Eldo V. Kuzhikandathil

Specification of a Dopaminergic Phenotype from Adult Human Mesenchymal Stem Cells

Dopamine (DA) neurons derived from stem cells are a valuable source for cell replacement therapy in Parkinson disease, to study the molecular mechanisms of DA neuron development, and for screening pharmaceutical compounds that target DA disorders. Compared with other stem cells, MSCs derived from the adult human bone marrow (BM) have significant advantages and greater potential for immediate clinical application. We report the identification of in vitro conditions for inducing adult human MSCs into DA cells. Using a cocktail that includes sonic hedgehog and fibroblast growth factors, human BM‐derived MSCs were induced in vitro to become DA cells in 12 days. Based on tyrosine hydroxylase (TH) expression, the efficiency of induction was determined to be ∼67%. The cells develop a neuronal morphology expressing the neuronal markers NeuN and β III tubulin, but not glial markers, glial fibrillary acidic protein and Olig2. As the cells acquire a postmitotic neuronal fate, they downregulate cell cycle activator proteins cyclin B, cyclin‐dependent kinase 2, and proliferating cell nuclear antigen. Molecular characterization revealed the expression of DA‐specific genes such as TH, Pitx3, Nurr1, DA transporter, and vesicular monoamine transporter 2. The induced MSCs also synthesize and secrete DA in a depolarization‐independent manner. The latter observation is consistent with the low expression of voltage gated Na+ and Ca2+ channels in the induced MSCs and suggests that the cells are at an immature stage of development likely representing DA neuronal progenitors. Taken together, the results demonstrate the ability of adult human BM‐derived MSCs to form DA cells in vitro.

Brain‐derived neurotrophic factor facilitates maturation of mesenchymal stem cell‐derived dopamine progenitors to functional neurons

The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson’s disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain‐derived neurotrophic factor (BDNF) in the maturation of the MSC‐derived DA progenitors. 9‐day induced MSCs show significant tropomyosin‐receptor‐kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca2+ imaging analyses and electrophysiology. BDNF‐treated cells showed the following: increases in intracellular Ca2+ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post‐synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC‐derived DA progenitors.

MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction

Alcohol addiction is a major social and health concern. Here, we determined the expression profile of microRNAs (miRNAs) in the nucleus accumbens (NAc) of rats treated with alcohol. The results suggest that multiple miRNAs were aberrantly expressed in rat NAc after alcohol injection. Among them, miR‐382 was down‐regulated in alcohol‐treated rats. In both cultured neuronal cells in vitro and in the NAc in vivo, we identified that the dopamine receptor D1 (Drd1) is a direct target gene of miR‐382. Via this target gene, miR‐382 strongly modulated the expression of DeltaFosB. Moreover, overexpression of miR‐382 significantly attenuated alcohol‐induced up‐regulation of DRD1 and DeltaFosB, decreased voluntary intake of and preference for alcohol and inhibited the DRD1‐induced action potential responses. The results indicated that miRNAs are involved in and may represent novel therapeutic targets for alcoholism.

Bioisosteric Heterocyclic Versions of 7-{[2-(4-Phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol: Identification of Highly Potent and Selective Agonists for Dopamine D3 Receptor with Potent in Vivo Activity

In the current report, we extend the SAR study on our hybrid structure 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol further to include heterocyclic bioisosteric analogues. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (Ki). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The highest binding affinity and selectivity for D3 receptors were exhibited by (-)-34 (Ki=0.92 nM and D2/D3=253). In the functional GTPgammaS binding assay, (-)-34 exhibited full agonist activity with picomolar affinity for D3 receptor with high selectivity (EC50=0.08 nM and D2/D3=248). In the in vivo rotational study, (-)-34 exhibited potent rotational activity in 6-OH-DA unilaterally lesioned rats with long duration of action, which indicates its potential application in neuroprotective treatment of Parkinsons disease.

Brain-derived neurotrophic factor regulates the expression of D1 dopamine receptors

We have previously demonstrated that the CAD catecholaminergic neuronal cell line is an appropriate model system to study the regulation of D1 dopamine receptor expression. In this report, we show that brain‐derived neurotrophic factor (BDNF) up‐regulates the expression of D1 dopamine receptor in CAD cells. In addition, by comparing D1 receptor mRNA expression in wild‐type, heterozygous and homozygous trkB knockout mice, we show that TrkB receptor signaling up‐regulates D1 receptor expression in vivo. In CAD cells expressing the TrkB receptor, BDNF increased D1 receptor mRNA in a time‐ and dose‐dependent manner with a fourfold increase in D1 receptor mRNA observed as early as 3 h with 10 ng/mL of BDNF. Using different classes and concentrations of kinase inhibitors, we determined that BDNF‐induced increase of D1 receptor mRNA is mediated by the phosphatidylinositol 3‐kinase signaling pathway. The increase required both new transcription and protein synthesis, as it was blocked by actinomycin D and cyclohexamide, respectively. Promoter deletion analysis identified a D1 promoter region necessary for mediating the effect of BDNF. These results provide novel evidence that D1 dopamine receptor expression is regulated by BDNF and its signaling pathway.

Identification and characterization of novel properties of the human D3 dopamine receptor

Among dopamine receptors, the function and properties of the D3 receptor subtype are poorly understood. Here we report the identification and characterization of two unique properties of the human D3 receptor. The D3 receptor exhibits a tolerance property wherein the magnitude of the second agonist-induced response is reduced by 60% compared to the first response and progressively decreases upon repeated agonist application. In addition, unlike the D2 dopamine receptor, the D3 receptor response terminates 15-fold more slowly upon agonist removal. Using D3/D2S chimeric receptors, we demonstrate that D3 receptor tolerance property is mediated by a novel conformational mechanism involving the D3 receptor second cytoplasmic region. The slow response termination rate property requires the third cytoplasmic region and is due to the high affinity of the D3 receptor for ligand as well as its unique G-protein signaling mechanism.

Multi-modal regulation of endogenous D1 dopamine receptor expression and function in the CAD catecholaminergic cell line

Dopamine receptors exhibit tissue‐ and cell type‐specific expression that is modulated during development, aging and in diseases such as Parkinsons. The molecular mechanisms regulating expression of dopamine receptors are not well understood, in part due to the lack of a model cell line that not only expresses endogenous dopamine receptors but also has the requisite regulatory mechanisms. Here, we demonstrate that the CAD catecholaminergic cell line expresses D1, D2, D3 and D5 dopamine receptor subtypes and associated signaling proteins. CAD cell differentiation induced by serum withdrawal increases the levels of D1 receptor mRNA by transcriptional up‐regulation. This increase is also mimicked by the neurotrophin NT3. Interestingly, the increase of D1 receptor mRNA does not result in increased levels of D1 receptor protein in differentiated CAD cells. Furthermore, while the D1 receptor protein is expressed in differentiated CAD cells, it loses its ability to activate adenylyl cyclase. We demonstrate that the post‐transcriptional regulation is not due to decreased D1 receptor mRNA stability or generation of a truncated D1 receptor mRNA, and that the down‐regulation of D1 receptor function in differentiated CAD cells is mediated by post‐translational mechanisms that decrease cell surface receptor expression by altering receptor processing and trafficking.

MicroRNA 142-3p Mediates Post-Transcriptional Regulation of D1 Dopamine Receptor Expression

The D1 dopamine receptor subtype is expressed in the brain, kidney and lymphocytes. D1 receptor function has been extensively studied and the receptor has been shown to modulate a wide range of physiological functions and behaviors. The expression of D1 receptor is known to change during development, disease states and chronic treatment; however, the molecular mechanisms that mediate the changes in D1 receptor expression under these circumstances are not well understood. While previous studies have identified extracellular factors and signaling mechanisms regulating the transcription of D1 receptor gene, very little is known about other regulatory mechanisms that modulate the expression of the D1 receptor gene. Here we report that the D1 receptor is post-transcriptionally regulated during postnatal mouse brain development and in the mouse CAD catecholaminergic neuronal cell line. We demonstrate that this post-transcriptional regulation is mediated by a molecular mechanism involving noncoding RNA. We show that the 1277 bp 3′untranslated region of D1 receptor mRNA is necessary and sufficient for mediating the post-transcriptional regulation. Using deletion and site-directed mutagenesis approaches, we show that the D1 receptor post-transcriptional regulation is specifically mediated by microRNA miR-142-3p interacting with a single consensus binding site in the 1277 bp 3′untranslated region of D1 receptor mRNA. Inhibiting endogenous miR-142-3p in CAD cells increased endogenous D1 receptor protein expression levels. The increase in D1 receptor protein levels was biologically significant as it resulted in enhanced D1 receptor-mediated signaling, determined by measuring the activation of both, adenylate cyclase and, the dopamine- and cAMP-regulated phosphoprotein, DARPP-32. We also show that there is an inverse correlation between miR-142-3p levels and D1 receptor protein expression in the mouse brain during postnatal development. This is the first study to demonstrate that the post-transcriptional regulation of D1 receptor expression is mediated by microRNA-induced translational suppression.

The novel antidyskinetic drug sarizotan elicits different functional responses at human D2-like dopamine receptors.

Sarizotan (EMD 128130) is a chromane derivative that exhibits affinity at serotonin and dopamine receptors. Sarizotan effectively suppresses levodopa-induced dyskinesia in primate and rodent models of Parkinsons disease, and tardive dyskinesia in a rodent model. Results from clinical trials suggest that sarizotan significantly alleviates levodopa-induced dyskinesia. The functional effects of sarizotan on individual dopamine receptor subtypes are not known. Here we report the functional effects of sarizotan on human D2-like dopamine receptors (D2S, D2L, D3, D4.2 and D4.4) individually expressed in the AtT-20 neuroendocrine cell line. Using the coupling of D2-like dopamine receptors to G-protein coupled inward rectifier potassium channels we determined that sarizotan is a full agonist at D3 and D4.4 receptors (EC50=5.6 and 5.4 nM, respectively) but a partial agonist at D2S, D2L and D4.2 receptors (EC50=29, 23 and 4.5 nM, respectively). Consistent with its partial agonist property, sarizotan is an antagonist at D2S and D2L receptors (IC50=52 and 121 nM, respectively). Using the coupling of D2-like dopamine receptors to adenylyl cyclase we determined that sarizotan is a full agonist at D2L, D3, D4.2 and D4.4 receptors (EC50=0.51, 0.47, 0.48 and 0.23 nM, respectively) but a partial agonist at D2S receptors (EC50=0.6 nM).

Soluble Interleukin-6 Receptor Induces Motor Stereotypies and Co-Localizes with Gp130 in Regions Linked to Cortico-Striato-Thalamo-Cortical Circuits

Soluble cytokine receptors are normal constituents of body fluids that regulate peripheral cytokine and lymphoid activity and whose levels are increased in states of immune activation. Soluble interleukin-6 receptor (sIL-6R) levels positively correlate with disease progression in some autoimmune conditions and psychiatric disorders. Particularly strong links between levels of sIL-6R and the severity of psychotic symptoms occur in schizophrenia, raising the possibility that sIL-6R is involved in this disease. However, there is no evidence that peripheral sIL-6R induces relevant behavioral disturbances. We showed that single subcutaneous injections of sIL-6R (0–1 µg), stimulated novelty stress-induced exploratory motor behaviors in male Balb/c mice within 20–40-min of injection. A progressive increase in vertical stereotypies was observed 40–80 min post injection, persisting for the remainder of the test session. Paralleling these stimulant-like effects, sIL-6R pre-treatment significantly enhanced stereotypy scores following challenge with GBR 12909. We found that peripherally administered sIL-6R crossed the blood-brain barrier, localizing in brain regions associated with cortico-striatal-thalamo-cortical (CSTC) circuits, which are putative neuroanatomical substrates of disorders associated with repetitive stereotypies. Peripherally administered sIL-6R co-localized with gp130, a transmembrane protein involved in IL-6 trans-signaling, in the nucleus accumbens, caudate-putamen, motor and infralimbic cortices, and thalamic nuclei, but not with gp130 in the ventral tegmental area, substantia nigra, or sensorimotor cortex,. The results suggest that peripheral sIL-6R can act as a neuroimmune messenger, crossing the blood brain barrier (BBB) to selectively target CSTC circuits rich in IL-6 trans-signaling protein, and inducing repetitive stereotypies. As such sIL-6R may represent a novel therapeutic agent for relevant psychiatric disorders.