TAAR1-dependent effects of apomorphine in mice.

G protein-coupled trace amine-associated receptor 1 (TAAR1) is expressed in several brain regions and modulates dopaminergic activity partially by affecting D2 dopamine receptor function. In vitro , the nonselective dopamine agonist apomorphine can activate mouse and rat TAAR1. The aim of the present study was to evaluate whether apomorphine activity at the rodent TAAR1 observed in in vitro studies contributes to its behavioral manifestation in mice. For this purpose, we compared the behavioral effects of a wide range of apomorphine doses in wild type (WT) and TAAR1 knockout (TAAR1-KO) mice. Apomorphine-induced locomotor responses (0.01–4.0 mg/kg) were tested in locomotor activity boxes, and stereotypic behavior at 5 mg/kg was tested by ethological methods. A gnawing test was used to analyze the effects of the highest dose of apomorphine (10 mg/kg). No statistically significant differences were observed between TAAR1-KO and WT mice following inhibitory pre-synaptic low doses of apomorphine. At higher doses (2.0–5.0 mg/kg), apomorphine-induced climbing behavior was significantly reduced in TAAR1 mutants relative to WT controls. Moreover, the lack of TAAR1 receptors decreased certain types of stereotypies (as reflected in by measures of the global stereotypy score, licking but not sniffing or gnawing) that were induced by high doses of apomorphine. These data indicate that apomorphine activity at TAAR1 contributes to some behavioral manifestations, particularly climbing, in rodents following high doses of this drug. The contribution of TAAR1 to apomorphine-induced climbing in rodents should be considered when apomorphine is used as a screening tool in the search for potential antipsychotics

Neuronal haemoglobin induces loss of dopaminergic neurons in mouse Substantia nigra, cognitive deficits and cleavage of endogenous α-synuclein

Parkinson's disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. alpha-synuclein (alpha-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote alpha-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells' vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, alpha and beta-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous alpha-syn C-terminal truncated species. Similar alpha-syn fragments were found in vitro in DA iMN9D cells over-expressing alpha and beta- globins when treated with pre-formed alpha-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells' loss in vivo and the formation of C-terminal alpha-syn fragments

Improved cognitive performance in trace amine-associated receptor 5 (TAAR5) knock-out mice

Trace amine-associated receptors (TAARs) are a family of G protein-coupled receptors present in mammals in the brain and several peripheral organs. Apart from its olfactory role, TAAR5 is expressed in the major limbic brain areas and regulates brain serotonin functions and emotional behaviours. However, most of its functions remain undiscovered. Given the role of serotonin and limbic regions in some aspects of cognition, we used a temporal decision-making task to unveil a possible role of TAAR5 in cognitive processes. We found that TAAR5 knock-out mice showed a generally better performance due to a reduced number of errors and displayed a greater rate of improvement at the task than WT littermates. However, task-related parameters, such as time accuracy and uncertainty have not changed significantly. Overall, we show that TAAR5 modulates specific domains of cognition, highlighting a new role in brain physiology

SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease.

International audience; Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson's disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system's functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD

SINEUPs: a novel toolbox for RNA therapeutics

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides

: SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs

Hit-to-Lead Optimization of Mouse Trace Amine Associated Receptor 1 (mTAAR1) Agonists with a Diphenylmethane-Scaffold: Design, Synthesis, and Biological Study

The trace amine-associated receptor 1 (TAAR1) is a G-protein-coupled receptors (GPCR) potently activated by a variety of molecules besides trace amines (TAs), including thyroid hormone-derivatives like 3-iodothyronamine (T1AM), catechol-O-methyltransferase products like 3-methoxytyramine, and amphetamine-related compounds. Accordingly, TAAR1 is considered a promising target for medicinal development. To gain more insights into TAAR1 physiological functions and validation of its therapeutic potential we recently developed a new class of thyronamine-like derivatives. Among them compound SG2 showed high affinity and potent agonist activity at mouse TAAR1. In the present work we describe design, the synthesis and SAR study of a new series of compounds (1-16) obtained by introducing specific structural changes at key points of our lead-compound SG2 skeleton. Five of the newly synthesized compounds displayed mTAAR1 agonist activity higher than both SG2 and T1AM. Selected diphenylmethane analogs, namely 1 and 2, showed potent functional activity in in vitro and in vivo models

Molecular characterization of Dipetalonema yatesi from the black-faced spider monkey (Ateles chamek) with phylogenetic inference of relationships among Dipetalonema of Neotropical primates

Species of the genus Dipetalonema are parasitic nematodes of the family Onchocercidae (Nematoda; Filarioidea) which infect the peritoneal cavity of Neotropical primates. Of these, six species have been taxonomically described, two of these have been reported infecting the black-faced spider monkey (Ateles chamek): Dipetalonema gracile and Dipetalonema yatesi. Description of Dipetalonema species have been based on morphological characteristics, and their phylogenetic relationships remain unresolved. A few molecular studies have been carried out in Dipetalonema spp. infecting Neotropical primates. Seven filarioid nematodes (6 females and one male) recovered from one A. chamek in the Peruvian Amazon rainforest were morphologically identified as D. yatesi and molecularly characterized. A multi-locus genetic analysis of nuclear ribosomal region (18S) and mitochondrial (cox1, 12S, and nad5) gene sequences supported D. yatesi as a distinct lineage and yielded a highly resolved phylogenetic lineage tree for this filarioid genus of Neotropical primates. Our results highlighted that Dipetalonema species are divided in two well-supported clades, one containing D. yatesi and D. caudispina, and the second containing D. robini, D. gracile, and D. graciliformis. Due to sequence ambiguities from GenBank entries, relationships among isolates of D. gracile and D. graciliformis cannot be fully resolved, which requires further investigation. However, this suggests that these could represent a species complex. Our study confirms that D. yatesi is a valid species and constitutes the first molecular phylogenetic analysis of this parasite in black-faced spider monkeys

Dopamine receptors in GtoPdb v.2023.1

Dopamine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Dopamine Receptors [373]) are commonly divided into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families, where the endogenous agonist is dopamine

Behavioral Effects of a Potential Novel TAAR1 Antagonist

The trace amine associated receptor 1 (TAAR1) is a G-protein coupled receptor expressed in the monoaminergic regions of the brain, and represents a potential novel therapeutic target for the treatment of neurological disorders. While selective agonists for TAAR1 have been successfully identified, only one high affinity TAAR1 antagonist has been described thus far. We previously identified four potential low potency TAAR1 antagonists through an in silico screen on a TAAR1 homology model. One of the identified antagonists (compound 22) was predicted to have favorable physicochemical properties, which would allow the drug to cross the blood brain barrier. In vivo studies were therefore carried out and showed that compound 22 potentiates amphetamine- and cocaine-mediated locomotor activity. Furthermore, electrophysiology experiments demonstrated that compound 22 increased firing of dopamine neurons similar to EPPTB, the only known TAAR1 antagonist. In order to assess whether the effects of compound 22 were mediated through TAAR1, experiments were carried out on TAAR1-KO mice. The results showed that compound 22 is able to enhance amphetamine- and cocaine-mediated locomotor activity, even in TAAR1-KO mice, suggesting that the in vivo effects of this compound are not mediated by TAAR1. In collaboration with Psychoactive Drug Screening Program, we attempted to determine the targets for compound 22. Psychoactive Drug Screening Program (PDSP) results suggested several potential targets for compound 22 including, the dopamine, norepinephrine and serotonin transporters; as well as sigma 1 and 2 receptors. Our follow-up studies using heterologous cell systems showed that the dopamine transporter is not a target of compound 22. Therefore, the biological target of compound 22 mediating its psychoactive effects still remains unknown