Gangliosides were viewed for many years as unique to the plasma membrane, but a number of intracellular mechanisms are now known to be mediated and modulated by these glycolipids. Our work has shown GM1 in particular to have a critical role regulating Ca2+ homeostasis in the nucleus and other compartments by virtue of its tight association with a sodium-calcium exchanger in the nuclear envelope. Calcium dysregulation is characteristic of the vulnerable dopaminergic (DA) neurons in Parkinson's disease (PD), along with mitochondrial impairment, oxidative stress, and aggregation of alpha-synuclein (1-syn). This has been attributed by Surmeier and others to the autonomous pacemaking property of DA neurons of the substantia nigra pars compacta (SNpc) which utilize L-type Ca2+ channels to mediate continuous Ca2+ influx. This is believed to result over time in homeostatic Ca2+ stress leading to mitochondrial dysfunction and reduced ATP synthesis. We propose that such homeostatic Ca2+ stress depresses GM1 synthesis in DA neurons which in turn promotes further Ca2+ dysregulation in a cyclic feed-forward mechanism leading to suppression of tyrosine hydroxylase (TH) phenotypic marker and eventually cell death. We accordingly hypothesize that gradual loss of gangliosides, especially GM1, in the vulnerable DA neurons is a hitherto unrecognized risk factor contributing to the pathophysiological mechanisms undermining DA neuron viability. Our hypothesis is lent credence by the clinical benefit (albeit modest) of GM1 administration to PD patients, analogous to that observed in the mouse and monkey MPTP models of PD. Administered GM1 thus appears to function as replacement therapy for an essential intracellular regulator;the modest benefit is likely due to limited GM1 permeability. The superior rescue effected in those models by LIGA-20, a membrane permeable analog of GM1, is testament to the vital role of intracellular GM1. Our hypothesis receives strong support in preliminary results showing spontaneous development of parkinsonian symptoms in GM1-null (GalNAcT-/-) knockout (KO) mice as well as heterozygotes (HT) with reduced level of GM1. These include physical impairment, elevation and aggregation of 1-syn, and specific loss of DA neurons (TH+) in SNpc. Prevention of parkinsonism by LIGA-20 was demonstrated and will be explored more fully with additional behavior testing, preservation of DA neuron viability, and suppression of 1-syn aggregation. A parallel study will determine whether LIGA-20 rescue succeeds following onset of parkinsonism, a question with obvious clinical implications. Those studies are designed to establish the KO and HT mice as physiologically relevant models for PD, and LIGA-20-like membrane permeable derivatives of GM1 as promising therapeutic agents. To provide further support for GM1 deficiency as etiologic factor in PD we will analyze SNpc sections from PD subjects and normal controls (provided by N.Y. Brain Bank of Columbia University) for evidence of GM1 deficiency in DA neurons. In addition, normal human samples of SNpc over a wide age range will be studied to determine whether, as stipulated by our hypothesis, such deficiency develops gradually over the years. Parallel study of mouse SNpc will also be carried out, based on preliminary evidence that such deficiency does develop in normal mice and more rapidly in the above heterozygotes. The mechanisms by which intracellular GM1 preserves DA neuronal viability will be explored with N27 cells, a neuronal cell line derived from DA neurons of rat SNpc and shown to be a useful model for study of PD related mechanisms. Utilizing siRNA knockdown of GM1 synthase we will determine the effect of GM1 deficiency on intracellular Ca2+ homeostasis and rescue efficacy of GM1 vs. LIGA-20 to test the relative importance of intracellular- vs. plasma membrane GM1 on Ca2+ regulation. A key feature of that study will be GM1 in the nuclear envelope (NE) of N27 cells where it binds to and promotes the activity of a Na/Ca exchanger (NCX). The ability of LIGA-20 to enter the cell, bind to NCX in the NE and restore Ca2+ homeostasis will be observed and quantified. Another recently demonstrated intracellular function of intracellular GM1 is interaction with a-syn to retain the latter in a non-aggregating conformation. This is particularly relevant to PD, and N27 cells are expected to provide a useful model for viewing aggregated a-syn and its dissolution. These studies en toto are designed to test the central hypothesis that intracellular GM1 is crucial for maintaining viability of DA neurons of SNpc, and its loss can result from and contribute to ultimately fatal Ca2+ dysregulation and a-syn aggregation.
This study is designed to further understanding of the role of gangliosides, especially GM1, in Parkinson's disease (PD). Our studies show that intracellular GM1, as opposed to that in the plasma membrane, modulates intracellular reactivities essential for viability of the vulnerable neurons in PD (dopaminergic neurons of the substantia nigra). These reactions include (a) regulation of calcium homeostasis through activation of a nuclear sodium-calcium exchange protein, and (b) interaction with alpha-synuclein to maintain it in non-aggregating conformation. Our study of a genetically modified mouse that lacks GM1 revealed spontaneous development of parkinsonian symptoms which could be prevented by LIGA-20, a membrane permeable analog of GM1. We propose gradual loss of GM1 in dopaminergic neurons with aging, giving rise to loss of these regulatory functions and neuron death. Loss of a sufficient portion of nigral neurons results eventually in PD. In addition to analyzing GM1 levels in brain samples from PD patients, we will study the mechanistic role of GM1 using the above genetically modified mice and N27 cells.
|Ledeen, Robert; Wu, Gusheng (2011) New findings on nuclear gangliosides: overview on metabolism and function. J Neurochem 116:714-20|
|Wu, Gusheng; Lu, Zi-Hua; Kulkarni, Neil et al. (2011) Mice lacking major brain gangliosides develop parkinsonism. Neurochem Res 36:1706-14|
|Wang, Jianfeng; Wu, Gusheng; Miyagi, Taeko et al. (2009) Sialidase occurs in both membranes of the nuclear envelope and hydrolyzes endogenous GD1a. J Neurochem 111:547-54|
|Wu, Gusheng; Xie, Xin; Lu, Zi-Hua et al. (2009) Sodium-calcium exchanger complexed with GM1 ganglioside in nuclear membrane transfers calcium from nucleoplasm to endoplasmic reticulum. Proc Natl Acad Sci U S A 106:10829-34|
|Wang, Jianfeng; Lu, Zi-Hua; Gabius, Hans-Joachim et al. (2009) Cross-linking of GM1 ganglioside by galectin-1 mediates regulatory T cell activity involving TRPC5 channel activation: possible role in suppressing experimental autoimmune encephalomyelitis. J Immunol 182:4036-45|
|Ledeen, Robert W; Wu, Gusheng (2008) Nuclear sphingolipids: metabolism and signaling. J Lipid Res 49:1176-86|
|Ledeen, Robert; Wu, Gusheng (2007) GM1 in the nuclear envelope regulates nuclear calcium through association with a nuclear sodium-calcium exchanger. J Neurochem 103 Suppl 1:126-34|
|Wu, Gusheng; Lu, Zi-Hua; Obukhov, Alexander G et al. (2007) Induction of calcium influx through TRPC5 channels by cross-linking of GM1 ganglioside associated with alpha5beta1 integrin initiates neurite outgrowth. J Neurosci 27:7447-58|
|Ledeen, Robert W; Wu, Gusheng (2007) Sodium-calcium exchangers in the nucleus: an unexpected locus and an unusual regulatory mechanism. Ann N Y Acad Sci 1099:494-506|
|Ledeen, Robert W; Wu, Gusheng (2006) Gangliosides of the nuclear membrane: a crucial locus of cytoprotective modulation. J Cell Biochem 97:893-903|
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