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Objective: Ghrelin is a stomach-derived peptide that increases food intake through the activation of hypothalamic AMP-activated protein kinase (AMPK). However, the molecular mechanisms initiated by the activation of the ghrelin receptor, which in turn lead to AMPK activation, remain unclear. Sirtuin 1 (SIRT1) is a deacetylase activated in response to calorie restriction that acts through the tumor suppressor gene p53. We tested the hypothesis that the central SIRT1/p53 pathway might be mediating the orexigenic action of ghrelin.Research Design and Methods: SIRT1 inhibitors, such as Ex527 and sirtinol, and AMPK activators, such as AICAR, were administered alongside ghrelin in the brain of rats and mice (wild-type versus p53 knockout [KO]). Their hypothalamic effects on lipid metabolism and changes in transcription factors and neuropeptides were assessed by Western blot and in situ hybridization.Results: The central pretreatment with Ex527, a potent SIRT1 inhibitor, blunted the ghrelin-induced food intake in rats. Mice lacking p53, a target of SIRT1 action, failed to respond to ghrelin in feeding behavior. Ghrelin failed to phosphorylate hypothalamic AMPK when rats were pretreated with Ex527, as it did in p53 KO mice. It is noteworthy that the hypothalamic SIRT1/p53 pathway seems to be specific for mediating the orexigenic action of ghrelin, because central administration of AICAR, a potent AMPK activator, increased food intake in p53 KO mice. Finally, blockade of the central SIRT1 pathway did not modify ghrelin-induced growth hormone secretion.Conclusions: Ghrelin specifically triggers a central SIRT1/p53 pathway that is essential for its orexigenic action, but not for the release of growth hormone. [ABSTRACT FROM AUTHOR]

Dravet syndrome (DS), an intractable childhood epileptic encephalopathy with a high fatality rate, is typically caused by loss-of-function mutations in one allele of SCN1A, which encodes NaV1.1, a 250-kDa voltage-gated sodium channel. In contrast to other epilepsies, pharmaceutical treatment for DS is limited. Here, we demonstrate that viral vector-mediated delivery of a codon-modified SCN1A open reading frame into the brain improves DS comorbidities in juvenile and adolescent DS mice (Scn1aA1783V/WT). Notably, bilateral vector injections into the hippocampus and/or the thalamus of DS mice increased survival, reduced the occurrence of epileptic spikes, provided protection from thermally induced seizures, corrected background electrocorticographic activity and behavioral deficits, and restored hippocampal inhibition. Together, our results provide a proof of concept for the potential of SCN1A delivery as a therapeutic approach for infants and adolescents with DS-associated comorbidities.

The coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane cell adhesion molecule (CAM). CAR is expressed in numerous mammalian tissues including the brain, heart, lung, and testes. In epithelial cells, CAR functions are typical of the quintessential roles of numerous CAMs. However, in the brain the multiple roles of CAR are poorly understood. To better understand the physiological role of CAR in the adult brain, characterizing its location is a primordial step to advance our knowledge of its functions. In addition, CAR is responsible for the attachment, internalization, and retrograde transport of canine adenovirus type 2 (CAV-2) vectors, which have found a niche in the mapping of neuronal circuits and gene transfer to treat and model neurodegenerative diseases. In this study, we used immunohistochemistry and immunofluorescence to document the global location of CAR in the healthy, young adult mouse brain. Globally, we found that CAR is expressed by maturing and mature neurons in the brain parenchyma and located on the soma and on projections. While CAR occasionally colocalizes with glial fibrillary acidic protein, this overlap was restricted to areas that are associated with adult neurogenesis., (Copyright © 2020 Wehbi, Kremer and Dopeso-Reyes.)

The field of gene therapy has recently witnessed a number of major conceptual changes. Besides the traditional thinking that comprises the use of viral vectors for the delivery of a given therapeutic gene, a number of original approaches have been recently envisaged, focused on using vectors carrying genes to further modify basal ganglia circuits of interest. It is expected that these approaches will ultimately induce a therapeutic potential being sustained by gene-induced changes in brain circuits. Among others, at present, it is technically feasible to use viral vectors to (1) achieve a controlled release of neurotrophic factors, (2) conduct either a transient or permanent silencing of any given basal ganglia circuit of interest, (3) perform an in vivo cellular reprogramming by promoting the conversion of resident cells into dopaminergic-like neurons, and (4) improving levodopa efficacy over time by targeting aromatic L-amino acid decarboxylase. Furthermore, extensive research efforts based on viral vectors are currently ongoing in an attempt to better replicate the dopaminergic neurodegeneration phenomena inherent to the progressive intraneuronal aggregation of alpha-synuclein. Finally, a number of incoming strategies will soon emerge over the horizon, these being sustained by the underlying goal of promoting alpha-synuclein clearance, such as, for instance, gene therapy initiatives based on increasing the activity of glucocerebrosidase. To provide adequate proof-of-concept on safety and efficacy and to push forward true translational initiatives based on these different types of gene therapies before entering into clinical trials, the use of non-human primate models undoubtedly plays an instrumental role.

Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene. Mutations in GBA1 gene lead to Gaucher's disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase activity, therefore promoting the aggregation of alpha-synuclein, a common neuropathological finding underlying Parkinson's disease (PD) and dementia with Lewy bodies. However, it is also worth noting that a direct link between GBA1 mutations and alpha-synuclein aggregation indicating cause and effect is still lacking, with limited experimental evidence to date. Bearing in mind that a number of strategies increasing GCase expression for the treatment of PD are currently under development, here we sought to analyze the baseline expression of GCase in the brain of Macaca fascicularis, which has often been considered as the gold-standard animal model of PD. Although as with other lysosomal enzymes, GCase is expected to be ubiquitously expressed, here a number of regional variations have been consistently found, together with several specific neurochemical phenotypes expressing very high levels of GCase. In this regard, the most enriched expression of GCase was constantly found in cholinergic neurons from the nucleus basalis of Meynert, dopaminergic cells in the substantia nigra pars compacta, serotoninergic neurons from the raphe nuclei, as well as in noradrenergic neurons located in the locus ceruleus. Moreover, it is also worth noting that moderate levels of expression were also found in a number of areas within the paleocortex and archicortex, such as the entorhinal cortex and the hippocampal formation, respectively.

Although it has long been widely accepted that dopamine receptor types D1 and D2 form GPCR heteromers in the striatum, the presence of D1-D2 receptor heteromers has been recently challenged. In an attempt to properly characterize D1-D2 receptor heteromers, here we have used the in situ proximity ligation assay (PLA) in striatal sections comprising the caudate nucleus, the putamen and the core and shell territories of the nucleus accumbens. Experiments were carried out in control macaques as well as in MPTP-treated animals (with and without dyskinesia). Obtained data support the presence of D1-D2 receptor heteromers within all the striatal subdivisions, with the highest abundance in the accumbens shell. Dopamine depletion by MPTP resulted in an increase of D1-D2 density in caudate and putamen which was normalized by levodopa treatment. Two different sizes of heteromers were consistently found, thus suggesting that besides individual heteromers, D1-D2 receptor heteromers are sometimes organized in macromolecular complexes made of a number of D1-D2 heteromers. Furthermore, the PLA technique was combined with different neuronal markers to properly characterize the identities of striatal neurons expressing D1-D2 heteromers. We have found that striatal projection neurons giving rise to either the direct or the indirect basal ganglia pathways expressed D1-D2 heteromers. Interestingly, macromolecular complexes of D1-D2 heteromers were only found within cholinergic interneurons. In summary, here we provide overwhelming proof that D1 and D2 receptors form heteromeric complexes in the macaque striatum, thus representing a very appealing target for a number of brain diseases involving dopamine dysfunction.

Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters.

Sildenafil (Viagra) is a selective inhibitor of phosphodiesterase type 5 (PDE5), which degrades cyclic guanosine monophosphate to the linear nucleotide. Sildenafil is acutely used in erectile dysfunction and chronically in pulmonary hypertension. Evidence in the last decade shows that sildenafil may have potential as a therapeutic option for Alzheimer's disease or other neurodegenerative disorders. The purpose of this work was to explore whether sildenafil crosses the blood-brain barrier. Pharmacokinetic properties of sildenafil in rodents were investigated using (11) C-radiolabeling followed by in vivo positron emission tomography (PET) and ex vivo tissue dissection and gamma counting. PET results in rats suggest penetration into the central nervous system. Ex vivo data in perfused animals suggest that trapping of [(11) C]sildenafil within the cerebral vascular endothelium limits accumulation in the central nervous system parenchyma. Peroral sildenafil administration to Macaca fascicularis and subsequent chemical analysis of plasma and cerebrospinal fluid (CSF) using liquid chromatography coupled with tandem mass spectrometry showed that drug content in the CSF was high enough to achieve PDE5 inhibition, which was also demonstrated by the significant increases in CSF cyclic guanosine monophosphate levels. Central actions of sildenafil include both relaxation of the cerebral vasculature and inhibition of PDE5 in neurons and glia. This central action of sildenafil may underlie its efficacy in neuroprotection models, and may justify the continued search for a PDE5 ligand suitable for PET imaging. Sildenafil interacts with phosphodiesterase type 5 (PDE5) expressed in the endothelium and/or smooth muscle cells of brain vessels and also crosses the blood-brain barrier to interact with PDE5 expressed in brain cells. At therapeutic doses, the concentration of sildenafil in the cerebrospinal fluid (CSF) is high enough to inhibit PDE5 in the neural cells (neurons and glia). In turn, the concentration of cGMP likely increases in parenchymal cells and, as shown in this report, in the CSF. Read the Editorial Highlight for this article on page 220. Cover Image for this issue: doi: 10.1111/jnc.13302., (© 2015 International Society for Neurochemistry.)

Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

Although type 1 cannabinoid receptors (CB1Rs) are expressed abundantly throughout the brain, the presence of type 2 cannabinoid receptors (CB2Rs) in neurons is still somewhat controversial. Taking advantage of newly designed CB1R and CB2R mRNA riboprobes, we demonstrate by PCR and in situ hybridization that transcripts for both cannabinoid receptors are present within labeled pallidothalamic-projecting neurons of control and MPTP-treated macaques, whereas the expression is markedly reduced in dyskinetic animals. Moreover, an in situ proximity ligation assay was used to qualitatively assess the presence of CB1Rs and CB2Rs, as well as CB1R-CB2R heteromers within basal ganglia output neurons in all animal groups (control, parkinsonian and dyskinetic macaques). A marked reduction in the number of CB1Rs, CB2Rs and CB1R-CB2R heteromers was found in dyskinetic animals, mimicking the observed reduction in CB1R and CB2R mRNA expression levels. The fact that chronic levodopa treatment disrupted CB1R-CB2R heteromeric complexes should be taken into consideration when designing new drugs acting on cannabinoid receptor heteromers.

Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTP-induced degeneration, our data clearly demonstrated that these neurons are not giving rise to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal projections.

Background and Purpose: Heteromerization of GPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer-selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositol GPCR 55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB2 receptor) in human neutrophils, we investigated the possible heteromerization of CB2 receptors with GPR55., Experimental Approach: The direct interaction of human GPR55 and CB2 receptors heterologously expressed in HEK293 cells was assessed by co-immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross-talk on signalling was investigated at downstream levels by label-free real-time methods (Epic dynamic mass redistribution and CellKey impedance assays), ERK1/2-MAPK activation and gene reporter assays., Key Results: GPR55 and CB2 receptors co-localized on the surface of HEK293 cells, co-precipitated in membrane extracts and formed heteromers in living HEK293 cells. Whereas heteromerization led to a reduction in GPR55-mediated activation of transcription factors (nuclear factor of activated T-cells, NF-κB and cAMP response element), ERK1/2-MAPK activation was potentiated in the presence of CB2 receptors. CB2 receptor-mediated signalling was also affected by co-expression with GPR55. Label-free assays confirmed cross-talk between the two receptors., Conclusions and Implications: Heteromers, unique signalling units, form in HEK293 cells expressing GPR55 and CB2 receptors. The signalling by agonists of either receptor was governed (i) by the presence or absence of the partner receptors (with the consequent formation of heteromers) and (ii) by the activation state of the partner receptor., (© 2014 The British Pharmacological Society.)

Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions of extracellular signals to give heteromer-selective ligand binding and signaling, opening new avenues in the development of potential drug targets in pharmacotherapy. The aim of the present paper was to check for cannabinoid CB1-GPR55 receptor heteromers in the central nervous system (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors, using bioluminescence resonance energy transfer and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical fingerprint consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in striatum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer fingerprint was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed results consistent with receptor-receptor interactions in both caudate and putamen nuclei of a non-human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB1 and GPR55 receptors form heteromers in this specific CNS region., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Reelin is an extracellular matrix protein expressed in several interneuron subtypes in the hippocampus and dentate gyrus. Neuronal nitric oxide synthase (nNOS) is also expressed by interneurons in these areas. We investigated whether reelin and nNOS are co-localized in the same population of hippocampal interneurons, and whether this colocalization is altered in the heterozygous reeler mouse. We found colocalization of nNOS in reelin-positive cells in the CA1 stratum radiatum and lacunosum moleculare, the CA3 stratum radiatum, and the dentate gyrus subgranular zone, molecular layer, and hilus. In heterozygous reeler mice, the colocalization of nNOS in reelin-positive cells was significantly decreased only in the subgranular zone and molecular layer. The coexpression of reelin and nNOS in several hippocampal regions suggests that reelin and nNOS may work synergistically to promote glutamatergic function, and the loss of this coexpression in heterozygous reeler mice may underlie some of the behavioral deficits observed in these animals.

Serotonin transporter clustering is an important feature for regulation of this transporter activity. We used immunocytochemistry to analyze alterations in serotonin transporter clustering in blood lymphocytes of reeler mice. Serotonin transporter immunolabelling is observed mostly as a patchy staining in lymphocytes membranes. Comparison of the number and size of serotonin transporter clusters in wild-type mice, heterozygous reeler mice, and homozygous reeler mice showed an increase in the number and size of clusters in heterozygous reeler mice, but only an increase in clusters size in homozygous reeler mice. Reelin is down-regulated in the brain of schizophrenia, autism, and mood disorders, and is also expressed in blood plasma. There is the possibility therefore that alterations in serotonin transporter clustering in blood lymphocytes associated with a decrease in reelin expression may be operative in some cardiovascular or immune system alterations showing comorbidity with these mental disorders.

Patterns of protein expression can be used to identify biomarkers of disease, prognosis or treatment response. Peripheral 5-HT2A and D3 receptors have been proposed as protein markers in schizophrenia. We investigated the possible parallel regulation of these candidate biomarkers in central nervous system (CNS) and peripheral blood cells by a comparative study of the effects of antipsychotic treatment on the expression of the receptors in both systems in rats. Acute (24 and 48 h) and subchronic (16 days) treatment of rats with olanzapine induced a significant decrease in 5-HT2A receptor density both in frontal cortex (Bmax=76.2%, 83.0% and 46.0% of control after 24 h, 48 h and 16 days of treatment, respectively; P<0.01) and blood platelets (Bmax approximately 55% of control at all times measured; P<0.01), without any changes in receptor affinity. Furthermore, olanzapine induced redistribution in 5-HT2A-like immunoreactivity and time-dependent remodelling of synaptic circuits involved in the activity of pyramidal and GABAergic neurons in frontoparietal motor cortex of treated rats, as assessed by immunohistochemical studies. D3 receptor mRNA levels increased significantly by 52.5% (P<0.01) and 21.1% (P<0.05) in nucleus accumbens, and by 53.4% (P<0.05) and 91.7% (P<0.01) in lymphocytes, after acute (24 h and 48 h) treatment with olanzapine, returning to levels similar to control after subchronic treatment (16 days). In conclusion, we observed in rats after olanzapine treatment: (1) parallelism in the regulation of 5-HT2A receptors in frontal cortex and in blood platelets; (2) parallelism in the regulation of D3 mRNA levels in nucleus accumbens and lymphocytes. These results endorse the interest in future studies aimed at validating these receptors as candidate biomarkers in schizophrenia.

The GABA-reelin cortical connection (i.e., the expression and secretion of reelin by GABAergic cortical neurons) has been shown to function not only in the adult cortex but also during tangential migration of GABAergic neuroblasts. Therefore, it is of interest to focus on the possibility that a synergic action of these compounds (understood as a topobiological effect, implying place- and time-dependent interactions) may have important implications in regulating developmental processes such as neuronal migration, dendritic sprouting, synaptogenesis, and axon pruning, as well as being involved in regulation of synaptic plasticity trough life. The present review summarizes the actual knowledge in this field and discusses the possible importance that a dysregulation of GABAergic and reelin systems may have as vulnerability factors for the etiology and pathophysiology of schizophrenia.