Gene Therapy Vector Encoding Neuropeptide Y and Its Receptor Y2 for Future Treatment of Epilepsy: Preclinical Data in Rats.

Gene therapy to treat pharmacoresistant temporal lobe epilepsy in humans is now being developed using an AAV vector (CG01) that encodes the combination of neuropeptide Y and its antiepileptic receptor Y2. With this in mind, the present study aimed to provide important preclinical data on the effects of CG01 on the duration of transgene expression, cellular tropism, and potential side effects on body weight and cognitive function. The CG01 vector was administered unilaterally into the dorsal and ventral hippocampus of adult male rats and expression of both transgenes was found to remain elevated without a sign of decline at 6 months post-injection. CG01 appeared to mediate expression selectively in hippocampal neurons, without expression in astrocytes or oligodendrocytes. No effects were seen on body weight as well as on short- or long-term memory as revealed by testing in the Y-maze or Morris water maze tests. Thus these data show that unilateral CG01 vector treatment as future gene therapy in pharmacoresistant temporal lobe epilepsy patients should result in stable and long-term expression predominantly in neurons and be well tolerated without side effects on body weight and cognitive function.

The dorsomedial hypothalamus and nucleus of the solitary tract as key regulators in a rat model of chronic obesity.

The surging obesity epidemic calls for a deeper understanding of central nervous system (CNS) mechanisms underlying the biologically defended level of body weight. Here, we analyzed global gene expression in four hypothalamic and two brainstem nuclei involved in energy homeostatic control of body weight in diet-induced obese (DIO) and lean rats. Male Sprague-Dawley rats were offered ad libitum chow, or a two-choice diet consisting of a high palatable high sugar/fat diet and chow for 40 weeks. At termination, the hypothalamic arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), paraventricular nucleus (PVN) and lateral hypothalamus area (LHA), as well as the brainstem area postrema (AP) and nucleus of the solitary tract (NTS), were isolated by laser capture microdissection (LCM) followed by mRNA sequencing. Global gene expression analyses revealed a total of 88 differentially expressed genes (DEGs) in DIO rats. Transcriptome changes were mainly observed in the DMH and NTS and associated with neuropeptide signaling and regulation of signaling transduction pathways, suggesting a key role of these brain regions in body weight regulation.

Modulating Dopamine Signaling and Behavior with Chemogenetics: Concepts, Progress, and Challenges.

For more than 60 years, dopamine (DA) has been known as a critical modulatory neurotransmitter regulating locomotion, reward-based motivation, and endocrine functions. Disturbances in DA signaling have been linked to an array of different neurologic and psychiatric disorders, including Parkinson's disease, schizophrenia, and addiction, but the underlying pathologic mechanisms have never been fully elucidated. One major obstacle limiting interpretation of standard pharmacological and transgenic interventions is the complexity of the DA system, which only appears to widen as research progresses. Nonetheless, development of new genetic tools, such as chemogenetics, has led to an entirely new era for functional studies of neuronal signaling. By exploiting receptors that are engineered to respond selectively to an otherwise inert ligand, so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), chemogenetics enables pharmacological remote control of neuronal activity. Here we review the recent, extensive application of this technique to the DA field and how its use has advanced the study of the DA system and contributed to our general understanding of DA signaling and related behaviors. Moreover, we discuss the challenges and pitfalls associated with the chemogenetic technology, such as the metabolism of the DREADD ligand clozapine N-oxide (CNO) to the D2 receptor antagonist clozapine. We conclude that despite the recent concerns regarding CNO, the chemogenetic toolbox provides an exceptional approach to study neuronal function. The huge potential should promote continued investigations and additional refinements to further expound key mechanisms of DA signaling and circuitries in normal as well as maladaptive behaviors.

The C-terminal flanking peptide of neuropeptide Y (NPY) is not essential for seizure-suppressant actions of prepro-NPY overexpression in male rats.

The full coding sequence of neuropeptide Y (NPY), prepro-NPY, is sequentially metabolized into three peptides; an N-terminus 28-amino acid signaling peptide, the NPY peptide itself (NPY1-36), and a 30-amino acid C-terminus peptide, known as the C-terminal flanking peptide of neuropeptide-Y (CPON). While the signaling peptide directs intracellular trafficking and NPY1-36 is well characterized, the biological function of CPON is unknown. This is noteworthy because CPON is co-stored and co-released along with NPY1-36 and could thus potentially serve important functions. To assess the role of CPON, we adapted a viral genetic approach using two different vector designs encoding NPY, but where the CPON coding sequence was excluded from one of the vectors. Thus, the effect of CPON was indirectly assessed. Male rats received intrahippocampal injections of either a vector encoding NPY1-39 whose metabolism yields NPY1-36 and not CPON, or a prepro-NPY vector encoding both NPY1-36 and CPON. A third vector encoding EGFP served as control. We subsequently studied to what extent CPON might affect seizure susceptibility and memory performance, respectively, to address two important questions to evaluate the potential of NPY gene therapy in epilepsy. Both NPY vectors, as compared to EGFP control, were found to be equally effective at suppressing acute kainate-induced seizures, and both did not influence learning and memory performance in the Morris water maze. Thus CPON itself does not appear to aid actions governed by vector-mediated overexpression of NPY1-36 within the hippocampus. Whether CPON serves other important functions remains to be determined.

Localization, distribution, and connectivity of neuropeptide Y in the human and porcine retinas-A comparative study.

Neuropeptide Y (NPY) is a peptide neurotransmitter abundantly expressed in the mammalian retina. Since its discovery, NPY has been studied in retinas of several species, but detailed characterization of morphology, cell-type, and connectivity has never been conducted in larger mammals including humans and pigs. As the pig due to size and cellular composition is a well-suited animal for retinal research, we chose to compare the endogenous NPY system of the human retina to that of pigs to support future research in this field. In the present study, using immunohistochemistry, confocal microscopy and 3D reconstructions, we found NPY to be expressed in GABAergic and calretinin-immunoreactive (-ir) amacrine cells of both species as well as parvalbumin-ir amacrine cells of humans. Furthermore, we identified at least two different types of medium- to wide-field NPY-ir amacrine cells. Finally, we detected likely synaptic appositions between the NPY-ir amacrine cells and melanopsin- and nonmelanopsin-ir ganglion cells, GABAergic and dopaminergic amacrine cells, rod bipolar cells, and horizontal cells, suggesting that NPY-ir cells play diverse roles in modulation of both image and non-image forming retinal signaling. These findings extend existing knowledge on NPY and NPY-expressing cells in the human and porcine retina showing a high degree of comparability. The extensive distribution and connectivity of NPY-ir cells described in the present study further highlights the potential importance of NPY signaling in retinal function.

Differential plastic changes in synthesis and binding in the mouse somatostatin system after electroconvulsive stimulation.

OBJECTIVE: Electroconvulsive therapy (ECT) is regularly used to treat patients with severe major depression, but the mechanisms underlying the beneficial effects remain uncertain. Electroconvulsive stimulation (ECS) regulates diverse neurotransmitter systems and induces anticonvulsant effects, properties implicated in mediating therapeutic effects of ECT. Somatostatin (SST) is a candidate for mediating these effects because it is upregulated by ECS and exerts seizure-suppressant effects. However, little is known about how ECS might affect the SST receptor system. The present study examined effects of single and repeated ECS on the synthesis of SST receptors (SSTR1-4) and SST, and SST receptor binding ([125I]LTT-SST28) in mouse hippocampal regions and piriform/parietal cortices. RESULTS: A complex pattern of plastic changes was observed. In the dentate gyrus, SST and SSTR1 expression and the number of hilar SST immunoreactive cells were significantly increased at 1 week after repeated ECS while SSTR2 expression was downregulated by single ECS, and SSTR3 mRNA and SST binding were elevated 24 h after repeated ECS. In hippocampal CA1 and parietal/piriform cortices, we found elevated SST mRNA levels 1 week after repeated ECS and elevated SST binding after single ECS and 24 h after repeated ECS. In hippocampal CA3, repeated ECS increased SST expression 1 week after and SST binding 24 h after. In the parietal cortex, SSTR2 mRNA expression was downregulated after single ECS while SSTR4 mRNA expression was upregulated 24 h after repeated ECS. CONCLUSION: Considering the known anticonvulsant effects of SST, it is likely that these ECS-induced neuroplastic changes in the SST system could participate in modulating neuronal excitability and potentially contribute to therapeutic effects of ECT.

MicroRNA 101b Is Downregulated in the Prefrontal Cortex of a Genetic Model of Depression and Targets the Glutamate Transporter SLC1A1 (EAAT3) in Vitro.

BACKGROUND: MicroRNAs (miRNAs) are small regulatory molecules that cause translational repression by base pairing with target mRNAs. Cumulative evidence suggests that changes in miRNA expression may in part underlie the pathophysiology and treatment of neuropsychiatric disorders, including major depressive disorder (MDD). METHODS: A miRNA expression assay that can simultaneously detect 423 rat miRNAs (miRBase v.17) was used to profile the prefrontal cortex (PFC) of a genetic rat model of MDD (the Flinders Sensitive Line [FSL]) and the controls, the Flinders Resistant Line (FRL). Gene expression data from the PFC of FSL/FRL animals (GEO accession no. GSE20388) were used to guide mRNA target selection. Luciferase reporter assays were used to verify miRNA targets in vitro. RESULTS: We identified 23 miRNAs that were downregulated in the PFC of the FSL model compared with controls. Interestingly, one of the identified miRNAs (miR-101b) is highly conserved between rat and human and was recently found to be downregulated in the PFC of depressed suicide subjects. Using a combination of in silico and in vitro analyses, we found that miR-101b targets the neuronal glutamate transporter SLC1A1 (also known as EAAC1 or EAAT3). Accordingly, both mRNA and protein levels of SLC1A1 were found to be upregulated in the PFC of the FSL model. CONCLUSIONS: Besides providing a list of novel miRNAs associated with depression-like states, this preclinical study replicated the human association of miR-101 with depression. In addition, since one of the targets of miR-101b appears to be a glutamate transporter, our preclinical data support the hypothesis of a glutamatergic dysregulation being implicated in the etiology of depression.

[Optogenetics brings hidden neural mechanisms into the light and could become a future therapy]. / Optogenetik bringer neurale mekanismer frem i lyset og kan blive en ny terapiform.

Optogenetics is an emergent technology that combines light-sensitive proteins derived from algae, so-called opsins, with genetics. Viral vectors encoding opsins are injected into selective brain regions whereby specific cell populations can be controlled with high precision light pulses delivered via implanted optical fibres. This review focuses on explaining basic principles of optogenetics and describes important insights into neuropsychiatric mechanisms provided by the technology.