α-Synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells

“α-Synuclein (α-Syn), a protein highly enriched in presynaptic terminals (Maroteaux et al., 1988), is implicated in normal brain function (George et al., 1989) as well as disease (reviewed by Perez and Hastings, 2004).”

“An active PP2A enzyme consists of a heterotrimer of the structural A subunit, a catalytic C subunit, and a regulatory B subunit (Dobrowsky et al., 1993).”

“The A and C subunits are ubiquitously expressed (Mayer et al., 1991) and form the catalytic complex (PP2A/C), which interacts with at least three different families of regulatory B subunits, as well as with certain tumor antigens (Mumby and Walter, 1993).”

“Control MN9D cells with low endogenous levels of α-Syn, included both untransfected parental MN9D cells and MN9D cells stably transfected with either pcDNA3 or GFP vector (Perez et al., 2002).”

“…protein 14-3-3 (Ostrerova et al., 1999), which binds to and regulates the activity of many cellular proteins. α-Syn interacts with and affects the activity of the enzymes phospholipase D (Ahn et al., 2002), protein kinase C and the extracellular regulated kinases (ERK) (Ostrerova et al., 1999).”

“Since it is well-documented that Ser40 dephosphorylation occurs almost exclusively by PP2A (Berresheim and Kuhn, 1994; Dunkley et al., 2004; Haavik et al., 1989; Leal et al., 2002) our findings indicate that (1) TH Ser40 is dephosphorylated by PP2A in dopaminergic cells, and (2) the Ser40 residue on TH remains accessi…”

“…kinase mediating Ser40 phosphorylation and because α-Syn is so strongly implicated in enzymatic inhibition, e.g. ERK2, PLD2, TH (Iwata et al., 2001; Jenco et al., 1998; Perez et al., 2002) we first hypothesized that the reduction in TH Ser40 phosphorylation was probably occurring by α-Syn…”

“Cell lysates prepared in 1% NP40 buffer containing protease inhibitors were sonicated for ∼5 seconds and particulates were eliminated by centrifugation at 15,000 g for 10 minute at 4°C as previously described (Perez et al., 1999).”

“…there is evidence that B subunits are associated with targeting the PP2A/C catalytic complexes to various intracellular sites such as microtubules (McCright et al., 1996; Sontag et al., 1995) and mitochondria (Ruvolo et al., 2002) suggesting that PP2A complexes are actively trafficked by their…”

“It is known that 14-3-3 can activate TH (Ichimura et al., 1988) by 14-3-3 binding first to TH Ser19 then to TH Ser40 (Kleppe et al., 2001).”

“…B subunits are associated with targeting the PP2A/C catalytic complexes to various intracellular sites such as microtubules (McCright et al., 1996; Sontag et al., 1995) and mitochondria (Ruvolo et al., 2002) suggesting that PP2A complexes are actively trafficked by their associated interacting…”

“, 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.”

“…S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al., 1986; Wu et al., 1992).”

“The regulation of TH activity in the short term depends on the phosphorylation of key seryl residues (Ser19, Ser31 and Ser40) in the amino-terminal regulatory domain (Campbell et al., 1986; Haycock, 1990).”

“The regulatory B subunits of PP2A are known to be temporally expressed during development (Csortos et al., 1996; Mayer et al., 1991; McCright and Virshup, 1995; Ruediger et al., 1991) and neuron-specific isoforms have also been identified (Mayer et al.”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“…PP2A may affect PP2A conformation or trafficking and subsequently contribute to its activation. α-Syn oxidative modification or aggregation occurs in neurodegenerative diseases such as Alzheimer’s disease, a condition in which PP2A is also implicated (Trojanowski and Lee, 1995; Zhao et al., 2003).”

“, 1991) and neuron-specific isoforms have also been identified (Mayer et al., 1991).”

“The MN9D dopaminergic cells express abundant TH (Choi et al., 1991) and synthesize DA (Choi et al.”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“The substrate specificity of PP2A appears to be determined by the regulatory B subunits (Cegielska et al., 1994; Csortos et al., 1996) and there is evidence that B subunits are associated with targeting the PP2A/C catalytic complexes to various intracellular sites such as microtubules (McCright et…”

“, 1995) and mitochondria (Ruvolo et al., 2002) suggesting that PP2A complexes are actively trafficked by their associated interacting proteins, one of which may be α-Syn.”

“ERK2, PLD2, TH (Iwata et al., 2001; Jenco et al., 1998; Perez et al., 2002) we first hypothesized that the reduction in TH Ser40 phosphorylation was probably occurring by α-Syn inhibition of PKA.”

“…protein 14-3-3 (Ostrerova et al., 1999), which binds to and regulates the activity of many cellular proteins. α-Syn interacts with and affects the activity of the enzymes phospholipase D (Ahn et al., 2002), protein kinase C and the extracellular regulated kinases (ERK) (Ostrerova et al., 1999).”

“α-Synuclein (α-Syn), a protein highly enriched in presynaptic terminals (Maroteaux et al., 1988), is implicated in normal brain function (George et al., 1989) as well as disease (reviewed by Perez and Hastings, 2004).”

“, 2002), which reduces TH activity both in dopaminergic cells (Haavik et al., 1989) and in rat striatum (Bevilaqua et al.”

“Of these, phosphorylated Ser40 is a major contributor to DA synthesis in vivo (Ramsey et al., 1996).”

“These constructs form the basis of the ‘bomb system’ for inducible transgene expression (Suhr et al., 1998; Suhr et al., 2001) and were generously provided by Fred Gage and Steve Suhr at the Salk Institute (La Jolla, CA, USA).”

“The MN9D dopaminergic cells express abundant TH (Choi et al., 1991) and synthesize DA (Choi et al., 1992).”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“The regulatory B subunits of PP2A are known to be temporally expressed during development (Csortos et al., 1996; Mayer et al., 1991; McCright and Virshup, 1995; Ruediger et al., 1991) and neuron-specific isoforms have also been identified (Mayer et al.”

“Generation of stably transfected inducible PC12 cell lines α-Syn was cloned into pcDNA3 as previously described (Stefanis et al., 2001) followed by subcloning into the SKSP shuttle cloning vector using HindIII-XhoI.”

“Low-passage number MN9D cells were grown as previously described (Ugarte et al., 2003).”

“2). α-Syn increases PP2A activity and binds to the PP2A catalytic domain We measured PP2A activity from the MN9D and induced PC12 cells using a well-established immunoprecipitation and phosphatase activity protocol (Begum and Ragolia, 1996).”

“Although multiple kinases can phosphorylate TH on Ser40 in vitro, including cyclic GMP-dependent protein kinase (Rodriguez-Pascual et al., 1999; Roskoski et al., 1987), CamKII (Campbell et al., 1986; Yamauchi and Fujisawa, 1981), 3523 α-Synuclein is an abundant presynaptic protein implicated in…”

“There is additional evidence that the binding of 14-3-3 to TH phosphorylated on Ser19 and Ser40 stabilizes its conformation and enhances TH activity (Bevilaqua et al., 2001).”

“It is known that 14-3-3 can activate TH (Ichimura et al., 1988) by 14-3-3 binding first to TH Ser19 then to TH Ser40 (Kleppe et al., 2001).”

“Furthermore, we treated cells with low to high dose okadaic acid (5 nM-1 μM) dissolved in DMSO (0.13 μM) (Garcia et al., 2002; Haavik et al., 1989) for 1 hour to assess the impact on PP2A inhibition in the presence of α-Syn overexpression and saw a similar effect.”

“α-Synuclein (α-Syn), a protein highly enriched in presynaptic terminals (Maroteaux et al., 1988), is implicated in normal brain function (George et al., 1989) as well as disease (reviewed by Perez and Hastings, 2004).”

“, 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al., 1986; Wu et al., 1992).”

“…PP2A may affect PP2A conformation or trafficking and subsequently contribute to its activation. α-Syn oxidative modification or aggregation occurs in neurodegenerative diseases such as Alzheimer’s disease, a condition in which PP2A is also implicated (Trojanowski and Lee, 1995; Zhao et al., 2003).”

“…Ser40 in vitro, including cyclic GMP-dependent protein kinase (Rodriguez-Pascual et al., 1999; Roskoski et al., 1987), CamKII (Campbell et al., 1986; Yamauchi and Fujisawa, 1981), 3523 α-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegenerative diseases.”

“It is well documented that dephosphorylation of TH Ser40 occurs almost exclusively by the phosphatase PP2A (Berresheim and Kuhn, 1994; Haavik et al., 1989; Leal et al., 2002), which reduces TH activity both in dopaminergic cells (Haavik et al.”

“Tebufenozide, which is a molt-inducing insecticide that mimics the action of ecdysone (Addison, 1996), was used for induction of α-Syn expression.”

“It is well documented that dephosphorylation of TH Ser40 occurs almost exclusively by the phosphatase PP2A (Berresheim and Kuhn, 1994; Haavik et al., 1989; Leal et al., 2002), which reduces TH activity both in dopaminergic cells (Haavik et al., 1989) and in rat striatum (Bevilaqua et al., 2003).”

“Although multiple kinases can phosphorylate TH on Ser40 in vitro, including cyclic GMP-dependent protein kinase (Rodriguez-Pascual et al., 1999; Roskoski et al., 1987), CamKII (Campbell et al., 1986; Yamauchi and Fujisawa, 1981), 3523 α-Synuclein is an abundant presynaptic protein implicated in…”

“Since it is well-documented that Ser40 dephosphorylation occurs almost exclusively by PP2A (Berresheim and Kuhn, 1994; Dunkley et al., 2004; Haavik et al., 1989; Leal et al., 2002) our findings indicate that (1) TH Ser40 is dephosphorylated by PP2A in dopaminergic cells, and (2) the Ser40 residue…”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“Membrane bound PP2A in brain is reportedly less active (Sim et al., 1998), thus the interaction of PP2A with α-Syn within the cytosol may serve to stimulate PP2A activity.”

“a-Synuclein is quite promiscuous in its binding properties, and may bind various proteins in neuronal cells, including components of dopamine metabolism, such as tyrosine hydroxylase (TH) and the dopamine transporter (DAT), modulating their function (Ostrerova et al. 1999; Perez et al. 2002; Wersinger and Sidhu 2003;…”

“TBP-1 [332,333], phospholipase D [334], protein phosphatase 2A [335], DAT [336], mitochondrial complex IV enzyme cytochrome oxidase”

“-Synuclein inhibits dopamine synthesis by inhibiting the expression and activity of tyrosine hydroxylase [154, 168–170, 204], likely via reducing the phosphorylation state of tyrosine hydroxylase and stabilizing dephosphorylated inactive tyrosine hydroxylase [168, 205–207].”

“…on effects of the protein on catecholamine synthesis, uptake, and vesicular accumulation (Lee et al., 2001; Lotharius et al., 2002; Perez et al., 2002; Wersinger et al., 2003; Peng et al., 2005), is that amount of transmitter released per vesicle fusion event (quantal size) might be decreased.”

“Mechanistically, α-Syn decreases TH activity by reducing its phosphorylation in vitro (Perez et al., 2002; Peng et al., 2005) as TH is activated by phosphorylation of seryl residues in the TH regulatory domain (Alerte et al.”

“There is also evidence that -syn can modulate expression of genes involved in DA synthesis (11) and can affect enzymes involved in chromatin remodeling (12) and signal transduction (13).”

“Regulation of dopamine biosynthesis[45‐46] Protein phosphatase 2A Inhibition of tyrosine hydroxylase Regulation of dopamine levels”

“In addition, α-synuclein has been shown to bind TH, preventing its phosphorylation and inhibiting its activation by promoting the activity of protein phosphatase 2A (PP2A) [35,40].”

“regulate the activity of several enzymes, including tyrosine hydroxylase, the rate-limiting enzyme in dopamine production [40,41],”

“Several lines of evidence suggest that a-syn can reduce the phosphorylation of TH, bind with TH in a dephosphorylated state, and maintain TH in an inactive form (Perez et al., 2002; Peng et al., 2005; Wu et al., 2009; Lou et al., 2010).”

“Much of the experimental evidence has, however, been obtained in vitro or in cultured cells (Jenco et al., 1998; Perez et al., 2002; Wersinger and Sidhu, 2003; Peng et al., 2005; Larsen et al., 2006; Tehranian et al., 2006; Fountaine et al., 2008; Liu et al., 2008), and similar roles have yet to be identified in the a…”

“The research literature suggests that AS staining in TH-positive neurons is problematic: the less TH immunoreactivity, the more AS antibody binds.(16,17) If that is correct, TH labeling is less in more advanced PD.”

“, 2003; Wersinger and Sidhu, 2003) and the key DA synthesis enzyme, tyrosine hydroxylase (Peng et al., 2005; Perez et al., 2002).”

“The widely used N27 neuronal cell model represents a homogeneous population of TH-positive dopaminergic cells and is highly useful for studying degenerative mechanisms in PD (Clarkson et al., 1999; Kaul et al., 2005a; J. Peng et al., 2005; Zafar et al., 2007; Zhang et al., 2007a; Lee et al., 2009).”

“In tissue culture models, alpha-synuclein has been shown to inhibit DA synthesis by regulating the activity of tyrosine hydroxylase, protein phosphotase 2A, and aromatic amino acid decarboxylase (Perez et al., 2002; Peng et al., 2005; Tehranian et al., 2006).”

“This small and highly conserved protein is enriched in presynaptic terminals and is implicated in both normal as well as pathogenic brain function [60-64].”

“This is particularly apparent considering the effects of α-synuclein expression on tyrosine hydroxylase expression and activity [367,372,386].”

“Our lab and other research groups have used N27 cells extensively to study the neurotoxic mechanisms related to Parkinson's disease (Clarkson et al., 1999; Miranda et al., 2004; Kaul et al., 2005; Peng et al., 2005; Kanthasamy et al., 2006).”

“-Synuclein binds a variety of proteins (Jenco et al., 1998; Peng et al., 2005) and lipid vesicles (Cole et al.”

“It is possible that decreased levels of TH can also be associated with the observed increase in the level of a-synuclein, because the a-synuclein has easy interactions between with TH, thus reducing its phosphorylation (Peng et al., 2005).”

“Dopamine reduction is observed with the decrease in TH and dopamine transporter expressions [18, 20–24].”

“It is known that α-syn inhibits tyrosine hydroxylase, the rate limiting enzyme of DA biosynthesis [163,164], and aromatic amino acid decarboxylase, the enzyme that converts L-Dopa to DA [165].”

“There is considerable evidence that the expression of PP2A, while the activity of the phosphatase is increased in parallel with a-syn expression (Peng et al., 2005; Perez et al., 2002).”

“Thus, elevated levels of α-synuclein increase the activity of the Akt inhibitors phosphatase PP2A [135] and RTP801 [136] while Akt phosphorylation is significantly diminished in dopaminergic SN neurons of PD patients [137].”

“It was also confirmed that α-syn significantly reduced AADC activity and phosphorylation in cells (Perez et al., 2002; Peng et al., 2005; Tehranian et al., 2006).”

“All steps were carried out at 4 C as described by Peng et al. (2005), but with minor modifications.”

“Recently -synuclein has been shown to activate the protein phosphatase 2A (Peng et al., 2005), a phosphatase involved in actin dephosphorylation.”

“…of dopamine levels by α-synuclein has been demonstrated as a consequence of the inhibition of the neurotransmitter synthesis through the regulation of tyrosine hydroxylase activity (Abeliovich et al., 2000; Peng, 2005) and the inhibition of the vesicular monoamine transporter-2 (Guo et al., 2008).”

“In these cells, a-synuclein may inhibit the activity of tyrosine hydroxylase [16,17] and affect melanin production as described previously [18,19].”

“Therefore, in the dopaminergic system, the physical and functional association between PKC , TH, and PP2A could be facilitated and/or regulated by chaperone proteins such as -synuclein (Kaul et al., 2005; Peng et al., 2005) and 14-3-3 (Ostrerova et al.”

“…the result of disinhibition of TH following αsyn knockdown. α-syn acts as a negative regulator of both TH and aromatic amino acid decarboxylase (AADC; Perez et al., 2002; Perez and Hastings, 2004; Yu et al., 2004; Peng et al., 2005; Tehranian et al., 2006; Wang et al., 2009; Benskey et al., 2016a).”

“Previously, we noted that α-Syn acts to inhibit TH phosphorylation and dopamine synthesis in vitro [31,33].”

“It is known to interact with a variety of proteins [13, 14] and also with lipid vesicles [15], and it may be involved in lipid metabolism [16, 17].”

“It is thought that alpha-syn plays a role in the regulation of dopamine biosynthesis via activation of protein phosphatase 2A that reduces tyrosine hydroxylase phosphorylation, the rate-limiting enzyme in dopamine production [158, 159], and reducing the amount of available active mitogen-activated protein kinases [160…”

“All steps were carried out at 4°C as previously described (46, 47), with minor modifications.”

“…et al., 2015; Sharma et al., 2015 Regulation of DA biosynthesis Protein phosphatase 2A Inhibition of tyrosine Hydroxylase Regulation of DA levels Peng et al., 2005; Yang et al., 2013 Suppression of apoptosis Protein C Kinase Deactivation of NFkB Neuroprotection Jin et al., 2011 SNARE,…”

“The decreases in TH phosphorylation at Ser40 are not because of decreases in PKA activity, but rather to increases in PP2A activity (Peng et al. 2005).”

“, 2010), which was modulated by the activity of protein phosphatase 2A (Peng et al., 2005).”

“Additionally, normalization of PP2A function has been hypothesized to be relevant in relation to modulation of tyrosine hydroxylase function [37, 38].”

“a-Synuclein was found to co-immunoprecipitate with PP2A [22] and we found that the expression of PP2A was modulated by a-synuclein (data not shown).”

“In vitro studies have shown that α Syn overexpression leads to downregulation of crucial enzymes involved in DA biosynthesis (tyrosine hydroxylase and aromatic amino acid decarboxylase), as well as an impairment of dopamine transporter (DAT) reuptake activity [125–127].”

“Given that a-Syn associates with PP2A,(115) perhaps the phosphorylation status of both proteins is mediated in a reciprocal way, via PP2A, a possibility proposed by others.(58,115,117,119) What connection might there be among the phosphorylation of a-Syn, TH activity, PP2A activity, and MAM? First, we propose that the…”

“By inhibition of the enzyme that dephosphorylates TH, called phosphor-protein phosphatase 2A (PP2A), asyn promotes the activity of the TH (47).”

“…| www.frontiersin.org 13 June 2020 | Volume 14 | Article 527 (Reynolds et al., 2014) without affecting the protein level of PP2A in cell lines (Peng et al., 2005), but on the other hand, increased oligomerization and phosphorylation of α-syn reduced the activity of PP2A (Lou et al., 2010;…”

“Of relevance to this, the activity/level of protein phosphatase 2A (PP2A), for which phospho-Akt is a substrate, is elevated by alpha synuclein (Peng et al. 2005) and in response to 6-OHDA (Ryu et al. 2005; Chung et al. 2007) in cultured cells.”

“Inhibitors for specificity included: 50 nM protein-phosphataseinhibitor 2 (PP1-specific), 3 nM fostriecin (PP2A/PP4-specific), 50 nM cantharidin (PP4-specific), and 10 nM okadaic acid as previously described (Peng et al. 2005; Lou et al. 2010; Wu et al. 2012).”

“Thus, pharmacological agents such as antipsychotics [12,33] or neuropathological events [27,34–37]”

“While α-Syn and the catalytic subunit of PP2A are known to interact (Peng et al., 2005), no one knows if α-Syn might also bind B subunits of PP2A to in some manner contribute to holoenzyme assembly or localization.”

“…hydroxylase (TH), DA transporter (DAT) and vesicular monoamine transporter-2 (VMAT2) function, aromatic amino acid decarboxylase and tetrahydrobiopterin (co-factor essential for TH function) (Baptista et al., 2003; Lee et al., 2001; Peng et al., 2005; Perez et al., 2002; Tehranian et al., 2006).”

“Protein phosphatase 2A (PP2A) is the main phosphatase responsible for dephosphorylating TH and regulating its activity (Haavik et al. 1989; Peng et al. 2005).”

“leading to deactivation of TH and reduction of dopamine levels (Peng et al. 2005; Lou et al. 2010).”

“and alpha-synuclein aggregation; both critical to the neuropathology of Parkinson’s disease [106–109].”

“α-Syn Disrupts Tyrosine Hydroxylase and/or Tyrosinase Activity The prevailing hypothesis(26,32-34,37,39) to describe the inverse correlation between α-syn expression and melanin production states that α-syn disrupts the activities of catalytic enzymes involved in melanin biosynthesis, namely, tyrosine hydroxylase (TH)…”

“5 in the midbrain/hindbrain junction, suggesting that a-SYN may play a yet undefined role in early brain development. a-SYN is reported to regulate DA synthesis by binding to and inhibiting tyrosine hydroxylase (TH) (Peng et al. 2005; Alerte et al. 2008).”

“In addition to its role in synaptic transmission, α-syn is also involved in the suppression of apoptosis [39], antioxidation [40] and even regulation of dopamine biosynthesis [41, 42].”

“rat) cells leads to enhanced PP2A activation [105].”

“…often contain Lewy Bodies, inclusions with abundant aSyn, a presynaptic protein that broadly contributes to dopamine regulation (Perez et al., 2002; Perez and Hastings, 2004; Peng et al., 2005; Tehranian et al., 2006; Porras and Perez, 2014; Benskey et al., 2016; Mor and Ischiropoulos, 2018).”

“…detection of tyrosine hydroxylase (TH; 1:500 mouse anti-TH; MAB318, RRID:AB 2201528; MilliporeSigma; Charalampopoulos et al. 2005; Peng et al. 2005; 1:250 Alexa Fluor 488 donkey anti-mouse IgG; 715-545-152, RRID: AB 2313584; Jackson Immunoresearch) and expression was quantified,…”

“Alpha-synuclein down-regulates dopamine transmission by inhibiting the activity of tyrosine hydroxylase, decreasing dopamine synthesis and acting as a negative regulator of DA release (Peng et al., 2005; Perez et al., 2002).”