Nina Rawal

Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a

The Wnts are a family of glycoproteins that regulate cell proliferation, fate decisions, and differentiation. In our study, we examined the contribution of Wnts to the development of ventral midbrain (VM) dopaminergic (DA) neurons. Our results show that β-catenin is expressed in DA precursor cells and that β-catenin signaling takes place in these cells, as assessed in TOPGAL [Tcf optimal-promoter β-galactosidase] reporter mice. We also found that Wnt-1, -3a, and -5a expression is differentially regulated during development and that partially purified Wnts distinctively regulate VM development. Wnt-3a promoted the proliferation of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of tyrosine hydroxylase-positive neurons. Instead, Wnt-1 and -5a increased the number of rat midbrain DA neurons in rat embryonic day 14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1+ precursors, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 mRNAs. In contrast, Wnt-5a primarily increased the proportion of Nurr1+ precursors that acquired a neuronal DA phenotype and up-regulated the expression of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled-8 (a Wnt inhibitor) blocked endogenous Wnts and the effects of Wnt-1 and -5a on proliferation and the acquisition of a DA phenotype in precursor cultures. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles.

Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice.

Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell-derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.

Dual effects of nicotine on dopamine neurons mediated by different nicotinic receptor subtypes.

Burst firing of dopaminergic neurons has been found to represent a particularly effective means of increasing dopamine release in terminal areas as well as activating immediate early genes in dopaminoceptive cells. Spontaneous burst firing is largely controlled by the level of activation of NMDA receptors in the ventral tegmental area (VTA) as a consequence of glutamate released from afferents arising mainly in the prefrontal cortex. Nicotine has been found to effectively increase burst firing of dopaminergic cells. This effect of nicotine may be due to an alpha 7 nicotinic receptor-mediated presynaptic facilitation of glutamate release in the VTA. By the use of in-vivo single-cell recordings and immunohistochemistry we here evaluated the role of alpha 7 nicotinic receptors in nicotine-induced burst firing of dopamine cells in the VTA and the subsequent activation of immediate early genes in dopaminoceptive target areas. Nicotine (0.5 mg/kg s.c.) was found to increase firing rate and burst firing of dopaminergic neurons. In the presence of methyllycaconitine (MLA, 6.0 mg/kg i.p.) nicotine only increased firing rate. Moreover, in the presence of dihydro-beta-erythroidine (DH beta E, 1.0 mg/kg i.p.), an antagonist at non-alpha 7 nicotinic receptors, nicotine produced an increase in burst firing without increasing the firing rate. Nicotine also increased Fos-like immunoreactivity in dopamine target areas, an effect that was antagonized with MLA but not with DH beta E. Our data suggest that nicotines augmenting effect on burst firing is, indeed, due to stimulation of alpha 7 nicotinic receptors whereas other nicotinic receptors seem to induce an increase in firing frequency.

Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism

Previously, we have shown that Wnt-5a strongly regulates dopaminergic neuron differentiation by inducing phosphorylation of Dishevelled (Dvl). Here, we identify additional components of the Wnt-5a-Dvl pathway in dopaminergic cells. Using in vitro gain-of-function and loss-of-function approaches, we reveal that casein kinase 1 (CK1) δ and CK1ϵ are crucial for Dvl phosphorylation by non-canonical Wnts. We show that in response to Wnt-5a, CK1ϵ binds Dvl and is subsequently phosphorylated. Moreover, in response to Wnt-5a or CK1ϵ, the distribution of Dvl changed from punctate to an even appearance within the cytoplasm. The opposite effect was induced by a CK1ϵ kinase-dead mutant or by CK1 inhibitors. As expected, Wnt-5a blocked the Wnt-3a-induced activation of β-catenin. However, both Wnt-3a and Wnt-5a activated Dvl2 by a CK1-dependent mechanism in a cooperative manner. Finally, we show that CK1 kinase activity is necessary for Wnt-5a-induced differentiation of primary dopaminergic precursors. Thus, our data identify CK1 as a component of Wnt-5a-induced signalling machinery that regulates dopaminergic differentiation, and suggest that CK1δ/ϵ-mediated phosphorylation of Dvl is a common step in both canonical and non-canonical Wnt signalling.

GSK-3β inhibition/β-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons

Wnts are important regulators of dopamine (DA) neuron differentiation in the developing ventral mesencephalon and could thus serve as potential tools in the treatment of Parkinsons disease. In this study, we investigate whether established intracellular Wnt signalling components could modulate the development of DA neurons. Two chemical inhibitors of glycogen synthase kinase (GSK)-3β, indirubin-3-monoxime and kenpaullone, were found to increase neuronal differentiation in ventral mesencephalon precursor cultures. In addition, the GSK-3β-specific inhibitor kenpaullone increased the size of the DA neuron population through conversion of precursors expressing the orphan nuclear receptor-related factor 1 into tyrosine hydroxylase positive neurons, thereby mimicking an effect of Wnts. We show that GSK-3β inhibitors stabilized β-catenin and that overexpression of β-catenin in ventral mesencephalic precursors resulted in increased DA differentiation. The three- to fivefold increase in DA differentiation of precursor cells by GSK-3β inhibitors suggests that such compounds could be used to improve stem/precursor cell therapy approaches in Parkinsons disease.

Parkin protects dopaminergic neurons from excessive Wnt/{beta}-catenin signaling

Parkinsons disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates beta-catenin protein levels in vivo. Stabilization of beta-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neurons in parkin null animals, suggesting that both increased stabilization and decreased degradation of beta-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and beta-catenin-induced cell death.

Nitric oxide is involved in nicotine-induced burst firing of rat ventral tegmental area dopamine neurons

In the present study, using single cell recordings in vivo and intracellular recordings in vitro from midbrain slices, the role of N-methyl-d-aspartate (NMDA) receptor signaling on firing activity in ventral tegmental area dopamine neurons elicited by nicotine was investigated in the rat. In accordance with previous studies, systemic nicotine (0.5 mg/kg s.c.) increased both firing rate and burst firing of dopamine neurons in vivo, and bath-applied nicotine (10 microM) increased firing rate in vitro. The competitive NMDA receptor antagonist CGP39551 (2.5 mg/kg i.p.) inhibited nicotines effects on burst firing and also attenuated the nicotine-induced increase in firing rate. Moreover, although the nitric oxide (NO)-synthase inhibitor N-nitro-l-arginine-methyl-ester (l-NAME; 5.0 mg/kg i.p.) had no effect on cell firing by itself, it prevented the response to nicotine in vivo. In contrast, l-NAME (100 microM) did not influence nicotines effect on dopamine cell firing in vitro, suggesting that the effect of l-NAME seen in vivo is dependent on presynaptic afferent input. The present study confirms previous results suggesting that the effect of systemically administered nicotine is in part presynaptic and mediated via NMDA receptors. The data also indicate that NO plays an important role in the previously demonstrated, indirect, glutamate-mediated excitation of these neurons by nicotine. By inference, our results provide additional support for the involvement of NO in nicotine dependence.

Inhibition of JNK increases survival of transplanted dopamine neurons in Parkinsonian rats

Parkinson’s disease (PD) affects approximately 2% of the population over 65 years of age. This neurodegenerative disorder is caused by a selective and progressive death of the dopaminergic (DA) neurons of the substantia nigra and subsequent striatal deficiency of dopamine, resulting in movement anomalies. Vast efforts are being made to develop cell replacement therapies using fetal transplantation and stem cell based approaches to improve treatment. Proof of concept for transplantation of human fetal mesencephalic tissue into the striatum of PD patients was obtained in 1987. However, several issues have limited the use of fetal transplants in a clinical setting, including the appearance of dyskinesias, ethical considerations, tissue preparation and storage, distribution and site of graft, immunosupression, and limited availability of fetal tissue. Approximately 250 000 DA cells reside in the adult human substantia nigra but only 20 000–40 000 DA neurons can be obtained from a single fetus, making it necessary to obtain tissue from up to 6–8 fetuses per patient. Reports have shown that only 3–5% of fetal DA neurons survive upon transplantation into rodent models of PD. Moreover, a similar problem has been described for the transplantation of human embryonic stem cell derived DA neurons. Thus, improving the survival of DA neurons during isolation as well as post-transplantation has become a central issue. Cell death during grafting can be observed throughout several phases: dissection and preparation of tissue, the implantation procedure, and immediately or long-term after graft injection. However, the largest death associated with transplantation of fetal tissue in rodent models of PD occurs during the first week post-transplantation. We therefore decided to examine the possibility of reducing DA cell death during this period. Caspase inhibitors have been reported to increase the survival of transplanted tissue and thereby result in functional improvement. Importantly, however, these compounds lack specificity for DA neurons, thereby increasing the total survival of the graft, including nonDA cells. In this regard, we examined the function of c-Jun-Nterminal kinase (JNK) and its role in early DA cell death. JNK is activated in response to cellular stress and DNA damage and can activate caspases, resulting in apoptosis. Activated JNK has been shown in neuronal cell death both in vitro and in vivo, and increased levels of phospho c-Jun, a downstream mediator of JNK signaling, have been reported in rodent models of PD. In order to assess whether inhibition of JNK could modulate the numbers of DA neurons, we treated ventral midbrain (VM) embryonic day (E) 13.5 and E14.5 precursor cultures with increasing doses of the JNK inhibitor SP600125 for 1 or 3 days in vitro (1 DIV or 3 DIV). Addition of SP600125 to the precursor cultures increased the number of DA neurons (THþ ) in a dose-dependent manner, with maximal effects at 15 mM. In the E13.5 cultures, 15 mM SP600125 increased the number of THþ neurons per field by nine-fold at 1DIV and by 13-fold at 3DIV. In the E14.5 cultures, when more DA neurons have been born, 15 mM SP600125 increased the number of THþ neurons per field by two-fold at 1DIV and by eight-fold at 3DIV (Figure 1a(i)). The biggest increase (13-fold) was observed in E13.5 cells cultured for 3DIV (Figure 1a(ii)) and the highest yield of THþ neurons was obtained from E14.5 tissue cultured for 1DIV (approximately 7.5 THþ cells/field). Upon culturing for 7DIV, the neurons develop into mature DA neurons, as assessed by immunoreactivity against the dopamine transporter protein (data not shown). The observation that the largest effect was observed in the E13.5 cultures, when few DA neurons have been generated, suggests that the increase in DA numbers might also be ascribed to effects on progenitors restricted to the DA lineage. The effect of SP600125 on E13.5 3DIV cultures was confirmed using a cell permeable JNK inhibitor peptide, which also resulted in a significant increase in the number of THþ neurons, compared to a control peptide (P-value o0.004) (3mM respectively, Calbiochem No. 420116, 420118). In order to address the specificity of SP600125, we analyzed the effects on the predominant cell populations present in the culture. We found that the total number of cells, as assessed by Hoechst 33258, was not altered (control: 195.6710.24, SP600125 197.1716.71), nor was the neuronal fraction, as assessed by bIII-tubulin/Hoechst 33258 (control: 0.4070.05, SP600125 0.5770.11). Interestingly, however, the number of DA neurons out of the total number of neurons, as assessed by TH/Tuj1, increased by 11-fold (Figures 1b(i) and b(ii)). Inhibition of the JNK pathway has been described to have anti-apoptotic effects on neuronal cells. We thus examined the effects of JNK inhibition in our precursor cultures and found that the number of active Caspase 3 immunoreactive cells was significantly reduced upon addition of 15 mM SP600125 (control: 23.4671.00, SP600125: 12.1071.69) (Figure 1c). In support of our findings, administration of SP600125 has been shown to protect DA neurons in an MPTP model of PD. In order to further address the effects of JNK inhibition on downstream signaling components, we treated E13.5 cultures with 15 mM SP600125 and observed a statistically significant reduction in the levels of phospho-c-Jun, a marker of activated apoptotic JNK signaling (Figure 1d). Combined, these results suggest that apoptotic JNK signaling is more relevant during the generation and maintenance of DA neurons than in the total neuronal pool. Since JNK inhibitors have been found to protect adult DA neurons in an MPTP model of PD, Letters to the Editor