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.

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.

Increased Endogenous GDNF in Mice Protects Against Age-Related Decline in Neuronal Cholinergic Markers.

Gradual decline in cholinergic transmission and cognitive function occurs during normal aging, whereas pathological loss of cholinergic function is a hallmark of different types of dementia, including Alzheimer's disease (AD), Lewy body dementia (LBD), and Parkinson's disease dementia (PDD). Glial cell line-derived neurotrophic factor (GDNF) is known to modulate and enhance the dopamine system. However, how endogenous GDNF influences brain cholinergic transmission has remained elusive. In this study, we explored the effect of a twofold increase in endogenous GDNF (Gdnf hypermorphic mice, Gdnf wt/hyper) on cholinergic markers and cognitive function upon aging. We found that Gdnf wt/hyper mice resisted an overall age-associated decline in the cholinergic index observed in the brain of Gdnf wt/wt animals. Biochemical analysis revealed that the level of nerve growth factor (NGF), which is important for survival and function of central cholinergic neurons, was significantly increased in several brain areas of old Gdnf wt/hyper mice. Analysis of expression of genes involved in cholinergic transmission in the cortex and striatum confirmed modulation of cholinergic pathways by GDNF upon aging. In line with these findings, Gdnf wt/hyper mice did not undergo an age-related decline in cognitive function in the Y-maze test, as observed in the wild type littermates. Our results identify endogenous GDNF as a potential modulator of cholinergic transmission and call for future studies on endogenous GDNF function in neurodegenerative disorders characterized by cognitive impairments, including AD, LBD, and PDD.

Chronic 2-Fold Elevation of Endogenous GDNF Levels Is Safe and Enhances Motor and Dopaminergic Function in Aged Mice.

Glial cell line-derived neurotrophic factor (GDNF) supports function and survival of dopamine neurons that degenerate in Parkinson's disease (PD). Ectopic delivery of GDNF in clinical trials to treat PD is safe but lacks significant therapeutic effect. In pre-clinical models, ectopic GDNF is effective but causes adverse effects, including downregulation of tyrosine hydroxylase, only a transient boost in dopamine metabolism, aberrant neuronal sprouting, and hyperactivity. Hindering development of GDNF mimetic increased signaling via GDNF receptor RET by activating mutations results in cancer. Safe and effective mode of action must be defined first in animal models to develop successful GDNF-based therapies. Previously we showed that about a 2-fold increase in endogenous GDNF expression is safe and results in increased motor and dopaminergic function and protection in a PD model in young animals. Recently, similar results were reported using a novel Gdnf mRNA-targeting strategy. Next, it is important to establish the safety of a long-term increase in endogenous GDNF expression. We report behavioral, dopamine system, and cancer analysis of five cohorts of aged mice with a 2-fold increase in endogenous GDNF. We found a sustained increase in dopamine levels, improvement in motor learning, and no side effects or cancer. These results support the rationale for further development of endogenous GDNF-based treatments and GDNF mimetic.

Increased Calcium Influx through L-type Calcium Channels in Human and Mouse Neural Progenitors Lacking Fragile X Mental Retardation Protein.

The absence of FMR1 protein (FMRP) causes fragile X syndrome (FXS) and disturbed FMRP function is implicated in several forms of human psychopathology. We show that intracellular calcium responses to depolarization are augmented in neural progenitors derived from human induced pluripotent stem cells and mouse brain with FXS. Increased calcium influx via nifedipine-sensitive voltage-gated calcium (Cav) channels contributes to the exaggerated responses to depolarization and type 1 metabotropic glutamate receptor activation. The ratio of L-type/T-type Cav channel expression is increased in FXS progenitors and correlates with enhanced progenitor differentiation to glutamate-responsive cells. Genetic reduction of brain-derived neurotrophic factor in FXS mouse progenitors diminishes the expression of Cav channels and activity-dependent responses, which are associated with increased phosphorylation of the phospholipase C-γ1 site within TrkB receptors and changes of differentiating progenitor subpopulations. Our results show developmental effects of increased calcium influx via L-type Cav channels in FXS neural progenitors.

Functional changes of AMPA responses in human induced pluripotent stem cell-derived neural progenitors in fragile X syndrome.

Altered neuronal network formation and function involving dysregulated excitatory and inhibitory circuits are associated with fragile X syndrome (FXS). We examined functional maturation of the excitatory transmission system in FXS by investigating the response of FXS patient-derived neural progenitor cells to the glutamate analog (AMPA). Neural progenitors derived from induced pluripotent stem cell (iPSC) lines generated from boys with FXS had augmented intracellular Ca2+ responses to AMPA and kainate that were mediated by Ca2+-permeable AMPA receptors (CP-AMPARs) lacking the GluA2 subunit. Together with the enhanced differentiation of glutamate-responsive cells, the proportion of CP-AMPAR and N-methyl-d-aspartate (NMDA) receptor-coexpressing cells was increased in human FXS progenitors. Differentiation of cells lacking GluA2 was also increased and paralleled the increased inward rectification in neural progenitors derived from Fmr1-knockout mice (the FXS mouse model). Human FXS progenitors had increased the expression of the precursor and mature forms of miR-181a, a microRNA that represses translation of the transcript encoding GluA2. Blocking GluA2-lacking, CP-AMPARs reduced the neurite length of human iPSC-derived control progenitors and further reduced the shortened length of neurites in human FXS progenitors, supporting the contribution of CP-AMPARs to the regulation of progenitor differentiation. Furthermore, we observed reduced expression of Gria2 (the GluA2-encoding gene) in the frontal lobe of FXS mice, consistent with functional changes of AMPARs in FXS. Increased Ca2+ influx through CP-AMPARs may increase the vulnerability and affect the differentiation and migration of distinct cell populations, which may interfere with normal circuit formation in FXS.