Anita Sidhu - US grants

The principle objective of this grant application is to delineate the mechanisms underlying modulation of D-1 receptor function and activity, at the molecular level. The research will test our central hypothesis that different tissue contain D-1 receptors, which couple differentially to multiple effectors, resulting in different physiological and pharmacological properties. The multiple signal transducing ability of D-1 receptors may be a result of these receptors coupling to not only Gs but also to Gi. Such couplings to multiple G proteins may also be important in understanding the mechanisms by which D-1 receptors modulate D-2 dopamine receptor function. The studies outlined in this grant will help define the role of D-1 dopamine receptors in diseases such as schizophrenia, Parkinson's disease, depression and drug addiction. Using the tools we developed for such studies, the D-1 dopamine receptors from rat retina, rat striatum and human SK-N-MC cells will be solubilized and purified. These receptors will be analyzed and compared with respect to their pharmacological properties, glycosylation, resolution into two subpopulations after elution from affinity columns and regulation of agonist high-affinity binding sites. The significance of glycosylation in physiological function and the linkage of D-1 receptors to phosphoinositide metabolism, will be tested in SK-N-MC cells. The ability of D-1 receptors from all three tissue sources to couple to different G proteins will be measured, using methods we developed, by reconstituting purified receptors with exogenous purified G proteins will be determined. The specificity and affinity of coupling for Gi protein subtypes will be assayed by reconstituting receptors with isolated Gil, Gi2 and Gi3. By reconstituting D-1 receptors with exogenous effectors of adenylate cyclase, the ability of these purified receptors to mediate physiological activity can be measured. The inactivation of Gi and Go by pertussis toxin-treatment of these effector systems prior to reconstitution, will permit an analysis of the impact of these G proteins in dampening D-1-mediated activation of adenylate cyclase. The ability of D-1/D-2 receptors to interact in striatal membranes will be examined by photoaffinity labeling and radioligand binding methods. The role of G proteins in promoting such interactions will be tested by reconstitution of crude soluble receptors with specific G proteins. The ability of D-1/D-2 receptors to interact, under these conditions, will be measured. Such studies will demonstrate whether specific G proteins mediate such D-1/D-2 interactions in membranes.

DESCRIPTION: (Applicant's Abstract) The process by which dopamine (DA) receptors are desensitized is important not only for understanding the regulatory events of DA action on its receptors, but also for determining the mechanisms underlying the progressively diminished response to agonist-based therapies seen in neurological diseases, such as Parkinson's disease. Such desensitization studies may also be useful in alleviation of harmful side- effects of current therapeutic procedures. The desensitization of human D-1 DA receptors in SK-N-MC neuroblastoma cells is homologous, yet the D-1 receptor is phosphorylated by both protein kinase A (PKA) and receptor-specific kinase. Desensitization of D-1 DA receptors results in rapid loss of adenylyl cyclase activity, with a slower loss in receptor binding sites. At the molecular level, D-1 receptor mRNA transcription is bimodal: a 60 percent increase in mRNA with 2 hours is followed by a 50 percent decrease, relative to control receptor mRNA levels. The mechanisms regulating the homologous, yet PKA- dependent desensitization, of these receptors will be studied in this grant at both the protein and mRNA level and their overall impact on neuronal function will be ascertained using SK-N-MC cells and GH4C1 cells expressing the cloned human D-1 and D-5 DA receptor. The function of putative D-1 receptor-specific kinase will also be analyzed in detail. The effects of DA on D-1 mRNA transcription will be studied, by analyzing both the 5' promoter regions and by measurements of mRNA stability, during the stages of up- and down-regulation of receptor message. Using nuclear run-on studies, the rate of transcription of receptor mRNA will be analyzed, during the up-and down-regualtion cycles. The requirements for receptor occupancy by agonist and functional Gs-receptor couplings will be examined in detail. The effects of inhibitory D-4 DA receptors in modulating the actions of D-1 receptors in SK-N-MC cells will be studied. Regulatory and transcription factors such as CREB and immediate early genes, will be analyzed and their role in promoting receptor activation and increases in total cellular mRNA, by DA, will be determined. These studies will help elucidate the role of DA on neuronal plasticity. In addition to biochemical analyses, electrophysiological studies will be conducted to analyze the role of DA and D-1/D-5 receptors on long term potentiation and neurite growth, especially after desensitization of receptors. The information obtained after tetanic stimulation of cells will be correlated with the results obtained from biochemical studies, and will provide unique insight to the DA-mediated processes underlying neuronal cell firing.

DESCRIPTION (verbatim from the application): In kidney, dopamine (DA) and Dl DA receptors regulate sodium excretion. The spontaneously hypertensive rat, SHR, and the DOCA- (deoxycorticosone acetate) salt rats are established animal models of hypertension. We have shown that Dl DA receptors in SHR are dysfunctional, being unable to couple to the G protein effector systems. The goal of this study is to analyze the linkage between oxidative stress, Dl DA receptors and hypertension in both SHR and the DOCA-salt rat models of hypertension. We have found increased oxidative stress in SHR kidney, compared to the normotensive rats, the Wistar-Kyoto (WKY) rat and Sprague-Dawley. Lipid peroxidation (LP) is increased, with release of toxic aldehydic products. One of these products, 4-hydroxynonenal, can directly modulate Dl DA receptors in WKY rat proximal tubule (PT) membranes, leading to loss of receptor coupling and function. Thus, increased oxidative stress and LP may be an important means by which the Dl DA receptor becomes dysfunctional. Other imbalances in the redox state of SHR PTs and kidney include: both elevated and depressed levels of nitric oxide synthase isoforms, increased NO production, depressed glutathione peroxidase activity and increased expression of Cu-Zn-superoxide dismutase and catalase. Using isolated tissues from SHR and DOCA-salt rat, and from their normotensive controls, we will analyze in detail the nature of the alterations in the redox state of the kidney from these animals, and will study the underlying mechanisms in PT cell culture systems. We will treat the SHR with different blockers of the renin-angiotensin system (RAS), which normalizes blood pressure, and will examine the effects of these blockers on the various indices of oxidative stress (LP, antioxidant enzymes, nitrite production and nitric oxide synthase levels). It is likely that RAS blockers will normalize the redox state of the SHR kidney, and that upon such normalization, Dl DA receptor function may also be normalized, restoring the ability of DA and the Dl DA receptors to excrete sodium.

DESCRIPTION (verbatim from the application): In kidney, dopamine (DA) and Dl DA receptors regulate sodium excretion. The spontaneously hypertensive rat, SHR, and the DOCA- (deoxycorticosone acetate) salt rats are established animal models of hypertension. We have shown that Dl DA receptors in SHR are dysfunctional, being unable to couple to the G protein effector systems. The goal of this study is to analyze the linkage between oxidative stress, Dl DA receptors and hypertension in both SHR and the DOCA-salt rat models of hypertension. We have found increased oxidative stress in SHR kidney, compared to the normotensive rats, the Wistar-Kyoto (WKY) rat and Sprague-Dawley. Lipid peroxidation (LP) is increased, with release of toxic aldehydic products. One of these products, 4-hydroxynonenal, can directly modulate Dl DA receptors in WKY rat proximal tubule (PT) membranes, leading to loss of receptor coupling and function. Thus, increased oxidative stress and LP may be an important means by which the Dl DA receptor becomes dysfunctional. Other imbalances in the redox state of SHR PTs and kidney include: both elevated and depressed levels of nitric oxide synthase isoforms, increased NO production, depressed glutathione peroxidase activity and increased expression of Cu-Zn-superoxide dismutase and catalase. Using isolated tissues from SHR and DOCA-salt rat, and from their normotensive controls, we will analyze in detail the nature of the alterations in the redox state of the kidney from these animals, and will study the underlying mechanisms in PT cell culture systems. We will treat the SHR with different blockers of the renin-angiotensin system (RAS), which normalizes blood pressure, and will examine the effects of these blockers on the various indices of oxidative stress (LP, antioxidant enzymes, nitrite production and nitric oxide synthase levels). It is likely that RAS blockers will normalize the redox state of the SHR kidney, and that upon such normalization, Dl DA receptor function may also be normalized, restoring the ability of DA and the Dl DA receptors to excrete sodium.

DESCRIPTION (provided by applicant): Oxidative stress is an important causative factor in the onset and maintenance of several neurodegenerative conditions, such as Alzheimer's disease and Parkinson's Disease (PD). While dopamine (DA)-replacement therapy can control the symptoms of PD, it can also cause severe dyskinesia in patients. Blockage of the D1 DA receptors with Dl-selective antagonists in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-lesioned primates, significantly improves dyskinesia, through unknown mechanisms. Autoxidation of DA is a major source of free radicals; activation of D1 receptors also triggers oxidative stress, and these effects are additive, such that the resulting damage produced in the postsynaptic cell is several fold greater than that elicited by sources of free radicals (hydrogen peroxide (H202)), which does not stimulate the D1 receptor. Several indices of oxidative stress, lipid peroxidation, nitrite production, nitric oxide synthases, neurofilament (NF)-kappaB nuclear translocation, are all elevated two to six fold higher with DA-mediated D1 receptor activation, than H202 alone. We will examine in detail the contribution of D1 receptor stimulation, through the use of agonists and antagonists, in causing oxidative stress in SK-N-MC human neuroblastoma cells, which endogenously express the D1 receptor and is representative of postsynaptic cells. We will examine the mechanism and functional consequences of D1 receptor stimulation on signaling pathways, as well as by selectively blocking parts of the oxidative stress cascade(s). The participation of D1 receptors and oxidative stress in cell death and apoptosis will also be measured. Since blockage of D1 receptors in the MPTP model of PD improves some of the symptoms of PD, we will investigate whether D1 receptors augment MPTP effects in SK-N-MC cells. Conversely, blockage of D1 receptors with antagonists may attenuate MPTP effects on the various indices of oxidative stress. A clear understanding of the effects of dopamine autoxidation and the participation of D1 DA receptors in inducing oxidative stress, is important for understanding patient response to agonist therapy in PD, and may aid in the design of novel therapeutic treatments.

DESCRIPTION (provided by applicant): Imbalances in dopamine (DA) receptor/G protein coupling dynamics are important in the onset and maintenance of several neuropathological diseases, such as schizophrenia, Parkinson's disease, drug abuse and attention deficit disorder. The D1-like receptors, D1 and D5, share similar structural, physiological and pharmacological homology. The functional attributes of these receptors in DA neurotransmission are largely unknown in diseased and normal states. We have shown that in transfected cells, these receptors can be functionally differentiated in that: D1 receptors couple to both G(s)alpha and G(o)alpha, while D5 couples to G(s)alpha and G(z)alpha. Moreover, D5 but not D1 receptors, inhibit phosphoinositide production. Moreover D1, but not D5, can inhibit adenyl cyclase activity, in the absence of receptor/G(s)alpha coupling. Through functional assays, we will examine the mechanism and functional consequences of D1 coupling to G(o)alpha, and D5 to G(z)alpha, in order to determine whether such coupling causes activation of alternate signaling pathways. Using progressively shorter synthetic peptides directed against specific amino acid motifs of intracellular loops of the D1 and D5 receptor, we will map the domains through which D1 couples to G(s)alpha/G(o)alpha and D5 to G(s)alpha/G(z)alpha. The ability of various peptides to block receptor/Galpha interactions will be examined through co-immunoprecipitation and functional assays. Deletion mutants will be constructed to demonstrate the participation of specific sites in receptor function. We will analyze the interactions between D1 and D5 receptors with their cognate G proteins, using a highly sensitive novel assay, fluorescence resonance energy transfer (FRET). Such FRET studies will enable us to determine in intact cells whether the receptors couple simultaneously to the two Galpha, or if such coupling occurs in a sequential manner. We will also examine interactions between synthetic peptides and G proteins, and determine whether receptor oligomerization is essential for dual coupling of D1 and D5 receptors to Galpha. A clear understanding of the mechanism and functional consequences of coupling of these receptors to different and diverse Galpha is important for defining the roles of these receptors in diseased and normal states, and may aid in the design of novel therapeutic treatments, to selectively activate or suppress specific signaling pathways.

DESCRIPTION (provided by applicant): The family of synucleins is abundantly present in presynaptic neurons and is associated with numerous neurodegenerative disease states, collectively termed synucleinopathies. One member of this family, (-synuclein, is present as the major component of Lewy bodies [LB] in LB variant of Alzheimer's disease, dementia with LBs, sporadic Parkinson's disease, multiple system atrophy and neurodegeneration with brain iron accumulation. Mutants of alpha-synuclein, the A30P and A53T alpha-synucleins, are present in LBs of certain genetic forms of PD. However, neither the primary normal function of alpha-synuclein nor its mode of disease inducing action is known. Understanding the molecular and functional correlates of alpha-synuclein would help in the understanding of both the normative, and aberrant activity of this protein that give rise to the formation of fibrillary aggregates and LBs, in a process that is accelerated by increased oxidative stress and inflammation found in diseases of the aged. We provide preliminary evidence to suggest that one possible novel role for (-synuclein is to regulate the activity of the presynaptic dopamine transporter [DAT]. This regulation of DAT activity is bimodal, causing both the increased activity of the transporter and attenuation of normal transporter activity, as is indexed by [31H] dopamine uptake assays. The modulation of DAT function proceeds through rapid trafficking of the transporter to and from the plasma membrane. The mode of action of the A30P and A53T in such dual regulation of DAT activity differs from that of (-synuclein, and they also differ from one another in a highly prominent manner. In this proposal we will study in detail the mechanisms which underline such bimodal regulation of transporter activity, using cells co-transfected with alpha-synuclein and its subtypes, and the DAT cDNA, as well as in primary cultured neurons. The ability of the alpha-synuclein and its A30P and A53T mutants, and the identity of the structural components, which participate in direct protein:protein complex formation will be analyzed thoroughly in normal growth conditional states, through a battery of studies to include, immunology, transporter assays, immunocytochemistry and FRET. We will examine the role of oxidative stress in causing dysfunction of the three variants of alpha-synuclein. Studies will be conducted to determine if changes in alpha-synuclein/DAT interactions will reduce oxidative stress and cell death.

Krebs'cycle

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): Emerging evidence indicate considerable overlap in the pathological features of tauopathies and synucleopathies. Hyperphosphorylated tau, a toxic precursor of neurofibrillary tangles of Alzheimer's disease [PD] and other taupathies, is also found in certain synucleopathies, while conversely, a-synuclein [a-Syn], a presynaptic protein linked to Parkinson's disease, is found not only in synucleopathies but also in taupathies. We have shown that the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP], induces hyperphosphorylation of Tau [p-Tau], with phosphorylation at multiple sites, in the presence of a-Syn, a protein linked to PD. The requirement for a-Syn in p-tau formation by MPTP was mandatory, since in a-Syn null mice and in transfected cells not expressing a-Syn, MPTP failed to induce p-Tau. Our data also shows that p-GSK-Sp, a protein linked to AD and hyperphasphorylation is activated by MPTP. Importantly, we have data in human post mortem tissue from striata of PD and PD+dementia patients, which mirror changes in protein levels we have observed in the MPTP in vitro and in vivo models. These combined data suggest a convergent pathomechanism for taupathies and synucleopathies. In this grant, we will examine the mechanisms by which MPTP and a-Syn induce p-Tau formation in the MPTP mouse model of parkinsonism, using A53T transgenic mice and a-syn overexpressor mice, and human post mortem tissues. We will determine whether aggregates of a-Syn and its A30P/A53T mutant alter protein degradative pathways and have different kinectics of p-Tau formation. We will assess the underlying mechanisms of various kinases known to hyperphosphorylate Tau, including protein kinase A, p- ERK and p-GSK-3(3. In particular, using specific inhibitors of kinases, we will reverse the MPTP-mediated degeneration in mice, as a novel method to treat synucleopathies. Since the A53T of a-synuclein has a greater propensity to aggregate compared to wild-type a-Syn, we will elucidate the mechanisms of activation of p-Tau formation in the transgenic mouse overexpressing the human form of the mutant. Parallel studies will be conducted in human post mortem tissues of PD and PD with dementia patients, to measure the clinical relevance of our findings. From such studies, it will be possible to understand the overlapping pathology and mechanisms of tauopathies and synucleopathies, and to develop common targeted therapies.

DESCRIPTION (provided by applicant): Serotonin [5-hydroxytryptamine, 5-HT] is a neurotransmitter, whose dysfunction is linked to the genesis of numerous psychiatric diseases, including mood disorders. The presynaptic serotonin transporters (SERTs) constitute the primary mechanism for re-uptake of 5-HT and termination of its signal in the synapse, and are high-affinity targets for addictive drugs including MDMA, cocaine and tricyclics, and SERT inhibitors constitute one of the largest groups of drugs used in the therapeutic treatment of depression. Little is known about the mode by which SERT function is regulated. Our preliminary data has identified a-synuclein, a presynaptic vesicle-associated protein of unknown function, as a modulator of SERT function, causing attenuation of normal transporter activity, indexed by diminished [3H]5HT uptake assays. The interaction between SERT and a-synuclein proceeds through the formation of stable protein:protein heteromeric complexes, and results in trafficking of SERT away from the cell surface of the plasma membrane. In this proposal we will study in detail the mechanisms which underline such regulation of transporter activity, using cells co-transfected with alpha-synuclein, and its subtypes, and the SERT cDNA, as well as in cultured primary neurons. In particular, the identity of the structural components which both facilitate and reverse a-synuclein effects on SERT function and trafficking, will be analyzed thoroughly, through a battery of studies to include, immunology, transporter assays, immunocytochemistry and FRET. We will also examine the participation of a-synuclein in both protein kinase C-mediated phosphorylation of SERT, and in transporter desensitization upon exposure to 5-HT. Our previous studies showing that a-synuclein can also modulate the function of the dopamine transporter, suggests that a primary function of this protein may be the regulation of monamine neurotransmitter homeostasis. Thus, our studies on SERT may have added heuristic and practical benefits for the understanding of other neurotransmitter transporters linked to mental illness and drug abuse. The identification of a-synuclein as a regulatory protein partner of SERT holds promise in the development of novel therapeutic strategies in the treatment of depression and drug addiction.

Aberrant norepinephrine [NE] neurotransmission in the human brain is linked to mood disorders, depression, drug addiction and neurodegenerative diseases. [unreadable][unreadable]-synuclein [[unreadable][unreadable]-Syn], a member of the synuclein family of proteins, is expressed in monoaminergic neurons, but its function in the brain is not known. We have shown that [unreadable].-Syn can modulate the function and trafficking of the NE transporter [NET], through interactions with the microtubule [MT] cytoskeleton. We show here that [unreadable][unreadable]-Syn can also regulate these NET activities. Such modulation by [unreadable][unreadable]-Syn is unique to NET. In a rat model of depression, there is overexpression of [unreadable][unreadable]-Syn, causing NET function and trafficking to be dysregulated and unresponsive to the effects of nocodazole [a MT destabilizing agent], probably due to tight binding of the [unreadable][unreadable]-Syn/NET complex to the MT cytoskeleton. Chronic treatment of these animals with desipramine [a NET blocker] reduces [unreadable][unreadable]-Syn protein expression, while increasing [unreadable].-Syn levels, permitting NET to be appropriately regulated by [unreadable].-Syn, with full restoration of nocodazole sensitivity. In postmortem brains from patients with depression, we also show that [unreadable][unreadable]-Syn is overexpressed, adding clinical relevance for a role for [unreadable][unreadable]-Syn in the genesis and maintenance of depression in humans. We hypothesize that [unreadable][unreadable]-Syn acts as a prodepressant, and that imbalances in [unreadable][unreadable]-Syn/[unreadable].-Syn expression levels is central to the genesis of depression. When overexpressed, [unreadable][unreadable]-Syn overrides the normative regulation of NET by [unreadable].-Syn. Therefore, targeting [unreadable][unreadable]-Syn expression levels may be key to controlling depression in humans. We will investigate here in detail the cellular and molecular mechanisms by which desipramine reduces [unreadable][unreadable]-Syn levels in both in vitro and in vivo models, which express either [unreadable][unreadable]-Syn alone or both [unreadable][unreadable]-Syn and NET, in the presence or absence of [unreadable].-Syn. We will also analyze the mechanisms by which other NET antidepressants decrease [unreadable][unreadable]-Syn expression in in vitro models. Finally, we will measure neurochemical and behavioral responses to NET antidepressants in [unreadable].-Syn overexpressing transgenic mice, as well as in [unreadable].-Syn knock-out mice. From these studies we will be able to assess the mechanisms by which [unreadable][unreadable]-Syn expression is regulated as well as ascertain the physiopathological relevance of sucrtain if DMI affects [unreadable][unreadable]-Syn promoter thereby altering its expression levels, we will conduct nuclear run-on assays, using isolated nuclei. Transcription and RNA labeling will be performed for 30 min in a reaction buffer containing 5 mM each of ATP, CTP, GTP, and 32P-UTP. RNA will be isolated using Trizol reagent. Nylon membranes will be slot-blotted with 2.5 [unreadable]g of cDNA sequences encoding [unreadable][unreadable]-Syn (nucleotides 49-432 of cDNA), or -actin (nucleotides 50-545 of cDNA), in separate wells. Hybridization will performed and autoradiographs will be quantified using Quantity OneTM quantitation software and [unreadable][unreadable]-Syn levels normalized against -actin. In addition to Desipramine, we will also examine the effects of several antidepressants in T470 cells that are structurally similar and dissimilar to Desipramine, to determine if the response elicited by DMI is selective for this compound or a characteristic feature of antidepressants. Some of these antidepressants include: imipramine, reboxetine, as well as structurally un-related antidepressants, trazadone and amoxapine. Research Plan for Specific Aim 2: For these series of studies, we will use two species [and speciesappropriate non-transgenic controls] of transgenic mice: [unreadable].-Syn knock-out mice expressing [unreadable][unreadable]-Syn only and [unreadable].-Syn overexpressing mice. Adult mice of bothA levels and stability, as well as their relative distribution and co-localization after treatment with the sntidepressant. The effect of other antidepressants on [unreadable][unreadable]-Syn expression that are structurally similar and dissimilar to DMI will also be examined. Specific Aim 2: To dissect the individual participation of [unreadable].-Syn and [unreadable][unreadable]-Syn in transgenic mice [[unreadable].-Syn knock-out mice and [unreadable].-Syn overexpressing mice] in regulating NET function and their response to DMI. Results from these studies will help dissect the contribution of each of the synucleins on NET trafficking and cytoskeletal protein interactions, before and after treatment with desipramine. Research Plan for SA 1: Our published and preliminary results show that DMI reduces [unreadable][unreadable]-Syn levels in the depressive rat model, WKY rat, and in endogenously expressing cells, such as the T-470 breast carcinoma cells. We will examine the molecular mechanisms by which DMI causes a reduction in [unreadable][unreadable]-Syn levels by conducting studies in the T470 breast cancer cells which express only [unreadable][unreadable]-Syn, but not [unreadable].-Syn. Thus, time-course and dose-response studies will be followed by Western blots to analyze [unreadable][unreadable]-Syn levels, after treatment with specific levels of DMI for specific time periods. Co-immunoprecipitation studies will be conducted to measure interactions between [unreadable][unreadable]-Syn and microtubule binding proteins [MAPs, tau] and microtubules [tubulin]. Protein stability will be analyzed by pulse-chase labeling using [3H]methionine followed by immunoprecipitation with [unreadable][unreadable]-Syn antibodies. mRNA levels will be assessed by real-time RTPCR and mRNA stability will be analyzed after blockade of RNA transcription by actinomycin D (10 ug/ml). To ascertain if DMI affects [unreadable][unreadable]-Syn promoter thereby altering its expression levels, we will conduct nuclear run-on assays, using isolated nuclei. Transcription and RNA labeling will be performed for 30 min in a reaction buffer containing 5 mM each of ATP, CTP, GTP, and 32P-UTP. RNA will be isolated using Trizol reagent. Nylon membranes will be slot-blotted with 2.5 [unreadable]g of cDNA sequences encoding [unreadable][unreadable]-Syn (nucleotides 49-432 of cDNA), or -actin (nucleotides 50-545 of cDNA), in separate wells. Hybridization will performed and autoradiographs will be quantified using Quantity OneTM quantitation software and [unreadable][unreadable]-Syn levels normaecific therapy. This grant is also relevant for the ARRA in that it will immediately create 3 new professional jobs.