Modulation of human α-synuclein aggregation by a combined effect of calcium and dopamine
Graphical abstract
Introduction
Parkinson's disease (PD) is the second most common progressive neurodegenerative disease after Alzheimer's (Lang and Lozano, 1998). PD is caused by deposition of aggregated α-syn into Lewy bodies (LBs) (McKeith et al., 1999) which causes substantial loss of dopaminergic neurons (DN) in the substantia nigra (SN) region of midbrain (Spillantini et al., 1997). α-syn is a 140 amino acid long acidic protein, predominately expressed in the brain. Endogenous α-syn exists as a helical tetramer of 58 kDa in neuronal and non-neuronal cells under non-denaturing conditions and its destabilization leads to its aggregation (Bartels et al., 2011). This tetrameric form of α-syn is resistant to aggregation but small structural perturbations caused by heating, posttranslational modifications, etc. lead to its destabilization making it prone to aggregation (Wang et al., 2011). However, in in-vitro conditions, monomeric α-syn has no defined secondary structure and belongs to the group of intrinsically disordered proteins (IDPs) (Dunker et al., 2001). It acquires an α-helical structure upon binding to lipid vesicles (Davidson et al., 1998). It has three domains, the N-terminal domain that interacts with lipids (Bussell and Eliezer, 2003), the middle β-amyloid forming domain and a less conserved negatively charged C-terminal that binds to Ca(II) and microtubule-associated proteins (Jensen et al., 2000). Its familial point mutants A30P, A53T, and E46K have been associated with the early onset of PD (Conway et al., 2000, Cookson, 2009). Recently, a novel disease causing H50Q mutation has been reported that has a similar disease causing mechanism as that of A53T and E46K (Appel-Cresswell et al., 2013, Kasten and Klein, 2013).
α-syn has been known to be involved in Ca(II) and DA signaling (Narayanan et al., 2005, Nielsen et al., 2001) and plays a role in the modulation of dopamine transporter function, synaptic plasticity and neurotransmitter release from presynaptic vesicles (Sidhu et al., 2004). It is also involved in calcium-mediated neuronal homeostasis, synaptic transmission, and cell survival including the pathogenesis of PD (Berridge, 1998, Mattson, 2007). Nielsen and coworkers using microdialysis showed that Ca(II) binds to the C-terminus of α-syn with an IC50 of about 2–300 μM in a reaction which is uninhibited by a 50 fold excess of Mg(II) with the stiochiometry of 0.5 Ca(II) per protein molecule. α-syn selectively binds to cytosolic Ca(II) but not Mg(II) which suggests that Ca(II) regulates its function (Nielsen et al., 2001) and this binding induces the formation of annular oligomers (Nath et al., 2011). It has been shown using mass spectrometry that Ca(II) binds to the isolated C-terminal fragments 112–140, 114–140, and 124–140 of human α-syn (de Laureto et al., 2006) and residues from 124–140 are essential for Ca(II)-induced annular oligomer formation (Lowe et al., 2004).
DA exerts its effect in the brain through DA receptors (D1–D5) and D4 receptor is involved in the regulation of intracellular Ca(II) levels (Rondou et al., 2010). Oxidative stresses accompanied with aging are also associated with PD and lead to Ca(II) dysregulation which affect normal cellular processes (Ischiropoulos and Beckman, 2003). The interaction of DA and its oxyradicals with α-syn leads to the formation of covalently linked dopamine quinone which are responsible for selective death of DN in SN (Conway et al., 2001, Galvin, 2006). Also, an interaction between cytoplasmic Ca(II), DA and α-syn present under elevated levels has been suggested to be responsible for evoking selective death of DN in SN (Mosharov et al., 2009). Therefore, it is of significance to investigate in detail the combinatorial effect of Ca(II) and DA on the conformation and aggregation of α-syn as well as the cytotoxicity of various aggregates of α-syn formed in the presence of Ca(II) and DA.
In the present work, we have investigated the conformational and aggregation properties of human α-syn and its familial mutants A30P, A53T, E46K and H50Q in the presence of Ca(II) and DA alone and in combination. We have also studied the effect of anti-aggregating agent epigallocatechin gallate (EGCG) on the aggregation of H50Q in the absence and presence of Ca(II) and DA. ITC has been employed to quantify the binding of Ca(II) to α-syn and its familial mutants as well as the binding of DA to α-syn. We have also monitored the effect of various aggregating species on the cytotoxicity of α-syn formed at different time intervals of incubation in the presence of Ca(II) as well as Ca(II) and DA together as well as the internalization and internalization kinetics of mature aggregating species into SHSY5Y cells using confocal microscopy and live cell confocal imaging. Our data reveal that binding of Ca(II) to α-syn is weak and endothermic in nature and DA does not show any appreciable binding. While Ca(II) enhances fibrillation of α-syn, DA promotes the formation of oligomers. However, the presence of Ca(II) and DA together shifts the fibril forming tendency of α-syn towards protofibril formation which are more cytotoxic in nature than the mature fibrils. EGCG inhibits the aggregation propensity of H50Q in the absence and presence of Ca(II) and DA. The findings put together suggest that an interplay between the concentrations of Ca(II), DA and soluble form of α-syn and its familial mutants as well as the binding of Ca(II) to α-syn and its familial mutants critically regulate the nature of various aggregating species responsible for the survival of DN.
Section snippets
Materials
HEPES buffer, pH standards, streptomycin sulfate, CaCl2 and Thioflavin T were purchased from Sigma-Aldrich Co. LLC., St. Louis, MO. Ampicillin, kanamycin, ethanol, ammonium sulfate and IPTG were purchased either from Sisco Research laboratory Pvt. Ltd. or from Merck and Alexa fluor (AF488) was purchased from Molecular Probes, Inc., Eugene, USA. All other chemicals used were of high quality analytical grade. A plasmid containing the full length genes of the human α-syn was a generous gift from
Effect of Ca(II) on α-syn structure
Human α-syn contains four tyrosine residues and no tryptophan residues. The intrinsic tyrosine fluorescence intensity of α-syn was found to increase with increasing concentrations of Ca(II) and the change in the fluorescence intensity though small suggests the interaction of Ca(II) with α-syn (Fig. 1A). No change was observed in fluorescence emission of free l-tyrosine in the presence of Ca(II) (Fig. S1A). ANS binding revealed that there is neither a significant increase nor a shift in ANS
Discussion
We have investigated the effect of Ca(II) and DA on the conformation and aggregation of human α-syn and its familial mutants and correlated the cytotoxicity and internalization of the various α-syn aggregating species formed in the presence of Ca(II) and DA during amyloid formation. We have also evaluated various thermodynamic parameters of Ca(II) and DA binding to α-syn and its familial mutants using ITC. Increase in the intrinsic tyrosine fluorescence intensity of α-syn in the presence of
Conclusion
Human α-syn and its familial mutants A30P, A53T and E46K bind to Ca(II) and the binding is weak and endothermic in nature while binding of DA to α-syn could not be detected. Our data suggest that the presence of appropriate molar ratios of DA, Ca(II) and α-syn could critically affect the nature of the aggregating species of α-syn and hence their cytotoxicity. Various aggregating species of human α-syn internalized into the cells and localized into the cytosolic area of the human neuroblastoma
Abbreviations
- α-syn
α-synuclein
- Ca(II)
calcium
- DA
dopamine
- SN
substantia nigra
- ITC
Isothermal Titration Calorimetry
- CD
circular dichroism
- LB
Lewy body
- IDP
intrinsically disordered protein
- PD
Parkinson's disease
- ThT
Thioflavin T
- BSA
bovine serum albumin
- DC
dopaminochrome
- ANS
1-anilino-8-naphthalene sulfonate
- DN
dopaminergic neurons
- AF 488
Alexa Fluor 488
- DTT
Dithiothreitol
- EGCG
(−)-epi-gallocatechine gallate
The following are the supplementary data related to this article.
Acknowledgment
We thank Prof. Peter Lansbury, Harvard Medical School, Cambridge, MA, USA for providing the human α-syn clone, Prof. Faizan Ahmad and Dr. Imtiyaz Hussain, Jamia Milia Islamia, New Delhi for providing access to dynamic light scattering instrument, Prof. Aparna Dixit for the use of fluorimeter, and Advanced Instrument Research facility, Jawaharlal Nehru University, New Delhi for the CD, TEM, confocal microscopy and live cell imaging facilities. Manish Kumar Jain acknowledges Council of Scientific
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