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

NMDA Receptors on Non-Dopaminergic Neurons in the VTA Support Cocaine Sensitization

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).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.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

Glial cell line-derived neurotrophic factor. : Expression patterns, neuronal transport, regulation, effects, and receptor dependence

Background: The mammalian nervous system depends upon trophic proteins for survival, growth and adult maintenance. These trophic factors have also been considered as potential therapeutic agents for treating human neurodegenerative disorders. In Parkinson's disease, there is a loss of midbrain dopaminergic (DA) neurons and a subsequent reduction of DA in caudate and putamen, which leads to significant motor symtomatology. In 1993, a new trophic factor was discovered for midbrain DA neurons, which had marked positive effects in vitro (Lin et al., 1993). This factor, termed glial cell line-derived neurotrophic factor (GDNF) is a distant member of the TGF-B superfamily and is the first of a new subfamily of such factors. Two additional members of this subfamily have recently been cloned and have been termed Neurturin (NTN) and Persephin (PSP). More recently, cell surface binding proteins which are part of the receptor complex for these three trophic factors have been cloned and have been termed GFRa-l, 2, and 3, respectively. The signaling mechanism for GDNF and NTN is thought to involve initial association with GFR a-l and GFRa-2, respectively with subsequent activation of the tyrosine kinase receptor c-Ret, although this hypothesis has been validated only in vitro. Aims: In the first part of my thesis, I sought to define the localization and ontogeny of GDNF expression and how expression might be regulated. In the second part, I studied effects of GDNF on peripheral and central neurons, using ganglia explant cultures and the l-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) mouse model of Parkinson's disease. I also studied if a specific retrograde transport system for GDNF was present in midbrain DA circuits. In the last part of my thesis, I attempted to study the importance of GFRa-1 for the effects of GDNF, by generating a GFRa-1 null mutation. Results: The first study in my thesis was to characterized GDNF gene mRNA expression during development and adulthood using in situ hybridization (paper 1). Based on the results from the initial characterizations of GDNF gene expression during both development and adulthood, the following experiments were undertaken: 1. Studying if the GDNF gene was regulated in adulthood using pilocarpine-induced status epilepticus in the rat (paper II). These experiments clearly indicated that GDNF might play a crucial role in the protection of neurons against excitotoxic stress. 2. Testing the function of the GDNF protein on other types of cells in an in vitro explant system (paper Ill). Our results from these explant studies, taken together with the expression data, indicated that GDNF was also a potent factor for other classes of neurons in various autonomic ganglia. 3. Examining if there were protective and regenerative effects of the GDNF protein on the nigrostriatal system. This is the system that degenerates in humans with Parkinson's disease. To investigate this, adult MPTP-ex posed mice were treated with GDNF. We showed that GDNF could both partially regenerate and protect adult dopaminergic neurons (paper IV). 4. Determining if GDNF acted directly upon the dopaminergic neurons as a "classic target derived factor," or indirectly, by using retrograde transport studies (paper V). In these experiments we showed that GDNF, initially placed in the striatum, can be retrogradely transported to midbrain DA neuron soma via a specific receptor-mediated mech anisms. 5. Determining the importance of GFRa-1 for the effects of GDNF in vivo during development. It has previously been shown that null mutations of GDNF (Moore et al. 1996, Pichel et al. 1996; Sanchez et al., 1996) and Ret (Schuchardt 1994) have a similar phenotype, charactenzed by absence of, or severe malformation, of the kidneys and loss of enteric ganglia. We demonstrated that null mutations of GFRa-l have a similar phenotype (paper Vl) documenting the critical role of this binding protein for the actions of GDNF. Andreas C. Tomac, 199

<i>Pten</i> ablation in DA cells affect the direct striatal output system by significantly increasing mRNA expression levels for DRD1 and pDyn in striatal medium spiny neurons.

<p>BDNF mRNA levels were significantly elevated in the ventral mesencephalon (A) Dopamie receptor D1 (DRD1) and D2 (DRD2) mRNA expression levels in the striatum of control (n = 9) and <i>Pten</i> KO animals (n = 13). DRD1 mRNA expression levels were significantly increased in KO animals as compared to controls. DRD2 mRNA levels were unchanged between the groups. (B) Changes in DRD1 expression levels were accompanied by an elevation in prodynorphin (pDyn) mRNA in <i>Pten</i> KO animals. Preproenkephalin (PPE) mRNA expression levels were unchanged. (C) BDNF mRNA levels were significantly increased in the ventral mesencephalon in KO animals. All data are mean±SEM. *p<0.05, Student's t-test.</p

<i>Pten</i> ablation shows neuroprotective effects in the intrastriatal 6OHDA mouse model.

<p>Animals were treated with a unilateral 6OHDA injection in the striatum, and perfused 4 weeks after the injection to analyze the effect of <i>Pten</i> ablation on the magnitude of the lesion. (A) The SNc from control animals (n = 4) was markedly affected by the lesion as reflected by the significant loss of TH positive profiles in the ipsilateral side to the lesion as compared to the contralateral side. The dopaminergic neurons in <i>Pten</i> KO (n = 6) animals were largely spared after the lesion when compared to control animals. (B) The neuroprotective effects related to <i>Pten</i> ablation were observed at the level of the striatal axonal projections as well. Depletion of TH positive terminals in the striatum was reduced across the striatum in control animals, while axon terminal degeneration was much lower in <i>Pten</i> KO animals. (C) Survival rates for TH-positive neurons were quantified by stereologic counts. The number of surviving neurons (presented as percent surviving neurons when compared to the side contralateral to the lesion) was significantly increased in <i>Pten</i> KO animals (n = 6) when compared to controls (n = 4). (D) Optical density measurement in the injected striatum revealed a nearly complete preservation of TH terminals in <i>Pten</i> KO animals. TH staining density was significantly lower in control 6OHDA-treated animals with the biggest difference observed in the medial portion of the striatum, in accordance with sterotaxic coordinated used for the lesion. (A) Scale bar, 250 µm and (B) 500 µm. All data are mean±SEM. *p<0.05, Student's t-test.</p

The morphological preservation of the <i>Pten</i> deficient nigrostriatal system after 6OHDA treatment correlates with functional recovery after the lesion.

<p>To determine the functionality of the nigrostriatal system after the lesion, we examined methamphetamine-induced rotations. Methamphetamine treatment increases the extracellular availability of endogenous DA in the striatum, and it causes animals with a partial DA depletion to rotate against the lesion side (ipsilateral), due to the imbalance in DA release between the striatae. Strong ipsilateral rotational behavior was observed in control animals (n = 8) after treatment with (+) methamphetamine HCl (2.5 mg/kg) at 14 and 28 days after the lesion. Ipsilateral rotational behavior was significantly reduced in <i>Pten</i> KO animals (n = 10). All data are mean±SEM. *p<0.05, Student's t-test.</p

Exposure to a novel environment does not enhance locomotor activity in <i>Pten</i> KO mice.

<p>Control (n = 11) and <i>Pten</i> KO (n = 15) animals were placed in activity chambers to measure the locomotor response to a novel environment. No significant differences were found in all parameters measured during a cummulative period of 30 minutes. Total distances traveled is shown as a representative measure. All data are mean±SEM. *p<0.05, Student's t-test.</p

Pten KO animals do not show differences in striatal TH fiber density measurements.

<p>(A, C) Optical density values did not reveal significant differences in striatal TH staining intensity between control (n = 4) and <i>Pten</i> KO animals (n = 4). (B) There were no significant differences in area measurements corresponding to rostral and medial striatum. A slight but significant increase in area was observed in the caudal striatal region of <i>Pten</i> KO animals as compared to controls. Scale bar, 500 µm. All data are mean±SEM. *p<0.05, Student's t-test.</p

The lack of PTEN in DA neurons results in a significant enlargement of the ventral midbrain area, attributable to an increase in the total number of DA neurons that are larger in size, and display more dendritic extensions.

<p>(A) The volume of the substantia nigra compacta (SNc), ventral tegmental area (VTA) and substantia nigra reticulata (SNr) was significantly larger in <i>Pten</i> KO animals (n = 4), as compared to controls (n = 4). The volume of all measured areas was at least 40% larger in KO animals, with the largest increment in size seen in the SNr. (B) The increase in volume of the SNc and VTA from <i>Pten</i> KO animals was accompanied by a significant increase in the number of TH positive neurons (B) and cell size (C) in both regions. (B) The mean number of TH positive profiles in <i>Pten</i> KO animals was 9401±105.1 in the SNc, and 4775±106.3 in the VTA, a 43% and 26% increase, respectively, when compared to controls. (C) The area of TH positive neurons was 47% and 39% larger in the SNc and VTA of <i>Pten</i> KO animals as compared to controls. (D) The increase in the total volume covered by the SNr was attributable to an increase in the number and caliber of TH-positive fibers present in <i>Pten</i> KO animals in parallel with an increase in the number and size of TH positive cells in the SNc and VTA. All data are mean±SEM. *p<0.05, Student's t-test.</p