Untranslated regions of brain-derived neurotrophic factor mRNA control its translatability and subcellular localization.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.

Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia.

Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2-3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A2AR), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A2AR with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF-A2AR crosstalk may regulate dopamine function in a therapeutically targetable manner.

Elevated expression of endogenous glial cell line-derived neurotrophic factor impairs spatial memory performance and raises inhibitory tone in the hippocampus.

Parvalbumin-positive interneurons (PV+) are a key component of inhibitory networks in the brain and are known to modulate memory and learning by shaping network activity. The mechanisms of PV+ neuron generation and maintenance are not fully understood, yet current evidence suggests that signalling via the glial cell line-derived neurotrophic factor (GDNF) receptor GFRα1 positively modulates the migration and differentiation of PV+ interneurons in the cortex. Whether GDNF also regulates PV+ cells in the hippocampus is currently unknown. In this study, we utilized a Gdnf "hypermorph" mouse model where GDNF is overexpressed from the native gene locus, providing greatly increased spatial and temporal specificity of protein expression over established models of ectopic expression. Gdnfwt/hyper mice demonstrated impairments in long-term memory performance in the Morris water maze test and an increase in inhibitory tone in the hippocampus measured electrophysiologically in acute brain slice preparations. Increased PV+ cell number was confirmed immunohistochemically in the hippocampus and in discrete cortical areas and an increase in epileptic seizure threshold was observed in vivo. The data consolidate prior evidence for the actions of GDNF as a regulator of PV+ cell development in the cortex and demonstrate functional effects upon network excitability via modulation of functional GABAergic signalling and under epileptic challenge.

Analysis of Acute and Chronic Methamphetamine Treatment in Mice on Gdnf System Expression Reveals a Potential Mechanism of Schizophrenia Susceptibility.

The increase in presynaptic striatal dopamine is the main dopaminergic abnormality in schizophrenia (SCZ). SCZ is primarily treated by modulating the activity of monoamine systems, with a focus on dopamine and serotonin receptors. Glial cell line-derived neurotrophic factor (GDNF) is a strong dopaminergic factor, that recently was shown to correlate with SCZ in human CSF and in striatal tissue. A 2-3-fold increase in GDNF in the brain was sufficient to induce SCZ-like dopaminergic and behavioural changes in mice. Here, we analysed the effect of acute, chronic, and embryonic methamphetamine, a drug known to enhance the risk of psychosis, on Gdnf and its receptors, Gfra1 and Ret, as well as on monoamine metabolism-related gene expression in the mouse brain. We found that acute methamphetamine application increases Gdnf expression in the striatum and chronic methamphetamine decreases the striatal expression of GDNF receptors Gfra1 and Ret. Both chronic and acute methamphetamine treatment upregulated the expression of genes related to dopamine and serotonin metabolism in the striatum, prefrontal cortex, and substantia nigra. Our results suggest a potential mechanism as to how methamphetamine elicits individual psychosis risk in young adults-variation in initial striatal GDNF induction and subsequent GFRα1 and RET downregulation may determine individual susceptibility to psychosis. Our results may guide future experiments and precision medicine development for methamphetamine-induced psychosis using GDNF/GFRa1/RET antagonists.

Opposing Spatially Segregated Function of Endogenous GDNF-RET Signaling in Cocaine Addiction.

Cocaine addiction is a serious condition with potentially lethal complications and no current pharmacological approaches towards treatment. Perturbations of the mesolimbic dopamine system are crucial to the establishment of cocaine-induced conditioned place preference and reward. As a potent neurotrophic factor modulating the function of dopamine neurons, glial cell line-derived neurotrophic factor (GDNF) acting through its receptor RET on dopamine neurons may provide a novel therapeutic avenue towards psychostimulant addiction. However, current knowledge on endogenous GDNF and RET function after the onset of addiction is scarce. Here, we utilized a conditional knockout approach to reduce the expression of the GDNF receptor tyrosine kinase RET from dopamine neurons in the ventral tegmental area (VTA) after the onset of cocaine-induced conditioned place preference. Similarly, after establishing cocaine-induced conditioned place preference, we studied the effect of conditionally reducing GDNF in the ventral striatum nucleus accumbens (NAc), the target of mesolimbic dopaminergic innervation. We find that the reduction of RET within the VTA hastens cocaine-induced conditioned place preference extinction and reduces reinstatement, while the reduction of GDNF within the NAc does the opposite: prolongs cocaine-induced conditioned place preference and increases preference during reinstatement. In addition, the brain-derived neurotrophic factor (BDNF) was increased and key dopamine-related genes were reduced in the GDNF cKO mutant animals after cocaine administration. Thus, RET antagonism in the VTA coupled with intact or enhanced accumbal GDNF function may provide a new approach towards cocaine addiction treatment.

Increased Physiological GDNF Levels Have No Effect on Dopamine Neuron Protection and Restoration in a Proteasome Inhibition Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease that comprises a range of motor and nonmotor symptoms. Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopamine neurons in vitro and in vivo, and intracranial delivery of GDNF has been tested in six clinical trials for treating PD. However, clinical trials with ectopic GDNF have yielded variable results, which could in part result from abnormal expression site and levels caused by ectopic overexpression. Therefore, an important open question is whether an increase in endogenous GDNF expression could be potent in reversing PD progression. Here, we tested the therapeutic potential of endogenous GDNF using mice in which endogenous GDNF can be conditionally upregulated specifically in cells that express GDNF naturally (conditional GDNF hypermorphic mice; GdnfcHyper ). We analyzed the impact of endogenous GDNF upregulation in both neuroprotection and neurorestoration procedures, and for both motor and nonmotor symptoms in the proteasome inhibitor lactacystin (LC) model of PD. Our results showed that upregulation of endogenous GDNF in the adult striatum is not protective in LC-induced PD model in mice. Since age is the largest risk factor for PD, we also analyzed the effect of deletion of endogenous GDNF in aged Gdnf conditional knock-out mice. We found that GDNF deletion does not increase susceptibility to LC-induced damage. We conclude that endogenous GDNF does not impact the outcome in the LC-induced proteasome inhibition mouse model of Parkinson's disease.

Myenteric Neurons Do Not Replicate in Small Intestine Under Normal Physiological Conditions in Adult Mouse.

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest part of the peripheral nervous system; moreover, abnormal ENS development and function are associated with multiple human pathologies. Data from several groups suggest that under normal physiological conditions in adult animals, enteric nerve cells do not replicate. A study by Kulkarni et al in 2017 challenged this view and proposed that nearly 70% of enteric neurons in the myenteric ganglia are born in 1 week. The authors of this study suggested that differences in DNA labelling times and DNA denaturation conditions might explain discrepancies with previous reports. Previous studies were carried out using different conditions and labelling techniques in various regions of the gastrointestinal tract; thus, conclusions have remained elusive. METHODS: Here, we have eliminated those variables by analyzing the whole small intestine using the reagents and conditions that Kulkarni et al used. To exclude variables related to immunohistochemistry, we carried out parallel experiments with "click chemistry"-based detection of DNA replication. RESULTS: Although proliferation was readily detected in the epithelium, we found no evidence of neuronal replication in the myenteric ganglia. CONCLUSIONS: We conclude that within 1 week under normal physiological conditions, myenteric neurons in the small intestine do not replicate.

Amphetamine enantiomers inhibit homomeric α7 nicotinic receptor through a competitive mechanism and within the intoxication levels in humans.

Amphetamine-type stimulants (ATS) are the second most consumed illicit drug worldwide and lack good treatments for associated substance use disorders, lagging behind other addictive drugs. For this reason, a deeper understanding of the pharmacodynamics of ATS is required. The present study seeks to determine amphetamine (AMPH) enantiomers' effects on the homomeric α7 nicotinic acetylcholine receptor (α7 nAChR). Here we have shown that AMPH enantiomers bind to the α7 nAChR and competitively inhibit acetylcholine responses. Our in silico docking analysis suggests that AMPH binds close to the ß7 strand of the B-loop of a chimera comprising of the human α7 nAChR and the acetylcholine binding protein from Lymnaea stagnalis. This may inhibit the required movement of the C-loop for channel opening, due to steric hindrance, providing a structural mechanism for its antagonist effect. Finally, we have shown that, in α7 nAChR full knockout mice, the behavioral response to D-AMPH is attenuated, providing direct evidence for the role of α7 nAChRs on the physiological response to D-AMPH. Importantly, D-AMPH exerts these effects at concentrations predicted to be pharmacologically relevant for chronic methamphetamine users and during binges. In conclusion, our data present new findings that implicate the α7 nAChR on the pharmacodynamics of ATS, which may be important for behavioral responses to these drugs, indicating a potential role for α7 nAChRs in ATS substance-use disorders.