlyT1 autoradiography. In conclusion, we demonstrated the validity of viral vector-mediated delivery of Cre to loxP targeted transgenic mice as a novel strategy to investigate target gene function in selected subregions of the adult brain, which provides a valuable technique to investigate gene function both in normal physiology and in disease models.

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.

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.

Autism spectrum disorders (ASD) are a group of conditions characterized by impairments in social interaction and presence of repetitive behavior. These complex neurological diseases are among the fastest growing developmental disorders and cause varying degrees of lifelong disabilities. There have been a lot of ongoing research to unravel the pathogenic mechanism of autism. Computational methods have come to the scene as a promising approach to aid the physicians in studying autism. In this paper, we present an efficient method to predict autism-related candidate genes (autism genes in short) by integrating protein interaction network and miRNA-target interaction network. We combine the two networks by a new technique relying on shortest path calculation. To demonstrate the high performance of our method, we run several experiments on three different PPI networks extracted from the BioGRID database, the HINT database, and the HPRD database. Three supervised learning algorithms were employed, i.e., the Bayesian network and the random tree and the random forest. Among them, the random forest method performs better in terms of precision, recall, and F-measure. It shows that the random forest algorithm is potential to infer autism genes. Carrying out the experiments with five different lengths of the shortest paths in the PPI networks, the results show the advantage of the method in studying autism genes based on the large scale network. In conclusion, the proposed method is beneficial in deciphering the pathogenic mechanism of autism.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The diacylglycerol kinases family shows a high degree of molecular diversity and distinct expression patterns in thebrain, suggesting an importance of these enzymes for brain function. A likely mechanism of action is the regulationof protein kinase C (PKC) pathways. The expression of one diacylglycerol kinase isoform, iota, is increased in thecortex of AA rats bred for high alcohol preference. The autoradiogram shows the expression of this isoform in therat brain mapped by in situhybridization. Regions of high signal intensity are coded in red colors and of low intensityin blue. For further information on this topic, see the article by W Sommer et alon pages 103–108.

Background: Central neuropeptide Y (NPY) is known to control feeding and stress responses. Recently, it has been suggested that NPY also has a role in regulation of alcohol consumption. Methods: NPY and NPY receptor expression in genetically selected alcohol-preferring (AA), alcohol-nonpreferring (ANA), and Wistar rats were investigated. Expression was assessed using in situ hybridization histochemistry with riboprobes specific for preproNPY, Y1, and Y2 receptors. Effects of central NPY administration on ethanol self-administration were also examined in AA, ANA, and Wistar rats by using oral operant self-administration. Results: NPY mRNA expression was higher in ANA than in both AA and Wistar rats in the hippocampal CA region and dentate gyrus, whereas AA and Wistar did not differ from each other. No differences in NPY expression were found in the other regions analyzed: cingulate cortex, medial nucleus of the amygdala, arcuate, and paraventricular nuclei of the hypothalamus. Y1 receptor mRNA expression did not differ between the three lines. Y2 expression was higher in the dentate gyrus of both AA and ANA rats than in Wistar subjects. In the medial amygdala, Y2 mRNA was reduced in the AA line, compared to both ANA and Wistar rats. NPY injected intracerebroventricularly (1.5–3.0 nmol) did not affect operant ethanol self-administration in any of the three lines examined. Conclusion: The NPY system seems to differ in several respects between rat lines with different levels of alcohol preference. Differences observed within the hippocampus could be related to behavioral traits other than alcohol intake but it is also possible that elevated hippocampal expression of NPY in the ANA rats contributes to the low alcohol intake of this line. Aberrant NPY expression and/function within the amygdala complex could contribute to alcohol preference and constitute an anatomic substrate of the effects of NPY expression on alcohol intake observed previously in genetically modified animals.