PTEN deletion enhances survival, neurite outgrowth and function of dopamine neuron grafts to MitoPark mice.

Clinical trials in Parkinson's disease have shown that transplants of embryonic mesencephalic dopamine neurons form new functional connections within the host striatum, but the therapeutic benefits have been highly variable. One obstacle has been poor survival and integration of grafted dopamine neurons. Activation of Akt, a serine/threonine kinase that promotes cell survival and growth, increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. Because the lipid phosphatase, phosphatase and tensin homolog (PTEN) inhibits Akt, we generated a mouse with conditional knock-out of PTEN in dopamine neurons, leading to constitutive expression of Akt in these neurons. Ventral mesencephalic tissue from dopamine phosphatase and tensin homologue knock-out or control animals was then transplanted bilaterally into the dopamine depleted striata of MitoPark mice that express a parkinsonian phenotype because of severe respiratory chain dysfunction in dopamine neurons. After transplantation into MitoPark mice, PTEN-deficient dopamine neurons were less susceptible to cell death, and exhibited a more extensive pattern of fibre outgrowth compared to control grafts. Voltammetric measurements demonstrated that dopamine release and reuptake were significantly increased in the striata of animals receiving dopamine PTEN knock-out transplants. These animals also displayed enhanced spontaneous and drug-induced locomotor activity, relative to control transplanted MitoPark mice. Our results suggest that disinhibition of the Akt-signalling pathway may provide a valuable strategy to enhance survival, function and integration of grafted dopamine neurons within the host striatum and, more generally, to improve survival and integration of different forms of neural grafts.

Attenuated response to methamphetamine sensitization and deficits in motor learning and memory after selective deletion of ß-catenin in dopamine neurons.

In the present study, we analyzed mice with a targeted deletion of ß-catenin in DA neurons (DA-ßcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-ßcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-ßcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-ßcat KO mice. This study demonstrates that ablation of ß-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that ß-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.

Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice.

Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5 kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.

Gene expression patterns for GDNF and its receptors in the human putamen affected by Parkinson's disease: a real-time PCR study.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta superfamily, is a potent trophic factor for dopaminergic neurons of the ventral midbrain, which are known to degenerate during Parkinson's disease (PD). The neuroprotective, neurorestorative, and stimulatory properties of GDNF has prompted numerous suggestions that this trophic factor may be a potential therapeutic tool to treat PD, and it has also been widely speculated that altered GDNF expression levels may be involved in the pathophysiology of the disease. In this study, we have investigated if mRNA expression levels for GDNF and/or its receptors are altered during PD in the human putamen, a target area for dopamine neurons of the substantia nigra compacta. Expression levels were analyzed with quantitative real-time reverse transcriptase polymerase reaction (RT qPCR) in post-mortem tissues from PD patients and aged matched controls. Primer pairs specific for GDNF (isoforms I and II), and its receptor molecules, GFRalpha1 and cRET were utilized. GDNF, cRET and GFRalpha1 mRNA expression was clearly detected in the putamen of control and Parkinson's disease patients. A modest but significant upregulation of GDNF mRNA levels (Isoform I) was observed in the putamen of Parkinson's disease patients with a marked loss of nigral neurons. No significant changes were observed for the expression of cRet and GFRa1. These data suggest that the extensive loss of dopaminergic neurons in the substantia nigra, and concomitant loss of striatal dopamine, may induce compensatory changes in the expression of target derived GDNF, but not its receptor system.

L-Dopa induced dyskinesias in Parkinsonian mice: Disease severity or L-Dopa history.

In Parkinson's disease, the efficacy of l-Dopa treatment changes over time, as dyskinesias emerge with previously beneficial doses. Using MitoPark mice, that models mitochondrial failure in dopamine (DA) neurons and mimics the progressive loss of dopamine observed in Parkinson's disease, we found that the severity of DA denervation and associated adaptations in striatal neurotransmission at the time of initiation of l-Dopa treatment determines development of l-Dopa induced dyskinesias. We treated 20-week, and 28-week old MitoPark mice with l-Dopa (10mg/kg i.p. twice a day) and found locomotor responses to be significantly different. While all MitoPark mice developed sensitization to l-Dopa treatment over time, 28-week old MitoPark mice with extensive striatal DA denervation developed abnormal involuntary movements rapidly and severely after starting l-Dopa treatment, as compared to a more gradual escalation of movements in 20-week old animals that started treatment at earlier stages of degeneration. Our data support that it is the extent of loss of DA innervation that determines how soon motor complications develop with l-Dopa treatment. Gene array studies of striatal neurotransmitter receptors revealed changes in mRNA expression levels for DA, serotonin, glutamate and GABA receptors in striatum of 28-week old MitoPark mice. Our results support that delaying l-Dopa treatment until Parkinson's disease symptoms become more severe does not delay the development of l-Dopa-induced dyskinesias. MitoPark mice model genetic alterations known to impair mitochondrial function in a subgroup of Parkinson patients and provide a platform in which to study treatments to minimize the development of dyskinesia.

NMDA receptors on non-dopaminergic neurons in the VTA support cocaine sensitization.

BACKGROUND: The initiation of behavioral sensitization to cocaine and other psychomotor stimulants is thought to reflect N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic plasticity in the mesolimbic dopamine (DA) circuitry. The importance of drug induced NMDAR mediated adaptations in ventral tegmental area (VTA) DA neurons, and its association with drug seeking behaviors, has recently been evaluated in Cre-loxp mice lacking functional NMDARs in DA neurons expressing Cre recombinase under the control of the endogenous dopamine transporter gene (NR1(DATCre) mice). METHODOLOGY AND PRINCIPAL FINDINGS: Using an additional NR1(DATCre) mouse transgenic model, we demonstrate that while the selective inactivation of NMDARs in DA neurons eliminates the induction of molecular changes leading to synaptic strengthening, behavioral measures such as cocaine induced locomotor sensitization and conditioned place preference remain intact in NR1(DATCre) mice. Since VTA DA neurons projecting to the prefrontal cortex and amygdala express little or no detectable levels of the dopamine transporter, it has been speculated that NMDA receptors in DA neurons projecting to these brain areas may have been spared in NR1(DATCre) mice. Here we demonstrate that the NMDA receptor gene is ablated in the majority of VTA DA neurons, including those exhibiting undetectable DAT expression levels in our NR1(DATCre) transgenic model, and that application of an NMDAR antagonist within the VTA of NR1(DATCre) animals still blocks sensitization to cocaine. CONCLUSIONS/SIGNIFICANCE: These results eliminate the possibility of NMDAR mediated neuroplasticity in the different DA neuronal subpopulations in our NR1(DATCre) mouse model and therefore suggest that NMDARs on non-DA neurons within the VTA must play a major role in cocaine-related addictive behavior.

Expression pattern of NuIP gene in adult mouse brain.

We previously reported the identification of the Nurr1 interacting protein (NuIP) that was demonstrated to modulate the transcriptional activity of Nurr1, the orphan nuclear receptor required for midbrain dopaminergic neuron differentiation. NuIP was also cloned by others and referred to as a small G protein signaling modulators. The open reading frame of NuIP predicts a protein with an N-terminal RUN domain (RPIP8, UNC-14, and NESCA) and a C-terminal TBC domain (Tre-2, Bub2, and Cdc16) both of which are found in proteins of the GTPase activating protein (GAP) family, involved in the GTPase signaling pathway. To characterize the NuIP gene product, we developed a polyclonal antibody. Since NuIP gene is expressed most abundantly in adult and the level of expression during development is below the detection limit of immunohistochemistry, we now report the expression pattern of NuIP in adult mouse brain compared with the expression pattern of Nurr1 protein. Many regions co-expressed Nurr1 and NuIP including cortex, hippocampus, substantia nigra, and the cerebellum. However, there are also regions that exclusively express NuIP such as striatum, septum, globus pallidus, and the reticular thalamic nucleus. We also find that NuIP protein expresses mainly in NeuN-positive (neuronal nuclei) neurons but can be detected in GFAP-positive (glial fibrillary acidic protein) glial cells in hippocampus. Interestingly, NuIP is expressed in high levels in midbrain dopaminergic neurons including ventral tegmental area (VTA) and substantia nigra (SN) dopaminergic neurons but is not expressed or expressed in low levels in other dopaminergic neurons such as olfactory bulb and hypothalamus. Overall, the expression pattern of NuIP in adult mouse brain suggests that it may be involved in motor activity control in basal ganglia as well as higher central nervous system (CNS) functions such as cognition and memory in cortex and hippocampus.

Selective deletion of PTEN in dopamine neurons leads to trophic effects and adaptation of striatal medium spiny projecting neurons.

The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson's disease, and other neurodegenerative disorders.

Alterations in prodynorphin, proenkephalin, and GAD67 mRNA levels in the aged human putamen: correlation with Parkinson's disease.

A real-time quantitative PCR approach was used to quantify mRNA levels corresponding to the neuropeptides enkephalin, dynorphin, and the 67-kDa isoform of glutamic acid decarboxylase (GAD67) in the human putamen from young and aged individuals as well as from aged patients affected by Parkinson's disease (PD). cDNA-specific primers were designed to amplify GAD67, proenkephalin (pENK), prodynorphin (pDYN), and the housekeeping genes glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and guanine nucleotide binding protein, beta-peptide 2-like I (GNB2LI). GAPDH and GNB2LI mRNA levels were similarly expressed among the groups and were therefore used as endogenous reference genes. Normalized data showed that mRNA levels for both pENK and pDYN were reduced in the putamen of aged controls and aged individuals affected by PD, compared with young controls. In addition, we showed that GAD67 mRNA levels did not change during aging and PD. Further analyses showed no differences in mRNA levels, for pENK, pDYN, or GAD67 mRNA, between PD patients and aged matched controls. These findings contrast with animal models of parkinsonism, for which expression of pDYN, pENK, and GAD67 mRNA has been reported to change after striatal dopamine denervation. Compensatory mechanisms and regional differences within the human putamen as well as the severity index of the disease, clinical diagnosis, and response to phalmacological therapy are possible reasons for these results. The present study suggests that alteration of neuropeptide pathways in the human putamen may be involved in the functional deterioration of parts of the extrapyramidal system during aging.

Characterization of a mouse strain expressing Cre recombinase from the 3' untranslated region of the dopamine transporter locus.

Dopamine (DA) neurotransmission has been implicated in several neurological and psychiatric disorders. The dopamine transporter (DAT) is highly expressed in dopaminergic neurons of the ventral mesencephalon and regulates neurotransmission by transporting DA back into the presynaptic terminals. To mediate restricted DNA recombination events into DA neurons using the Cre/loxP technology, we have generated a knockin mouse expressing Cre recombinase under the transcriptional control of the endogenous DAT promoter. To minimize interference with DAT function by preservation of both DAT alleles, Cre recombinase expression was driven from the 3' untranslated region (3'UTR) of the endogenous DAT gene by means of an internal ribosomal entry sequence. Crossing this murine line with a LacZ reporter showed colocalization of DAT immunocytochemistry and beta-galactosidase staining in all regions analyzed. This knockin mouse can be used for generating tissue specific knockouts in mice carrying genes flanked by loxP sites, and will facilitate the analysis of gene function in dopaminergic neurons.