Anita Sidhu

α-Synuclein contributes to GSK-3β-catalyzed Tau phosphorylation in Parkinson’s disease models

We have shown in the parknsonism‐inducing neurotoxin MPP+/MPTP model that α‐Synuclein (α‐Syn), a presynaptic protein causal in Parkinsons disease (PD), contributes to hyperphosphorylation of Tau (p‐Tau), a protein normally linked to tauopathies, such as Alzheimers disease (AD). Here, we investigated the kinase involved and show that the Tau‐specific kinase, glycogen synthase kinase 3β (GSK‐3β), is robustly activated in various MPP+/MPTP models of Parkinsonism (SH‐SY5Y cotransfected cells, mesencephalic neurons, transgenic mice overexpressing α‐Syn, and postmortem striatum of PD patients). The activation of GSK‐3β was absolutely dependent on the presence of α‐Syn, as indexed by the absence of p‐GSK‐3β in cells lacking α‐Syn and in α‐Syn KO mice. MPP+ treatment induced translocation and accumulation of p‐GSK‐3β in nuclei of SHSY5Y cells and mesencephalic neurons. Through coimmunoprecipitation (co‐IP), we found that α‐Syn, pSer396/ 404‐Tau, and p‐GSK‐3β exist as a heterotrimeric complex in SH‐SY5Y cells. GSK‐3β inhibitors (lithium and TDZD‐8) protected against MPP+‐induced events in SHSY5Y cells, preventing cell death and p‐GSK‐3β formation, by reversing increases in α‐Syn accumulation and p‐Tau formation. These data unveil a previously unappreciated role of α‐Syn in the induction of p‐GSK‐3β, and demonstrate the importance of this kinase in the genesis and maintenance of neurodegenerative changes associated with PD.—Duka, T., Duka, V., Joyce, J. N., Sidhu, A. α‐Synuclein contributes to GSK‐3β‐catalyzed Tau phosphorylation in Parkinsons disease models. FASEB J. 23, 2820–2830 (2009). www.fasebj.org

The D1 dopamine receptor in the rat brain: Quantitative autoradiographic localization using an iodinated ligand

The distribution of dopamine D1 receptors in the rat, labeled with [125I]SCH 23982, was studied using a quantitative in-vitro light-microscopic autoradiographic method. The binding of [125I]SCH 23982 to slide-mounted tissue sections and membrane preparations of prefrontal cortex was saturable, specific and of high affinity. Scatchard analysis revealed a Kd of 1.15 +/- 0.47 nM and Bmax of 8.76 +/- 0.34 fmol/mg tissue in prefrontal cortex membranes and a Kd of 1.27 +/- 0.14 nM and Bmax of 67.6 +/- 3.75 fmol/mg tissue in slide-mounted tissue sections at the level of the striatum. [125I]SCH 23982 was found to predominantly label D1 receptors, but a small fraction of the binding was to serotonin receptors. D1 receptors were found throughout the forebrain and were concentrated in the substantia nigra pars reticulata, accumbens nucleus, caudate putamen, entopeduncular nucleus, olfactory tubercle and the major island of Calleja. [125I]SCH 23982 binding to serotonin receptors was concentrated in the cortices, dorsal raphe, central gray, anterior hypothalamic area and the molecular cell layer of the cerebellum. Knowledge of the distribution of D1 receptors may increase our understanding of the role of D1 receptors in central nervous system dopaminergic function. Furthermore, data on the potential sites of interaction of [125I]SCH 23982 with serotonin receptors may help to understand the complex physiology and pharmacology of the primarily D1 selective compound.

Modulation of dopamine transporter function by α-synuclein is altered by impairment of cell adhesion and by induction of oxidative stress

Human α‐synuclein accumulates in dopaminergic neurons as intraneuronal inclusions, Lewy bodies, which are characteristic of idiopathic Parkinsons disease (PD). Here, we suggest that modulation of the functional activity of the dopamine transporter (DAT) by α‐synuclein may be a key factor in the preferential degeneration of mesencephalic dopamine (DA)‐synthesizing neurons in PD. In cotransfected Ltk‐, HEK 293, and SK‐N‐MC cells, α‐synuclein induced a 35% decrease in [3H]DA uptake. Biotinylated DAT levels were decreased by 40% in cotransfected cells relative to cells expressing only DAT. DAT was colocalized with α‐synuclein in mesencephalic neurons and cotransfected Ltk‐ cells. Coimmunoprecipitation studies showed the existence of a complex between α‐synuclein and DAT, in specific rat brain regions and cotransfected cells, through specific amino acid motifs of both proteins. The attenuation of DAT function by α‐synuclein was cytoprotective, because DA‐mediated oxidative stress and cell death were reduced in cotransfected cells. The neurotoxin MPP+ (1‐methyl‐4‐phenylpyridinium), oxidative stress, or impairment of cell adhesion ablated the α‐synuclein‐mediated inhibition of DAT activity, which caused increased uptake of DA and increased biotinylated DAT levels, in both mesencephalic neurons and cotransfected cells. These studies suggest a novel normative role for α‐synuclein in regulating DA synaptic availability and homeostasis, which is relevant to the pathophysiology of PD.

Elevated tauopathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia.

Parkinsons disease (PD), a progressive neurodegenerative disease, results in abnormal accumulation of insoluble alpha-synuclein (alpha-Syn) in dopaminergic neurons. Here we examined tauopathic changes and the alpha-Syn/p-GSK-3beta/proteasome pathway in postmortem striata and inferior frontal gyri (IFG) from patients with PD and PD with dementia (PDD). In both PD and PDD, alpha-Syn levels were high, especially the insoluble form of this protein; in PDD, insoluble alpha-Syn levels were persistently higher than PD across both brain regions. Levels of p-GSK-3beta phosphorylated at Tyr 216, which hyperphosphorylates Tau to produce toxic pathological forms of p-Tau, were higher in striata of both PD and PDD compared to controls, but were unaltered in IFG. While proteasomal activity was unchanged in striatum of PD and PDD, such activity was diminished in the IFG of both PD and PDD. A decrease in 19S subunit of the proteasomes was seen in IFG of PDD, while lower levels of 20S subunits were seen in striatum and IFG of both PD and PDD patients. Parkin levels were similar in PD and PDD, suggesting lack of involvement of this protein. Most interestingly, tauopathic changes were noted only in striatum of PD and PDD, with increased hyperphosphorylation seen at Ser262 and Ser396/404; increases in Ser202 levels were seen only in PD but not in PDD striatum. We were unable to detect any tauopathy in IFG in either PD or PDD despite increased levels of alpha-Syn, and decreased proteasomal activity, and is probably due to lack of increase in p-GSK-3beta in IFG. Unlike Alzheimers disease where tauopathy is more globally observed in diverse brain regions, our data demonstrates restricted expression of tauopathy in brains of PD and PDD, probably limited to dopaminergic neurons of the nigrostriatal region.

Coupling of dopamine receptor subtypes to multiple and diverse G proteins.

The family of five dopamine receptors subtypes activate cellular effector systems through G proteins. Historically, dopamine receptors were thought to only stimulate or inhibit adenylyl cyclase, by coupling to either Gsα or Giα, respectively. Recent studies in transfected cells, reviewed here, have shown that multiple and highly diverse signaling pathways are activated by specific dopamine receptor subtypes. This multiplicity of signaling responses occurs through selective coupling to distinct G proteins and each of the receptors can interact with more than one G protein. Although some of the multiple coupling of dopamine receptors to different G proteins occurs from within the same family of G proteins, these receptors can also couple to G proteins belonging to different families. Such multiple interactions between receptors and G proteins elicits functionally distinct physiological effects which acts to enhance and subsequently suppress the original receptor response, and to activate apparently distinct signaling pathways. In the brain, where coexpression of functionally distinct receptors in heterogenous cells further adds to the complexity of dopamine signaling, minor alterations in receptor/G protein coupling states during either development or in adults, may underlie the imbalanced signaling seen in dopaminergic‐linked diseases such as schizophrenia, Parkinsons disease and attention deficit hyperactivity disorder.

Neurodegeneration in Niemann-Pick type C disease mice

Abstract. Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disorder associated with intracellular cholesterol and glycolipid trafficking defects. Two separate genes, NPC1 and NPC2, have been linked to NP-C. NPC1 encodes a polytopic membrane-bound protein with a putative sterol-sensing domain. NPC2 has been recently identified as epididymal secretory glycoprotein 1. The NPC1 protein functions in the vesicular redistribution of endocytosed lysosomal cargo, but how its inactivation leads to neurodegeneration is not known. The neurological symptoms of NP-C typically appear after a period of normal early development and reflect progressive degeneration of widespread brain regions. Here we have delineated the pattern of neurodegeneration in NP-C mice, whose genetic defect has been shown to be an inactivating mutation of the mouse NPC1 gene. The results reveal a spatially and temporally specific pattern of degeneration of nerve fibers followed by degeneration of neuronal cell bodies beginning as early as day 9 and continuing throughout life. We have recently showed that in the primate brain, the NPC1 protein is localized predominantly within perisynaptic astrocytic processes. The present observations suggest that a functional disturbance in NPC1 could disrupt vesicular transport of cholesterol, glycolipids and possibly other endocytic cargo in glia, which is critical for maintaining the integrity of neurons.

Highly Potent and Specific GSK-3β Inhibitors That Block Tau Phosphorylation and Decrease α-Synuclein Protein Expression in a Cellular Model of Parkinson's Disease

Research by Klein and co‐workers suggests that the inhibition of GSK‐3β by small molecules may offer an important strategy in the treatment of a number of central nervous system (CNS) disorders including Alzheimers disease, Parkinsons disease, and bipolar disorders. Based on results from kinase‐screening assays that identified a staurosporine analogue as a modest inhibitor of GSK‐3β, a series of 3‐indolyl‐4‐indazolylmaleimides was prepared for study in both enzymatic and cell‐based assays. Most strikingly, whereas we identified ligands having poor to high potency for GSK‐3β inhibition, only ligands with a Ki value of less than 8 nM, namely maleimides 18 and 22, were found to inhibit Tau phosphorylation at a GSK‐3β‐specific site (Ser 396/404). Accordingly, maleimides 18 and 22 may protect neuronal cells against cell death by decreasing the level of α‐Syn protein expression. We conclude that the GSK‐3β inhibitors described herein offer promise in defending cells against MPP+‐induced neurotoxicity and that such compounds will be valuable to explore in animal models of Parkinsons disease as well as in other Tau‐related neurodegenerative disease states.

Hyperphosphorylated Tau in an α-synuclein-overexpressing transgenic model of Parkinson's disease.

Although clinically distinct diseases, tauopathies and synucleinopathies share a common genesis and mechanisms, leading to overlapping degenerative changes within neurons. In human postmortem striatum of Parkinson’s disease (PD) and PD with dementia, we have recently described elevated levels of tauopathy, indexed as increased hyperphosphorylated Tau (p‐Tau). Here we assessed tauopathy in striatum of a transgenic animal model of PD, overexpressing human α‐synuclein under the platelet‐derived growth factor promoter. At 11 months of age, large and progressive increases in p‐Tau in transgenic mice, hyperphosphorylated at sites reminiscent of Alzheimer’s disease, were noted, along with elevated levels of α‐synuclein and glycogen synthase kinase 3β phosphorylated at Tyr216 (p‐GSK‐3β), a major kinase involved in the hyperphosphorylation of Tau. Differential Triton X‐100 extraction of striata showed the presence of aggregated α‐synuclein in the transgenic mice, along with p‐Tau and p‐GSK‐3β, which was also confirmed through immunohistochemistry. After p‐Tau formation, both Tau and microtubule‐associated protein 1 (MAP1) dissociated from the cytoskeleton, consistent with the diminished ability of these cytoskeleton‐binding proteins to bind microtubules. Increases in free tubulin and actin were also noted, indicative of cytoskeleton remodeling and destabilization. In vivo magnetic resonance imaging of the transgenic animals showed a reduction in brain volume of transgenic mice, indicating substantial atrophy. From immunohistochemical studies, α‐synuclein, p‐Tau and p‐GSK‐3β were found to be overexpressed and co‐localized in large inclusion bodies, reminiscent of Lewy bodies. The elevated state of tauopathy seen in these platelet‐derived growth factor–α‐synuclein mice provides further confirmation that PD may be a tauopathic disease.

An Inflammatory Pathomechanism for Parkinsons Disease

Parkinsons disease (PD) is a slowly progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc), striatal dopamine deficiency and appearance of Lewy bodies. Inflammatory and immune, or even autoimmune, stigmata, have been described in post-mortem brains of PD patients. Although disputed in humans, a reactive astrocytosis and a lymphocytic infiltration in the SNpc have been observed in animal models of PD, which need further examination. This review summarizes the current knowledge on brain inflammation in humans with PD, and how inflammation and/or (auto)immune reactions within the SNpc could be linked to other pathophysiological mechanisms that have been hypothesized for the etiology of PD, such as oxidative stress, exposure to neurotoxins, and post-infectious or post-traumatic injuries. In particular, we discuss how microglial cells could be activated during the course of PD, and present a new hypothesis that PD-linked protein (alpha-synuclein, in particular) aggregates could be implicated in their activation, to induce a chronic and sustained inflammation involved in the progression, at least, of the disease. The current status of anti-inflammatory agents, either already tried in PD clinical trials or putatively usefull as adjuvant therapies for PD, is also discussed.

Paraquat, but Not Maneb, Induces Synucleinopathy and Tauopathy in Striata of Mice through Inhibition of Proteasomal and Autophagic Pathways

SNCA and MAPT genes and environmental factors are important risk factors of Parkinsons disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.