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**Provisional chapter**

#### **Phosphorylation-Mediated Control of Stress Responses Induced by Nanosecond Pulsed Electric Fields Induced by Nanosecond Pulsed Electric Fields**

**Phosphorylation-Mediated Control of Stress Responses**

DOI: 10.5772/intechopen.69782

Ken-ichi Yano and Keiko Morotomi-Yano Ken-ichi Yano and Keiko Morotomi-Yano Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69782

#### **Abstract**

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Exposure of living organisms to short electric pulses is widely utilized in the life sciences, for example, for DNA transfection. Recent advances in electrical engineering have enabled the production of extremely short electric pulses in the range of nanoseconds, namely, nanosecond pulsed electric fields (nsPEFs). nsPEFs are increasingly recognized as a novel means for cancer therapy, because of their ability to induce cell death. Recent studies have demonstrated that nsPEFs act as cellular stress and activate two independent signaling pathways that involve phosphorylation of translation initiation factors and lead to suppression of general protein synthesis. eIF2α phosphorylation is one of the key reactions in stress-induced translational suppression and is rapidly induced by nsPEFs. Concomitantly, PERK and GCN2, both of which are stress-responsive protein kinases, are activated in nsPEF-exposed cells. Furthermore, nsPEFs cause a reduction in 4E-BP1 phosphorylation, which is controlled by mTORC1 and constitutes an alternative mechanism for translational suppression, independent of eIF2α phosphorylation. In accordance with elevated eIF2α phosphorylation and decreased 4E-BP1 phosphorylation, general protein synthesis is acutely suppressed after nsPEF exposure. These findings demonstrate that nsPEFs induce two independent signaling pathways for translational suppression, further highlighting a unique feature of nsPEFs as a novel means for life sciences.

**Keywords:** stress response, electroporation, pulsed electric field, eIF2α, PERK, GCN2, 4E-BP1, translational suppression, protein synthesis

#### **1. Introduction**

Living cells respond to various environmental stimuli by activating distinct sets of intracellular reactions. Cellular responses to external stimuli generally involve signal transduction that is

mediated by protein phosphorylation and eventually leads to modulation of various cellular events, such as metabolism, gene expression, proliferation, and cell death [1].

Some physical and chemical stimuli have adverse physiological effects and are known as cellular stress. In addition to adverse external stimuli, endogenous deleterious events are also regarded as cellular stress, such as accumulation of misfolded proteins in the endoplasmic reticulum (ER). Cells respond to these cellular stresses by inducing various intracellular reactions, which are collectively referred to as stress responses. Timely induction of stress responses is critical for maintenance of cellular physiology, and its dysregulation is frequently observed in various diseases, such as cancer [2], neurodegenerative disorders [3], and inflammatory diseases [4], indicating the importance of stress responses in physiological and pathological processes.

Currently, diverse forms of physical stimuli are utilized as tools for various biological and clinical applications. Among these physical stimuli, pulsed electric fields (PEFs) have been proven particularly useful, because different biological effects can be achieved, depending on the duration of the electric pulses. PEFs in the range of milliseconds to microseconds primarily act on the cell membrane and generate membrane pores, which are suited for introduction of exogenous macromolecules, such as plasmid DNA, into living cells [5, 6]. Thus, these PEFs are widely used for DNA transfection [7].

Recent advances in electric engineering allow us to generate ultrashort PEFs in the range of nanoseconds, which are referred to as nanosecond PEFs (nsPEFs). Although nsPEFs do not generate membrane pores suitable for DNA transfection, they have been proven to be useful for cancer therapy, because of their ability to induce cell death [8, 9]. Furthermore, nsPEFs have been shown to induce stress responses that are mediated by phosphorylation of multiple translation initiation factors and eventually lead to transient suppression of general protein synthesis. For these reasons, nsPEFs have received considerable attention as a potential therapeutic method with a novel mechanism of action. This review is intended to provide an overview of the stress responses induced by nsPEFs. First, two major mechanisms for stress responses in human cells are explained. Phosphorylation-mediated control of two translation initiation factors, eIF2α and 4E-BP1, is critical in these pathways. Second, an outline of the biological actions of PEFs is provided, with particular emphasis on nsPEFs. Finally, stress responses induced by nsPEFs are described in detail.

#### **2. Stress responses in human cells**

#### **2.1. Overview of stress response**

The fundamental aspects of cellular stress responses are highly conserved among eukaryotes from yeast to humans. **Figure 1** shows a simplified scheme for eukaryotic stress responses. Under normal physiological conditions, cells continuously undergo protein synthesis, and the rate of protein synthesis is primarily regulated at the translation initiation step. When cells sense stress, they rapidly activate signal transduction that involves phosphorylation-mediated Phosphorylation-Mediated Control of Stress Responses Induced by Nanosecond Pulsed Electric ... http://dx.doi.org/10.5772/intechopen.69782 99

**Figure 1.** General principle of stress responses in eukaryotic cells. When a cell is exposed to stress, a stress-sensing protein is activated, inducing signal transduction mediated by protein phosphorylation. The stress-induced signal is transduced to downstream effector proteins. Stress responses generally lead to inhibition of cap-dependent translation initiation and consequent suppression of general protein synthesis. Stress responses often cause additional changes, such as alteration of gene expression.

control of translation initiation factors. Stress-induced alterations in the phosphorylation status of translation initiation factors reduce translation initiation and thereby result in attenuation of general protein synthesis. Because protein synthesis requires significant amounts of energy and materials, transient suppression of general protein synthesis conserves cellular resources and is thus beneficial for cells under stress. For these reasons, stress-induced suppression of general protein synthesis serves as a mechanism for survival. Once the stress ends, translation capacity is rapidly recovered by dephosphorylation of the translation factors. Intriguingly, persistent activation of stress responses is often associated with the induction of cell death, suggesting that timely induction and attenuation of stress responses are both critical for cell survival [10–12]. Although eukaryotic cells share a fundamental stress response mechanism [13, 14], as described below, human cells possess more intricate stress responses, including at least two distinct pathways involving phosphorylation of multiple proteins.

#### **2.2. Stress response mediated by eIF2α phosphorylation**

mediated by protein phosphorylation and eventually leads to modulation of various cellular

Some physical and chemical stimuli have adverse physiological effects and are known as cellular stress. In addition to adverse external stimuli, endogenous deleterious events are also regarded as cellular stress, such as accumulation of misfolded proteins in the endoplasmic reticulum (ER). Cells respond to these cellular stresses by inducing various intracellular reactions, which are collectively referred to as stress responses. Timely induction of stress responses is critical for maintenance of cellular physiology, and its dysregulation is frequently observed in various diseases, such as cancer [2], neurodegenerative disorders [3], and inflammatory diseases [4], indicating the importance of stress responses in physiological and pathological

Currently, diverse forms of physical stimuli are utilized as tools for various biological and clinical applications. Among these physical stimuli, pulsed electric fields (PEFs) have been proven particularly useful, because different biological effects can be achieved, depending on the duration of the electric pulses. PEFs in the range of milliseconds to microseconds primarily act on the cell membrane and generate membrane pores, which are suited for introduction of exogenous macromolecules, such as plasmid DNA, into living cells [5, 6]. Thus, these PEFs

Recent advances in electric engineering allow us to generate ultrashort PEFs in the range of nanoseconds, which are referred to as nanosecond PEFs (nsPEFs). Although nsPEFs do not generate membrane pores suitable for DNA transfection, they have been proven to be useful for cancer therapy, because of their ability to induce cell death [8, 9]. Furthermore, nsPEFs have been shown to induce stress responses that are mediated by phosphorylation of multiple translation initiation factors and eventually lead to transient suppression of general protein synthesis. For these reasons, nsPEFs have received considerable attention as a potential therapeutic method with a novel mechanism of action. This review is intended to provide an overview of the stress responses induced by nsPEFs. First, two major mechanisms for stress responses in human cells are explained. Phosphorylation-mediated control of two translation initiation factors, eIF2α and 4E-BP1, is critical in these pathways. Second, an outline of the biological actions of PEFs is provided, with particular emphasis on nsPEFs. Finally, stress

The fundamental aspects of cellular stress responses are highly conserved among eukaryotes from yeast to humans. **Figure 1** shows a simplified scheme for eukaryotic stress responses. Under normal physiological conditions, cells continuously undergo protein synthesis, and the rate of protein synthesis is primarily regulated at the translation initiation step. When cells sense stress, they rapidly activate signal transduction that involves phosphorylation-mediated

events, such as metabolism, gene expression, proliferation, and cell death [1].

processes.

98 Protein Phosphorylation

are widely used for DNA transfection [7].

responses induced by nsPEFs are described in detail.

**2. Stress responses in human cells**

**2.1. Overview of stress response**

#### *2.2.1. Stress-induced eIF2α phosphorylation and translational suppression*

Translation initiation is a critical rate-limiting step in protein synthesis, and eukaryotic translation initiation factor 2 (eIF2) plays an essential role in this process [15]. eIF2 binds to guanine nucleotides, such as GDP and GTP. During initiation of translation, an eIF2-bound guanine nucleotide needs to cycle between GDP and GTP (see **Figure 2A** for details of GDP-GTP cycling in translation initiation). eIF2 consists of three subunits, α, β, and γ, and phosphorylation of the α subunit of eIF2 (eIF2α) is induced by various forms of stress [14, 16]. Phosphorylation of eIF2α at serine 51 interferes with GDP-GTP exchange on eIF2 [17] and consequently suppresses translation initiation (**Figure 2B**). The role of eIF2α phosphorylation in inhibition of translation initiation is highly conserved among eukaryotes, and the site of stress-induced phosphorylation (serine 51) is conserved in yeast and humans [13, 14].

**Figure 2.** Suppression of translation initiation by eIF2α phosphorylation. (A) Role of GDP-GTP exchange on eIF2 in translation initiation. eIF2 is a trimer composed of three subunits, α, β, and γ, and binds to guanine nucleotides, such as GTP and GDP. For translation initiation, GTP-bound eIF2 recruits the initiator methionyl-tRNA (Met-tRNAi) and in turn forms the 43S preinitiation complex with the small ribosomal subunit and the initiation factors eIF1, eIF1A, and eIF3. The 43S preinitiation complex is recruited to the 5′ end of mRNA, which is marked with a cap structure, and scans the 5′ untranslated region of mRNA for the initiation codon. During this process, GTP on eIF2 is hydrolyzed to GDP, and GDPbound eIF2 is released from the translation machinery. eIF2B, which has guanine exchange activity, replaces GDP on eIF2 with GTP, and GTP-bound eIF2 enters a new round of translation initiation [18, 19]. (B) Phosphorylation-mediated suppression of GDP-GTP exchange on eIF2. Under stressed conditions, serine 51 of eIF2α is rapidly phosphorylated by a stress-responsive protein kinase. Phosphorylation of eIF2α transforms eIF2 from a substrate into an inhibitor of eIF2B. eIF2B stalls on phosphorylated eIF2 and thus cannot exert its guanine exchange activity. Consequently, eIF2α phosphorylation leads to an increase in the GDP-bound form of eIF2, which is inactive for translation initiation, and results in attenuation of general protein synthesis [14, 16, 17].

Although initiation of translation of most mRNA species is dependent on their 5′ cap and is profoundly affected by eIF2α phosphorylation, a subset of mRNA species is actively translated under stressed conditions via alternative translation mechanisms that are independent of the cap structure. Approximately 2.5% of total mRNA is estimated to be preferentially translated under stressed conditions [20, 21], permitting synthesis of a subset of proteins that play critical roles in control of the stress response. For example, initiation of translation of ATF4 (activating transcription factor 4) mRNA is increased by ER stress caused by accumulation of unfolded proteins in the ER. The 5′ UTR of ATF4 mRNA contains two small open reading frames (upstream open reading frames [uORFs]), which play a critical role in initiation of translation under stressed conditions. ATF4 mRNA encodes a transcription factor that induces gene expression for adaptive responses. Thus, stress-induced eIF2α phosphorylation results in both suppression of general protein synthesis to conserve cellular resources and elevated translation of specific mRNA species for adaptive responses [22, 23].

In human cells, phosphorylation of eIF2α is induced by a wide variety of exogenous as well as endogenous stresses, such as amino acid deprivation, UV irradiation, and accumulation of unfolded proteins in the ER. Although these stresses are sensed by individual mechanisms, multiple stress-induced events converge on a single reaction, namely, eIF2α phosphorylation. Thus, eIF2α phosphorylation integrates various stress-induced signals. The stress-induced signaling pathway in human and mammalian cells that involves eIF2α phosphorylation and downstream ATF4 induction is referred to as the integrated stress response (**Figure 3**) [12, 24].

#### *2.2.2. Stress-responsive protein kinases*

Although initiation of translation of most mRNA species is dependent on their 5′ cap and is profoundly affected by eIF2α phosphorylation, a subset of mRNA species is actively translated under stressed conditions via alternative translation mechanisms that are independent of the cap structure. Approximately 2.5% of total mRNA is estimated to be preferentially translated under stressed conditions [20, 21], permitting synthesis of a subset of proteins that play critical roles in control of the stress response. For example, initiation of translation of ATF4 (activating transcription factor 4) mRNA is increased by ER stress caused by accumulation of unfolded proteins in the ER. The 5′ UTR of ATF4 mRNA contains two small open reading frames (upstream open reading frames [uORFs]), which play a critical role in initiation of translation under stressed conditions. ATF4 mRNA encodes a transcription factor that induces gene expression for adaptive responses. Thus, stress-induced eIF2α phosphorylation results in both suppression of general protein synthesis to conserve cellular resources and

**Figure 2.** Suppression of translation initiation by eIF2α phosphorylation. (A) Role of GDP-GTP exchange on eIF2 in translation initiation. eIF2 is a trimer composed of three subunits, α, β, and γ, and binds to guanine nucleotides, such as GTP and GDP. For translation initiation, GTP-bound eIF2 recruits the initiator methionyl-tRNA (Met-tRNAi) and in turn forms the 43S preinitiation complex with the small ribosomal subunit and the initiation factors eIF1, eIF1A, and eIF3. The 43S preinitiation complex is recruited to the 5′ end of mRNA, which is marked with a cap structure, and scans the 5′ untranslated region of mRNA for the initiation codon. During this process, GTP on eIF2 is hydrolyzed to GDP, and GDPbound eIF2 is released from the translation machinery. eIF2B, which has guanine exchange activity, replaces GDP on eIF2 with GTP, and GTP-bound eIF2 enters a new round of translation initiation [18, 19]. (B) Phosphorylation-mediated suppression of GDP-GTP exchange on eIF2. Under stressed conditions, serine 51 of eIF2α is rapidly phosphorylated by a stress-responsive protein kinase. Phosphorylation of eIF2α transforms eIF2 from a substrate into an inhibitor of eIF2B. eIF2B stalls on phosphorylated eIF2 and thus cannot exert its guanine exchange activity. Consequently, eIF2α phosphorylation leads to an increase in the GDP-bound form of eIF2, which is inactive for translation initiation, and

elevated translation of specific mRNA species for adaptive responses [22, 23].

results in attenuation of general protein synthesis [14, 16, 17].

100 Protein Phosphorylation

In human and mammalian cells, eIF2α is phosphorylated by four serine/threonine protein kinases, which are differentially activated by stress [14, 25]. These protein kinases show structural homology in their catalytic domains and are considered to exist in a monomeric form under unstressed conditions. Upon stress induction, they undergo homodimerization to become catalytically active, followed by autophosphorylation for full activation [26, 27].

PERK (protein kinase RNA-like endoplasmic reticulum kinase) is an ER transmembrane protein, and its N-terminal domain resides in the ER lumen and plays a role in sensing unfolded proteins. The C-terminal region of PERK is located in the cytoplasm and contains a kinase domain. PERK is activated by accumulation of unfolded proteins in the ER and in turn phosphorylates eIF2α.

GCN2 (general control nonderepressible 2) is critical for translational suppression under amino acid deprivation [28]. GCN2 binds to uncharged transfer RNAs and exerts its catalytic

**Figure 3.** Integrated stress response. Eukaryotic cells respond to various stresses by inducing eIF2α phosphorylation that leads to suppression of general protein synthesis. Human and mammalian cells possess four stress-responsive protein kinases: PERK, GCN2, PKR, and HRI. These protein kinases are differentially activated by stress and in turn phosphorylate eIF2α. eIF2α phosphorylation and downstream ATF4 induction are known as the integrated stress responses, because different cellular reactions induced by various external cues converge to these reactions. eIF2α phosphorylation inhibits cap-dependent initiation of translation and consequently suppresses general protein synthesis. A complex of GADD34 and PP1 dephosphorylates eIF2α and serves as a negative feedback mechanism for the integrated stress response.

activity for eIF2α phosphorylation. In addition to amino acid deprivation, GCN2 has been reported to be activated by UV irradiation [29] and proteasome inhibition [30], although the activation mechanisms remain elusive.

PKR (double-stranded RNA-dependent protein kinase) was originally identified as a protein kinase activated by double-stranded RNA, which emerges during viral infection [31]. Phosphorylation of eIF2α by PKR interferes with translation of viral mRNA and thus serves as an antiviral mechanism. In addition, activation of PKR is involved in the pathology of obesity [32] and cancer [33], suggesting various physiological roles of PKR.

Heme-regulated inhibitor (HRI) has physiological roles particularly in erythroid tissues [34]. HRI is activated by heme deprivation and phosphorylates eIF2α to reduce globin synthesis under low-iron conditions [35].

#### *2.2.3. Recovery from eIF2α phosphorylation-mediated translational suppression*

Once stressed conditions end, eIF2α must be dephosphorylated to restore general protein synthesis. GADD34 (growth arrest and DNA damage-inducible protein 34) is a critical regulator of eIF2α dephosphorylation, and its activity in the relief of stress responses is controlled at both the transcriptional and translational levels. Expression of *GADD34* gene is low under normal physiological conditions and is activated by various forms of stress. GADD34 mRNA is translated by a cap-independent mechanism, in which uORFs in the 5′ UTR of GADD34 mRNA play critical roles [36]. GADD34 protein forms a complex with protein phosphatase 1 (PP1), yielding a catalytically active protein phosphatase that specifically catalyzes dephosphorylation of eIF2α to relieve translational suppression [37]. Thus, GADD34 constitutes a negative feedback mechanism for eIF2α-mediated translational suppression.

#### **2.3. Stress response mediated by 4E-BP1 phosphorylation**

Human cells have an alternative mechanism for stress-induced translational suppression, which involves 4E-binding protein 1 (4E-BP1) [38]. As mentioned above, most mRNA species are translated in a cap-dependent manner. eIF4E binds to the cap structure of mRNA and in turn recruits eIF4G and other translation initiation factors, resulting in formation of an active translation initiation complex on the 5′ end of mRNA. 4E-BP1 serves as a negative regulator of this process. In unstressed conditions, 4E-BP1 is highly phosphorylated at multiple sites, suppressing its inhibitory activity. Under energy deprivation and other stressed conditions, phosphorylation of 4E-BP1 is substantially decreased, and 4E-BP1 competes with eIF4G for binding to eIF4E, thereby inhibiting translation initiation (**Figure 4**) [38].

Phosphorylation of 4E-BP1 is primarily controlled by mTORC1 (mammalian target of rapamycin complex 1), which is a member of the phosphatidylinositol 3-kinase-related family of kinases [39]. Under normal physiological conditions, mTORC1 is catalytically active and suppresses the inhibitory activity of 4E-BP1 by phosphorylation. The kinase activity of mTORC1 is regulated by several cellular proteins, one of which is AMP-activated protein kinase (AMPK). AMPK senses the cellular energy status and negatively regulates mTORC1 activity [40]. AMPK, mTORC1, and 4E-BP1 constitute a stress-responsive mechanism for translational

**Figure 4.** Control of translation initiation mediated by 4E-BP1 phosphorylation. Human cells possess an alternative mechanism for translational suppression, which is distinct from the eIF2α phosphorylation-mediated integrated stress response. eIF4E and other translation initiation factors form an active complex for initiation of translation at the cap structure of mRNA. 4E-BP1 serves as a negative regulator for the complex formation of eIF4E and other factors. (A) Under normal conditions, 4E-BP1 is highly phosphorylated by mTORC1 and sequestered from the translation initiation complex. (B) Under stressed conditions, such as energy deprivation, mTORC1 kinase activity is reduced, resulting in decreased 4E-BP1 phosphorylation, allowing inhibition of eIF4G-eIF4E binding.

suppression, independent of eIF2α phosphorylation. Thus, two mechanisms, the eIF2αmediated integrated stress response and the 4E-BP1-mediated mechanism, function in translational suppression in human cells (**Figure 4**).

#### **3. Pulsed electric fields as a novel physical tool in the life sciences**

#### **3.1. Effects of pulsed electric fields on living organisms**

activity for eIF2α phosphorylation. In addition to amino acid deprivation, GCN2 has been reported to be activated by UV irradiation [29] and proteasome inhibition [30], although the

PKR (double-stranded RNA-dependent protein kinase) was originally identified as a protein kinase activated by double-stranded RNA, which emerges during viral infection [31]. Phosphorylation of eIF2α by PKR interferes with translation of viral mRNA and thus serves as an antiviral mechanism. In addition, activation of PKR is involved in the pathology of obe-

Heme-regulated inhibitor (HRI) has physiological roles particularly in erythroid tissues [34]. HRI is activated by heme deprivation and phosphorylates eIF2α to reduce globin synthesis

Once stressed conditions end, eIF2α must be dephosphorylated to restore general protein synthesis. GADD34 (growth arrest and DNA damage-inducible protein 34) is a critical regulator of eIF2α dephosphorylation, and its activity in the relief of stress responses is controlled at both the transcriptional and translational levels. Expression of *GADD34* gene is low under normal physiological conditions and is activated by various forms of stress. GADD34 mRNA is translated by a cap-independent mechanism, in which uORFs in the 5′ UTR of GADD34 mRNA play critical roles [36]. GADD34 protein forms a complex with protein phosphatase 1 (PP1), yielding a catalytically active protein phosphatase that specifically catalyzes dephosphorylation of eIF2α to relieve translational suppression [37]. Thus, GADD34 constitutes a

Human cells have an alternative mechanism for stress-induced translational suppression, which involves 4E-binding protein 1 (4E-BP1) [38]. As mentioned above, most mRNA species are translated in a cap-dependent manner. eIF4E binds to the cap structure of mRNA and in turn recruits eIF4G and other translation initiation factors, resulting in formation of an active translation initiation complex on the 5′ end of mRNA. 4E-BP1 serves as a negative regulator of this process. In unstressed conditions, 4E-BP1 is highly phosphorylated at multiple sites, suppressing its inhibitory activity. Under energy deprivation and other stressed conditions, phosphorylation of 4E-BP1 is substantially decreased, and 4E-BP1 competes with eIF4G for

Phosphorylation of 4E-BP1 is primarily controlled by mTORC1 (mammalian target of rapamycin complex 1), which is a member of the phosphatidylinositol 3-kinase-related family of kinases [39]. Under normal physiological conditions, mTORC1 is catalytically active and suppresses the inhibitory activity of 4E-BP1 by phosphorylation. The kinase activity of mTORC1 is regulated by several cellular proteins, one of which is AMP-activated protein kinase (AMPK). AMPK senses the cellular energy status and negatively regulates mTORC1 activity [40]. AMPK, mTORC1, and 4E-BP1 constitute a stress-responsive mechanism for translational

sity [32] and cancer [33], suggesting various physiological roles of PKR.

*2.2.3. Recovery from eIF2α phosphorylation-mediated translational suppression*

negative feedback mechanism for eIF2α-mediated translational suppression.

binding to eIF4E, thereby inhibiting translation initiation (**Figure 4**) [38].

**2.3. Stress response mediated by 4E-BP1 phosphorylation**

activation mechanisms remain elusive.

102 Protein Phosphorylation

under low-iron conditions [35].

Pulsed electric fields (PEFs) refer to high-voltage electric pulses, which are milliseconds, microseconds, and nanoseconds in duration. PEFs have different effects on living organisms depending on pulse duration (**Figure 5**). PEFs with duration of milliseconds to microseconds primarily act on the cell membrane and cause pore formation. These membrane pores are suitable for transfer of macromolecules, such as plasmid DNA and drugs [5, 6]. Therefore, exposure of living cells to these PEFs is called electroporation and is commonly used for DNA transfection [7]. In addition, these PEFs are used for the introduction of antitumor drugs, which is called electrochemotherapy [41, 42]. Because PEFs with duration of milliseconds to microseconds primarily act on the cell membrane, these PEFs often induce cell death via cell membrane damage.

Recent advances in electrical engineering have enabled the generation of high-voltage electric pulses for ultrashort periods in the nanosecond range, which are called nanosecond PEFs

**Figure 5.** Comparison between electroporation and nsPEF action. PEFs have different effects on living organisms depending on pulse duration. (*Left*) PEFs with duration of milliseconds to microseconds are widely used for electroporation, because these PEFs primarily act on the cell membrane and generate membrane pores suited for macromolecule transfer.  Furthermore, nsPEFs have been suggested to directly affect intracellular components. Although nsPEFs are unsuitable for DNA transfection and cancer electrochemotherapy by electroporation, these PEFs can induce various cellular responses, including cell death induction.

(nsPEFs). It has become increasingly evident that nsPEFs have unique biological actions distinct from electroporation. The pulse duration of nsPEFs is too short to generate membrane pores large enough for entry of macromolecules. Thus, nsPEFs are generally unsuitable for DNA transfection and cancer electrochemotherapy. Instead, nsPEFs produce very small membrane pores that allow passage of small molecules, such as ions and water [43–45]. Accordingly, exposure of cultured human cells to nsPEFs causes Ca2+ influx and membrane blebbing due to ion imbalance across the cell membrane [46–48].

Whereas millisecond-to-microsecond PEFs primarily act on the cell membrane, previous theoretical studies have strongly suggested that nsPEFs exert their effects on both the cell membrane and intracellular components [49, 50]. In accordance, extensive biochemical analyses have proven that nsPEFs elicit various intracellular responses, as described below.

#### **3.2. Cellular responses to nsPEFs**

Recent studies have revealed that nsPEFs elicit different intracellular responses in a manner dependent on nsPEF intensity. **Figure 6** summarizes the relationship between nsPEF intensity and intracellular responses in human cells. Relatively weak nsPEFs do not cause morphological changes observable under a microscope, growth retardation, or cell death. However, cells rapidly respond to such stimuli by activating multiple intracellular signal pathways, including MAPK pathways [51, 52] and AMPK pathway [53]. Intracellular signaling is mediated by sequential phosphorylation of proteins in these pathways, leading to expression of downstream genes [51, 52]. When moderate-intensity nsPEFs are used, two independent mechanisms for stress responses are activated [54]. Intense nsPEFs efficiently induce cell death in vitro [8] as well as in vivo [9].

**Figure 6.** Relationship between nsPEF intensity and cellular responses. Exposure of human cells to nsPEFs causes different cellular responses, depending on intensity of nsPEFs. Relatively weak nsPEFs activate several signal transduction pathways, such as MAPK pathways, and their downstream gene expression without affecting on cell viability. Moderate-intensity nsPEFs elicit stress responses and cause growth retardation. Intense nsPEFs induce either apoptotic or necrotic cell death in a cell-type-dependent manner and can be used for cancer therapy.

When cultured human cells are exposed to intense nsPEFs, cell-type dependency of cell death modes has been observed. For example, apoptosis is induced in HL-60 and Jurkat cells by intense nsPEFs [8, 55, 56], whereas necrotic cell death is elicited in several cell lines, including U937, K562, and HeLa S3 [56, 57], demonstrating that the cellular context determines the mode of cell death. Induction of necrosis by intense nsPEFs is a Ca2+-dependent process [48, 58], while nsPEF-induced apoptosis is largely unaffected by the presence or absence of Ca2+ [58].

Because of the ability of nsPEFs to induce efficient cell death, many studies have attempted to apply nsPEFs to cancer therapy and have demonstrated their effectiveness in several mouse models [9, 59, 60]. Furthermore, a human clinical trial of nsPEFs for cancer therapy has started [61]. For these reasons, nsPEFs have attracted much interest and are increasingly recognized as a novel method of cancer therapy.

#### **4. Induction of stress responses by nsPEFs**

(nsPEFs). It has become increasingly evident that nsPEFs have unique biological actions distinct from electroporation. The pulse duration of nsPEFs is too short to generate membrane pores large enough for entry of macromolecules. Thus, nsPEFs are generally unsuitable for DNA transfection and cancer electrochemotherapy. Instead, nsPEFs produce very small membrane pores that allow passage of small molecules, such as ions and water [43–45]. Accordingly, exposure of cultured human cells to nsPEFs causes Ca2+ influx and membrane blebbing due to

**Figure 5.** Comparison between electroporation and nsPEF action. PEFs have different effects on living organisms depending on pulse duration. (*Left*) PEFs with duration of milliseconds to microseconds are widely used for electroporation, because these PEFs primarily act on the cell membrane and generate membrane pores suited for macromolecule transfer.  Furthermore, nsPEFs have been suggested to directly affect intracellular components. Although nsPEFs are unsuitable for DNA transfection and cancer electrochemotherapy by electroporation, these PEFs can induce various

Whereas millisecond-to-microsecond PEFs primarily act on the cell membrane, previous theoretical studies have strongly suggested that nsPEFs exert their effects on both the cell membrane and intracellular components [49, 50]. In accordance, extensive biochemical analyses

Recent studies have revealed that nsPEFs elicit different intracellular responses in a manner dependent on nsPEF intensity. **Figure 6** summarizes the relationship between nsPEF intensity and intracellular responses in human cells. Relatively weak nsPEFs do not cause morphological changes observable under a microscope, growth retardation, or cell death. However, cells rapidly respond to such stimuli by activating multiple intracellular signal pathways, including MAPK pathways [51, 52] and AMPK pathway [53]. Intracellular signaling is mediated by sequential phosphorylation of proteins in these pathways, leading to expression of downstream genes [51, 52]. When moderate-intensity nsPEFs are used, two independent mechanisms for stress responses are activated [54]. Intense nsPEFs efficiently induce cell death

have proven that nsPEFs elicit various intracellular responses, as described below.

ion imbalance across the cell membrane [46–48].

cellular responses, including cell death induction.

104 Protein Phosphorylation

**3.2. Cellular responses to nsPEFs**

in vitro [8] as well as in vivo [9].

Previously, nsPEFs have been shown to activate two independent stress-responsive pathways, both of which are mediated by protein phosphorylation and result in translational suppression. First, nsPEFs induce eIF2α phosphorylation via two protein kinases, PERK and GCN2. Concomitant with eIF2α phosphorylation, GADD34 expression is elevated. Second, nsPEFs cause decreased phosphorylation of 4E-BP1, which is presumably controlled by mTORC1. Following induction of these phosphorylation-mediated stress responses, general protein synthesis is markedly reduced in nsPEF-exposed cells. **Figure 7** represents a summary of nsPEF-induced stress responses.

#### **4.1. eIF2α phosphorylation induced by nsPEFs**

Most mRNA species are translated in a cap-dependent manner, and eIF2 plays a critical role in this process. Under stressed conditions, the activity of eIF2 for translation initiation is suppressed by phosphorylation of its α subunit (eIF2α) [14]. eIF2α phosphorylation is considered to be a hallmark of induction of the integrated stress response and can be examined by

**Figure 7.** Stress responses induced by nsPEFs. nsPEFs affect multiple stress-responsive proteins in the two independent signaling pathways, both of which result in suppression of general protein synthesis. The first signaling pathway induced by nsPEFs is mediated by eIF2α phosphorylation. nsPEFs activate PERK and GCN2, which in turn phosphorylate eIF2α. eIF2α phosphorylation interferes GDP-GTP exchange in translation initiation, resulting in suppression of general protein synthesis. The second pathway induced by nsPEFs involves a reduction in 4E-BP1 phosphorylation. 4E-BP1 is highly phosphorylated by mTORC1 under normal conditions. nsPEFs cause a decrease in 4E-BP1 phosphorylation, leading to inhibition of eIF4G-eIF4E complex formation and consequent suppression of translation initiation. nsPEFs are known to activate AMPK, which is known to function as a negative regulator of mTORC1 under energy deprivation conditions. AMPK activation may account for the decrease in 4E-BP1 phosphorylation following nsPEF exposure. Direct effects of nsPEFs on mTORC1 will be examined by future research. Increased eIF2α phosphorylation and decreased 4E-BP1 phosphorylation serve as two independent mechanisms for the suppression of general protein synthesis.

Western blotting using an antibody specific to phosphorylated eIF2α. When cultured cells are exposed to nsPEFs, eIF2α phosphorylation can be detected, indicating that nsPEFs activate the integrated stress response [54]. nsPEF-induced eIF2α phosphorylation can be detected in all cell lines examined so far, which include HeLa S3, HCT116, Jurkat, and mouse embryonic fibroblasts (MEFs). In HeLa S3 cells, eIF2α phosphorylation is detectable within 1 min after nsPEF exposure, persists at high levels for 30 min, and decreases thereafter [54]. GADD34 is known to play a critical role in recovery from the integrated stress response. Under stressed conditions, GADD34 is positively controlled at the transcriptional and translational levels. Consistently, expression of GADD34 is significantly activated in nsPEF-exposed cells, suggesting that GADD34 is involved in recovery from the nsPEF-induced stress response [54].

As described above, cellular responses to nsPEFs are dependent on nsPEF intensity (**Figure 6**). Relatively mild nsPEFs activate several signal transduction pathways, such as MAPK pathways, but are insufficient to induce eIF2α phosphorylation. Moderate levels of nsPEF intensity are required for induction of eIF2α phosphorylation. Such nsPEFs also cause retardation in cell proliferation but not cell death [54]. Intense nsPEFs induce eIF2α phosphorylation and cell death. Currently, it remains unknown whether the nsPEF-induced stress response positively affects cell survival or facilitates cell death induction.

#### **4.2. Participation of PERK and GCN2 in nsPEF-induced eIF2α phosphorylation**

Human and mammalian cells have four stress-responsive protein kinases for eIF2α phosphorylation. These kinases differentially respond to various forms of stress, and at least one of them is activated for eIF2α phosphorylation [14]. Autophosphorylation is critical for the activation of these kinases and can be analyzed by Western blot analysis using antibodies against phosphorylated forms of these kinases. In nsPEF-exposed cells, PERK and GCN2 are activated, as shown by their autophosphorylation [54]. Experiments using PERK and GCN2 knockout cells suggest that these kinases play mutually compensatory roles in nsPEF-induced eIF2α phosphorylation. MEFs lacking either *PERK* or *GCN2* gene display nsPEF-induced eIF2α phosphorylation comparable to that in wild-type cells. However, double-knockout cells lacking both *PERK* and *GCN2* genes exhibit a significant reduction in nsPEF-induced eIF2α phosphorylation [54]. These observations suggest that PERK and GCN2 perform redundant functions in nsPEF-induced eIF2α phosphorylation.

Because PERK is well known to be activated by ER stress, the observation on the nsPEF-induced PERK activation raised the possibility that nsPEFs cause ER stress. To clarify this point, downstream events in the ER stress pathway were analyzed [54]. The signal pathway induced by ER stress generally leads to alterations of gene expression [62]. Transcription of *CHOP* (CCAAT-enhancer-binding protein homologous protein) gene is a major downstream event in the ER stress response. In addition, mRNA for XBP1 (X-box-binding protein 1) is known to undergo alternative splicing after ER stress induction [63]. However, quantitative RT-PCR analysis of *CHOP* and *XBP1* mRNAs demonstrated that nsPEF-exposed cells showed neither elevated CHOP expression nor altered XBP1 splicing. Furthermore, UV irradiation has been reported to induce activation of GCN2 and transcription of downstream genes, such as *GADD45* (growth arrest and DNA damage-inducible 45) [64, 65], but quantitative RT-PCR analysis showed no substantial changes in these mRNAs in nsPEF-exposed cells. These observations indicate that nsPEFs exert their effects in a manner that is distinct from ER stress or UV irradiation, although nsPEFs activate PERK and GCN2 [54].

#### **4.3. Decreased 4E-BP1 phosphorylation by nsPEFs**

Western blotting using an antibody specific to phosphorylated eIF2α. When cultured cells are exposed to nsPEFs, eIF2α phosphorylation can be detected, indicating that nsPEFs activate the integrated stress response [54]. nsPEF-induced eIF2α phosphorylation can be detected in all cell lines examined so far, which include HeLa S3, HCT116, Jurkat, and mouse embryonic fibroblasts (MEFs). In HeLa S3 cells, eIF2α phosphorylation is detectable within 1 min after nsPEF exposure, persists at high levels for 30 min, and decreases thereafter [54]. GADD34 is known to play a critical role in recovery from the integrated stress response. Under stressed conditions, GADD34 is positively controlled at the transcriptional and translational levels. Consistently, expression of GADD34 is significantly activated in nsPEF-exposed cells, suggesting that GADD34 is involved in recovery from the nsPEF-induced stress response [54].

phosphorylation serve as two independent mechanisms for the suppression of general protein synthesis.

106 Protein Phosphorylation

**Figure 7.** Stress responses induced by nsPEFs. nsPEFs affect multiple stress-responsive proteins in the two independent signaling pathways, both of which result in suppression of general protein synthesis. The first signaling pathway induced by nsPEFs is mediated by eIF2α phosphorylation. nsPEFs activate PERK and GCN2, which in turn phosphorylate eIF2α. eIF2α phosphorylation interferes GDP-GTP exchange in translation initiation, resulting in suppression of general protein synthesis. The second pathway induced by nsPEFs involves a reduction in 4E-BP1 phosphorylation. 4E-BP1 is highly phosphorylated by mTORC1 under normal conditions. nsPEFs cause a decrease in 4E-BP1 phosphorylation, leading to inhibition of eIF4G-eIF4E complex formation and consequent suppression of translation initiation. nsPEFs are known to activate AMPK, which is known to function as a negative regulator of mTORC1 under energy deprivation conditions. AMPK activation may account for the decrease in 4E-BP1 phosphorylation following nsPEF exposure. Direct effects of nsPEFs on mTORC1 will be examined by future research. Increased eIF2α phosphorylation and decreased 4E-BP1

As described above, cellular responses to nsPEFs are dependent on nsPEF intensity (**Figure 6**). Relatively mild nsPEFs activate several signal transduction pathways, such as MAPK pathways, but are insufficient to induce eIF2α phosphorylation. Moderate levels of nsPEF intensity are required for induction of eIF2α phosphorylation. Such nsPEFs also cause retardation in cell proliferation but not cell death [54]. Intense nsPEFs induce eIF2α phosphorylation and cell death. Currently, it remains unknown whether the nsPEF-induced stress response posi-

Human and mammalian cells have four stress-responsive protein kinases for eIF2α phosphorylation. These kinases differentially respond to various forms of stress, and at least one

**4.2. Participation of PERK and GCN2 in nsPEF-induced eIF2α phosphorylation**

tively affects cell survival or facilitates cell death induction.

In addition to the eIF2α-mediated response, a distinct mechanism involving 4E-BP1 phosphorylation is known to play a critical role in stress-induced translational suppression [38]. Under normal physiological conditions, 4E-BP1 is highly phosphorylated by mTORC1 [39], and hyperphosphorylation suppresses its inhibition of cap-dependent translation initiation. Stressed conditions, particularly energy deprivation, reduce mTORC1 activity and result in decreased 4E-BP1 phosphorylation. 4E-BP1 at low phosphorylation status interferes with assembly of translation factors on the cap structure of mRNAs and thereby suppresses general protein synthesis (**Figure 4**). In nsPEF-exposed cells, 4E-BP1 phosphorylation is rapidly decreased [54], suggesting that nsPEFs activate stress responses mediated by 4E-BP1 phosphorylation. The decrease in 4E-BP1 phosphorylation is indistinguishable between wild-type cells and *PERK/GCN2* double-knockout cells, supporting the idea that nsPEFs activate two independent mechanisms.

The decrease in 4E-BP1 phosphorylation following nsPEF exposure suggests that nsPEFs cause a reduction in the catalytic activity of mTORC1. A previous study demonstrated that AMPK is rapidly activated by nsPEFs [53]. AMPK functions as an energy sensor and is activated by elevated intracellular AMP levels, which are primarily caused by energy deprivation [40]. The catalytic activity of mTORC1 is well known to be negatively regulated by AMPK [39]. A previous study has demonstrated that, concomitant with AMPK activation, nsPEFs induce phosphorylation of AMPK substrates, such as acetyl-CoA carboxylase-2 [53], suggesting that nsPEF-activated AMPK also phosphorylates other substrates, including mTORC1. Although mTORC1 catalytic activity has not been examined in nsPEF-exposed cells yet, the above observations suggest that AMPK downregulates mTORC1, leading to reduced 4E-BP1 phosphorylation in nsPEF-exposed cells (**Figure 7**).

#### **4.4. Suppression of general protein synthesis by nsPEFs**

As described above, nsPEFs cause elevated eIF2α phosphorylation and decrease 4E-BP1 phosphorylation, both of which are known to be involved in suppression of general protein synthesis. To test whether exposure to nsPEFs actually leads to translational suppression, measurement of protein synthesis rates was required. To this end, metabolic labeling of newly synthesized proteins with radioactive amino acids was employed [54]. **Figure 8** shows an outline of metabolic labeling using 35S-labeled amino acids. Using this method, suppression of general protein synthesis in nsPEF-exposed cells was demonstrated [54]. After nsPEF exposure, overall protein synthesis quickly decreased, and maximum suppression of protein synthesis was observed at 30 min. Protein synthesis in nsPEF-exposed cells recovered to approximately 80% within 2 h. When cells were treated with UV irradiation, general protein synthesis decreased gradually for several hours. Compared to UV irradiation, nsPEFs cause acute translational suppression, and recovery is more rapid than in UV-irradiated cells.

**Figure 8.** Measurement of protein synthesis rates by metabolic labeling of newly synthesized proteins with radioactive amino acids. A rate of protein synthesis can be measured as incorporation of radioactive amino acids into cellular proteins. Following appropriate treatment, such as nsPEF exposure, cells are incubated in culture medium containing 35S-labeled methionine and cysteine. During incubation, cells use radioactive amino acids to synthesize proteins, yielding <sup>35</sup>S-labeled proteins. Following preparation of whole-cell lysate, the protein fraction is separated from the free amino acids, and the radioactivity incorporated into the proteins is quantified by liquid scintillation counting.

#### **5. Conclusion**

Exposure of cultured human cells to nsPEFs elicits two distinct stress responses, both of which are controlled by phosphorylation of translation initiation factors. nsPEFs rapidly induce eIF2α phosphorylation and concomitant activation of the stress-responsive kinases, PERK and GCN2. In addition, nsPEFs cause decreased 4E-BP1 phosphorylation and AMPK activation, which appear to constitute a stress response pathway involving mTORC1. nsPEFs elicit acute suppression of general protein synthesis via two reactions for inhibition of translation initiation. Collectively, these findings clearly indicate that nsPEFs act as a novel form of cellular stress and suppress general protein synthesis.

Although the identification of key events in nsPEF-induced stress responses has significantly advanced our understanding of the biological effects of nsPEFs, several critical questions remain to be elucidated. First, the site of action of nsPEFs for eIF2α-mediated stress response is currently unclear. Because PERK and GCN2 are the most upstream molecules in their signaling pathways, nsPEFs may act directly on these kinases, causing eIF2α phosphorylation. Second, the decrease in 4E-BP1 phosphorylation and the activation of AMPK strongly suggest that mTORC1 participates in the nsPEF-induced stress response, because energy deprivation sequentially causes AMPK activation, reduced mTORC1 activity, and consequent decreased phosphorylation of 4E-BP1. To test this idea, the relationships among AMPK, mTORC1, and 4E-BP1 in nsPEF-exposed cells should be investigated in detail. Furthermore, analysis of effects of nsPEFs on cellular energy levels is also important for understanding how nsPEFs control 4E-BP1 phosphorylation.

Finally, the significance of nsPEF-induced stress responses for cell survival should be determined. Suppression of general protein synthesis under stressed conditions conserves biological resources and is regarded as an important mechanism for cell survival [2]. However, prolonged activation of stress responses often has an opposite effect, facilitating the induction of cell death, presumably because elimination of overstressed cells is beneficial for the body [10, 14]. Currently, it remains unclear whether nsPEF-induced stress responses serve as a prosurvival mechanism or serve to facilitate cell death induction. Future efforts will focus on understanding the contribution of nsPEF-induced stress responses to cell survival and death. Previous studies have revealed the unique effects of nsPEFs as a novel form of cellular stress. More detailed understanding of the molecular mechanisms and biological importance of nsPEF-induced stress responses will pave the way toward more effective applications of this novel technology in a wide range of biomedical sciences.

#### **Acknowledgements**

[40]. The catalytic activity of mTORC1 is well known to be negatively regulated by AMPK [39]. A previous study has demonstrated that, concomitant with AMPK activation, nsPEFs induce phosphorylation of AMPK substrates, such as acetyl-CoA carboxylase-2 [53], suggesting that nsPEF-activated AMPK also phosphorylates other substrates, including mTORC1. Although mTORC1 catalytic activity has not been examined in nsPEF-exposed cells yet, the above observations suggest that AMPK downregulates mTORC1, leading to reduced 4E-BP1

As described above, nsPEFs cause elevated eIF2α phosphorylation and decrease 4E-BP1 phosphorylation, both of which are known to be involved in suppression of general protein synthesis. To test whether exposure to nsPEFs actually leads to translational suppression, measurement of protein synthesis rates was required. To this end, metabolic labeling of newly synthesized proteins with radioactive amino acids was employed [54]. **Figure 8** shows an outline of metabolic labeling using 35S-labeled amino acids. Using this method, suppression of general protein synthesis in nsPEF-exposed cells was demonstrated [54]. After nsPEF exposure, overall protein synthesis quickly decreased, and maximum suppression of protein synthesis was observed at 30 min. Protein synthesis in nsPEF-exposed cells recovered to approximately 80% within 2 h. When cells were treated with UV irradiation, general protein synthesis decreased gradually for several hours. Compared to UV irradiation, nsPEFs cause acute translational suppression, and recovery is more rapid than in UV-irradiated cells.

Exposure of cultured human cells to nsPEFs elicits two distinct stress responses, both of which are controlled by phosphorylation of translation initiation factors. nsPEFs rapidly induce eIF2α phosphorylation and concomitant activation of the stress-responsive kinases,

**Figure 8.** Measurement of protein synthesis rates by metabolic labeling of newly synthesized proteins with radioactive amino acids. A rate of protein synthesis can be measured as incorporation of radioactive amino acids into cellular proteins. Following appropriate treatment, such as nsPEF exposure, cells are incubated in culture medium containing 35S-labeled methionine and cysteine. During incubation, cells use radioactive amino acids to synthesize proteins, yielding <sup>35</sup>S-labeled proteins. Following preparation of whole-cell lysate, the protein fraction is separated from the free amino

acids, and the radioactivity incorporated into the proteins is quantified by liquid scintillation counting.

phosphorylation in nsPEF-exposed cells (**Figure 7**).

108 Protein Phosphorylation

**5. Conclusion**

**4.4. Suppression of general protein synthesis by nsPEFs**

This work was supported by JSPS KAKENHI grant numbers 26350540 and 16H02311 and by a grant from the SEI Group CSR Foundation.

#### **Author details**

Ken-ichi Yano\* and Keiko Morotomi-Yano

\*Address all correspondence to<EMAIL_ADDRESS>
Institute of Pulsed Power Science, Kumamoto University, Kumamoto, Japan

#### **References**

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112 Protein Phosphorylation

**Provisional chapter**

#### **Modification of α-Synuclein by Phosphorylation: A Pivotal Event in the Cellular Pathogenesis of Parkinson's Disease Modification of α-Synuclein by Phosphorylation: A Pivotal Event in the Cellular Pathogenesis of Parkinson's Disease**

DOI: 10.5772/intechopen.70405

Indrani Datta and Kavina Ganapathy Indrani Datta and Kavina Ganapathy Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70405

#### **Abstract**

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Post-translational protein modifications play an important role in generating the large diversity of the proteome in comparison to the relatively small number of genes; phosphorylation being the most widespread. Phosphorylation of proteins regulates important molecularswitches for several cellular events and abnormal phosphorylation events are associated in many neurodegenerative diseases. In Parkinson's disease (PD) the main hallmark is the accumulation of cytoplasmic inclusions, Lewy bodies (LBs), consisting of α-synuclein (α-Syn) and ubiquitin. There's another key observation which is increasingly gaining prominence is a modified-form of α-Syn; the phospho α-Syn serine129 (pSyn). The significance of pSyn has gained importance in PD because its accumulation is distinctly enhanced in the diseased condition. The revelation of the involvement of pSyn on α-Syn aggregation, LB formation and neurotoxicity is crucial to understanding the pathogenesis and progression of PD. Since some *in vitro* and *in vivo* studies have indicated that pSyn is an early event preceding apoptosis, some important questions now needs to be explored in reference to the physiological functions regulated by phosphorylation, such as dopamine synthesis, vesicle mobilization, regulation of synaptic proteins, and synaptic plasticity. An investigation of the role of enzymes on the phosphorylation and clearance of α-Syn and region-specific susceptibility is required to be determined; to identify viable targets for new therapeutics.

**Keywords:** α-synuclein, phospho α-synuclein serine129, PD, phosphorylation, kinases, aggregation, biomarker, neurotoxicity, Lewy bodies

#### **1. Introduction**

Parkinson's disease (PD) is the second most common diagnosed neurodegenerative disease [1] with a prevalence of about 1% at the age of 65 and of 4–5% by the age of 85 [2]. The clinical

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

manifestations of classical PD are rest tremor, rigidity, bradykinesia, and postural imbalance. The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) together with a distinct decrease in striatal dopamine, and the occurrence of cytoplasmic eosinophilic inclusions called Lewy bodies (LBs) are considered the pathological hallmarks of PD [3]. There are also reports of other neuronal cell losses in locus coeruleus and olfactory lobe during the development of the disease [4]. It has however been identified that the clinical manifestation of motor symptoms appears with the loss of DA neurons in the midbrain [5]. According to Rodriguez-Oroz et al. [6], the anatomical-functional basis of the main clinical manifestations is related to the low level of dopamine concentration in contralateral striatum and the malfunction of dopamine circuits. Current medications for PD supplement dopamine (L-DOPA), or activate DA receptors (DA-receptor agonist), or inhibit the degradation of DA (monoamine oxidase B inhibitor and catechol-O-methyltransferase inhibitor), bringing about temporary abatement of motor symptoms but failing to delay or halt disease progression. The etiology of PD is still unknown: it comprises familial (fPD) forms accounting for less than 10% of all PD cases, and the far more common sporadic (sPD) form. The striking feature in both fPD and sPD is α-synuclein (α-Syn) aggregation with ubiquitin that eventually progresses to form LBs. Three missense mutations (A53T, A30P, and E46K) [7–9] of α-Syn are known to cause autosomal dominant fPD [10], probably through a gain-of-function mechanism. Moreover, overexpression of human α-Syn in mice results in progressive loss of DA terminals in the basal ganglia and accumulation of LB-like structures in neurons [11]. Mechanisms that might control α-Syn aggregation in sPD are not clear, but may include transcription factor dysregulation [12] and the inability of normal degradation pathways to function adequately [13]. El-Agnaf et al. [14] detected α-Syn species in live-human sPD and fPD patient plasma and cerebrospinal fluid.

#### **2. α-Syn structure and function**

Syn are a vertebrate-specific family of abundant neuronal proteins. They consist of three closely related members, α-,β-,and γ-Syn, of which α-Syn has been the prime focus ever since mutations in it were recognized as a basis for fPD. Syn is a highly conserved protein with a molecular weight of approximately 14 kDa, comprising 140 amino acids [15]. This heatresistant, soluble, acidic protein is abundant in the presynaptic terminals of central nervous system (CNS) neurons expressed pre-dominantly in the neocortex, hippocampus, SNpc, thalamus, and cerebellum [16–18]. Unlike α- and β-synuclein, γ-Syn is not concentrated in presynaptic terminals [19] and is largely found in the peripheral nervous system (PNS).

α-Syn is composed of three distinct domains: an N-terminal amphipathic repeat region that can form α-helices; a hydrophobic central segment; and a C-terminal acidic region (**Figure 1**). The highly conserved N-terminal domain (residues 1–65) includes 6 copies of an unusual 11 amino acid repeat that display variations of a KTKEGV consensus sequence. It is unordered in solution, but can shift to a α-helical conformation [20] comprising two distinct α-helices Modification of α-Synuclein by Phosphorylation: A Pivotal Event in the Cellular Pathogenesis... http://dx.doi.org/10.5772/intechopen.70405 117

manifestations of classical PD are rest tremor, rigidity, bradykinesia, and postural imbalance. The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) together with a distinct decrease in striatal dopamine, and the occurrence of cytoplasmic eosinophilic inclusions called Lewy bodies (LBs) are considered the pathological hallmarks of PD [3]. There are also reports of other neuronal cell losses in locus coeruleus and olfactory lobe during the development of the disease [4]. It has however been identified that the clinical manifestation of motor symptoms appears with the loss of DA neurons in the midbrain [5]. According to Rodriguez-Oroz et al. [6], the anatomical-functional basis of the main clinical manifestations is related to the low level of dopamine concentration in contralateral striatum and the malfunction of dopamine circuits. Current medications for PD supplement dopamine (L-DOPA), or activate DA receptors (DA-receptor agonist), or inhibit the degradation of DA (monoamine oxidase B inhibitor and catechol-O-methyltransferase inhibitor), bringing about temporary abatement of motor symptoms but failing to delay or halt disease progression. The etiology of PD is still unknown: it comprises familial (fPD) forms accounting for less than 10% of all PD cases, and the far more common sporadic (sPD) form. The striking feature in both fPD and sPD is α-synuclein (α-Syn) aggregation with ubiquitin that eventually progresses to form LBs. Three missense mutations (A53T, A30P, and E46K) [7–9] of α-Syn are known to cause autosomal dominant fPD [10], probably through a gain-of-function mechanism. Moreover, overexpression of human α-Syn in mice results in progressive loss of DA terminals in the basal ganglia and accumulation of LB-like structures in neurons [11]. Mechanisms that might control α-Syn aggregation in sPD are not clear, but may include transcription factor dysregulation [12] and the inability of normal degradation pathways to function adequately [13]. El-Agnaf et al. [14] detected α-Syn species in live-human sPD and fPD patient plasma and

Syn are a vertebrate-specific family of abundant neuronal proteins. They consist of three closely related members, α-,β-,and γ-Syn, of which α-Syn has been the prime focus ever since mutations in it were recognized as a basis for fPD. Syn is a highly conserved protein with a molecular weight of approximately 14 kDa, comprising 140 amino acids [15]. This heatresistant, soluble, acidic protein is abundant in the presynaptic terminals of central nervous system (CNS) neurons expressed pre-dominantly in the neocortex, hippocampus, SNpc, thalamus, and cerebellum [16–18]. Unlike α- and β-synuclein, γ-Syn is not concentrated in presynaptic terminals [19] and is largely found in the peripheral nervous system (PNS).

α-Syn is composed of three distinct domains: an N-terminal amphipathic repeat region that can form α-helices; a hydrophobic central segment; and a C-terminal acidic region (**Figure 1**). The highly conserved N-terminal domain (residues 1–65) includes 6 copies of an unusual 11 amino acid repeat that display variations of a KTKEGV consensus sequence. It is unordered in solution, but can shift to a α-helical conformation [20] comprising two distinct α-helices

cerebrospinal fluid.

116 Protein Phosphorylation

**2. α-Syn structure and function**

**Figure 1.** Schematic representation of α-Syn amino acid amino acid sequence (1–140) with amphipathic NH2 -terminal, non-amyloid component (NAC) region and acidic tail –COOH terminal. Arrows in the N-terminal, points to the three pathogenic mutations, and P in a red circle of NAC and C-terminal, points to the sites of phosphorylations.

interrupted by a short break [21]. α-Syn binds strongly to negatively charged phospholipids and becomes α-helical [22, 23], suggesting that the protein may normally be associated with the membrane [24]. This N-terminal domain includes the three sites of the fPD mutations A30P, E46K, and A53T (**Figure 1**).

The hydrophobic central segment of α-Syn (non-amyloid component (NAC), residues 66–95) (**Figure 1**) [18] is the second major component of brain amyloid plaques in Alzheimer's disease (AD) [18, 24]. This region consists of three repeats including the highly amyloidogenic part of the molecule that is responsible for α-Syn's ability to undergo a conformational change from random coil to β-sheet structure [25] and to form Aβ-like protofibrils and fibrils [24, 25]. These properties differentiate α-Syn from β-Syn and γ-Syn, which fail to form co-polymers with α-Syn [24]. The NAC region carries a phosphorylation site on Ser87 [26].

The acidic C-terminal domain (residues 96–140) of α-Syn has a strong negative charge composed primarily of acidic amino acids [20], but has no known structural elements. It consists of an acidic domain rich in proline residues (residues 125–140) that seems critical for the chaperone-like activity of α-Syn [27], as demonstrated by deletion mutants of the C-terminal region in which the α-Syn chaperone activity is lost [27–29]. In contrast to the amphipathic N-terminal and hydrophobic NAC regions, which are highly conserved between species, the C-terminal region is variable in size and in sequence [28–31]. This region is also organized in random structure in most conditions and contains several phosphorylation sites: Ser129, Tyr125, Tyr133, and Tyr136 [32] (**Figure 1**).

Although the normal functions of α-Syn are still being defined, several studies have shown that this protein has a key role to play in membrane-associated processes at the presynaptic level such as formation and maintenance of synaptic vesicle pools (**Figure 2**), regulation of lipid metabolism, and Ca2+ homeostasis [31–33]. Greten-Harrison et al. [34] using αβγ-Syn knockout mice has reported that deletion of Syns causes alterations in synaptic structure and transmission, age-dependent neuronal dysfunction, as well as diminished survival. *In vivo* and *in vitro* studies showed that abrogation of Syn expression decreased excitatory synapse size by ∼30%, revealing that Syns are important determinants of presynaptic terminal size [34]. Younger synuclein-null mice show better basic transmission in comparison to older mice that showed a pronounced deterioration. Interestingly, it is further reported that the late onset phenotypes in Syn-null mice were not due to a loss of synapses or neurons but rather a reflection of specific changes in synaptic protein composition and axonal structure.

**Figure 2.** Schematic representation of roles of α-Syn under physiological and pathological conditions. In physiological condition, (1) α-Syn maintains synaptic functions by associating with (2) vesicle formation, (3) trafficking and (4) docking. It also associates with (5) recycling of synaptic vesicle and (6) dopamine storage. The post-translational modification of α-Syn such as (7) phosphorylation and dephosphorylation, leads to activation and deactivation of the protein. This protein undergoes (8) lysosome autophagy (9) and (10) proteosomal degradation directed by ubiquitination. However, in pathological conditions (1) due to uncertain stimulus, α-Syn undergoes (2) misfolding, (3) mutation or (4) phosphorylation leading to the (5) aggregation of the protein affecting the (6) vesicle formation, trafficking and docking, (7) impaired lysosomal autophagy, (8) and (9) ubiquitination and inhibition of proteosomal degradation. This in turn results in (10) LB formation and (11) apoptosis.

Chandra et al. [21] found selective decreases in two small synaptic signaling proteins, complexins, and 14-3-3 proteins, in α,β double-KO mice. In 2000, Abeliovich et al. [2] have shown that mice lacking α-Syn display functional deficits in the nigrostriatal dopamine system. The α-Syn−/− mice were reported to be viable and fertile, exhibited intact brain (Aβ) architecture, and possessed normal complement of DA cell bodies, fibers, and synapses; however they displayed a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine [2]. Further Drolet et al.'s [35] work showed that mice lacking α-Syn have an attenuated loss of striatal dopamine following prolonged chronic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration. In addition, ultrastructural analysis and imaging studies have shown reduced synaptic vesicle density at the active zone, and imaging further reveals a defect in the re-clustering of synaptic vesicles after endocytosis [36]. Increased levels of α-Syn thus produce specific, physiological defects in synaptic vesicle recycling that precedes detectable neuropathology.

α-Syn also has been suggested to function as a chaperone protein *in vivo*, because it appears capable of interacting with a variety of ligands and cellular proteins apart from lipids [35, 37, 38], thus modifying their activities. The N-terminal portion of α-Syn shares 40% amino acid homology with molecular chaperone 14-3-3 [36], suggesting that the two proteins could sub-serve the same function. Eliezer et al. [23] had showed that the removal of the C-terminal acidic tail of α-Syn abolished its chaperone activity. In contrast, some reports indicate that the C-terminal acidic tail is indeed necessary but not sufficient for the chaperone function of α-Syn [28, 29]. In normal physiological conditions, α-Syn exists in monomeric form and is recognized and cleared via the ubiquitin-proteasome system (UPS) and chaperone-mediated autophagy (CMA) pathways [39] (**Figure 2**). In the pathological state, misfolding or mutations of α-Syn (A30P/A53T) lead to the formation of pathologically modified species that bind with several cytoplasmic proteins and ultimately aggregate into LBs in the DA neuronal cells [40] (**Figure 2**). This aberrant level of α-Syn is also cited in idiopathic PD subjects [41].

#### **2.1. Phosphorylation of α-synuclein at serine129**

Although the normal functions of α-Syn are still being defined, several studies have shown that this protein has a key role to play in membrane-associated processes at the presynaptic level such as formation and maintenance of synaptic vesicle pools (**Figure 2**), regulation of lipid metabolism, and Ca2+ homeostasis [31–33]. Greten-Harrison et al. [34] using αβγ-Syn knockout mice has reported that deletion of Syns causes alterations in synaptic structure and transmission, age-dependent neuronal dysfunction, as well as diminished survival. *In vivo* and *in vitro* studies showed that abrogation of Syn expression decreased excitatory synapse size by ∼30%, revealing that Syns are important determinants of presynaptic terminal size [34]. Younger synuclein-null mice show better basic transmission in comparison to older mice that showed a pronounced deterioration. Interestingly, it is further reported that the late onset phenotypes in Syn-null mice were not due to a loss of synapses or neurons but rather a reflection of specific changes in synaptic protein composition and axonal structure.

**Figure 2.** Schematic representation of roles of α-Syn under physiological and pathological conditions. In physiological condition, (1) α-Syn maintains synaptic functions by associating with (2) vesicle formation, (3) trafficking and (4) docking. It also associates with (5) recycling of synaptic vesicle and (6) dopamine storage. The post-translational modification of α-Syn such as (7) phosphorylation and dephosphorylation, leads to activation and deactivation of the protein. This protein undergoes (8) lysosome autophagy (9) and (10) proteosomal degradation directed by ubiquitination. However, in pathological conditions (1) due to uncertain stimulus, α-Syn undergoes (2) misfolding, (3) mutation or (4) phosphorylation leading to the (5) aggregation of the protein affecting the (6) vesicle formation, trafficking and docking, (7) impaired lysosomal autophagy, (8) and (9) ubiquitination and inhibition of proteosomal degradation. This in turn

results in (10) LB formation and (11) apoptosis.

118 Protein Phosphorylation

Protein phosphorylation is a reversible post-translational modification of proteins that has an important role in regulating structural and functional properties of proteins in health and disease. It is primarily associated with signaling pathways and cellular processes in all aspects of cell biology such as cell-cycle progression, differentiation, apoptosis, metabolism, transcription, cytoskeletal arrangement, intercellular communication, motility and migration [42–44]. In eukaryotes, the amino acids that are most commonly reported to be phosphorylated are serine, threonine and tyrosine [45, 46] with few reports suggesting phosphorylation at arginine, lysine and cysteine residues [45, 47, 48].

In PD too, phosphorylation appears to play an important role in fibrillogenesis, LB formation, and neurotoxicity of α-Syn *in vivo* [26, 49–51]. The majority of α-Syn in inclusions and LBs isolated from patients with PD and other synucleinopathies is phosphorylated at Ser129 (S129-P) [26, 49, 51]. Overexpression of wild-type or mutant α-Syn in *in vivo* and *in vitro* models showed expression of immunopositive phospho α-Syn serine129 (pSyn) in their proteinaceous inclusions [52–54]. Mass spectrometric analysis of α-Syn isolated from patients with synucleinopathy lesions has also confirmed that the protein is phosphorylated on Ser129 [49, 50].  Moreover, the phosphorylated α-Syn in LBs is usually ubiquitinated [50, 51, 55]. The fact that most of the α-Syn is not phosphorylated under physiological conditions *in vivo* suggests that α-Syn phosphorylation at serine129 contributes to the pathology of the disease [49, 56, 57].

#### **2.2. Phosphorylation and CNS**

The central processing unit of the human body is its CNS consisting of specialized cells called neurons relaying electrical and chemical signals to all parts of our body [58]. However, the most abundant cell type in the CNS is the glial cells comprising of astrocytes, oligodendrocytes and microglia [59, 60]. In addition, it is interspersed with microvasculature that provides the nutrients and support to these CNS cells. The central theme in CNS function is equilibrium among these various cell types to maintain optimal synaptic strengths, neuronal firing rates, and neurotransmitter release. The regulation of these functions can be either through inside-out or outside-in stimuli and are strongly associated with several signaling pathways within these cells. Virtually every class of neuronal protein is regulated by phosphorylation and most types of extracellular signals, including neurotransmitters, hormones, light, electrical potential, extracellular matrix, neurotrophic factors, and cytokines, can produce diverse physiological effects by regulating the phosphorylation of specific phosphor-proteins in their target cells. These extracellular signals modify the activity of protein kinases and/or phosphatases either directly (e.g. receptors with kinase activity) or via cascades of enzymatic reactions (e.g. receptor → G protein → enzyme ~ second messenger ~ protein kinase).

#### **2.3. Kinases involved in α-synuclein phosphorylation**

α-Syn phosphorylation can be induced by several kinases. Serine129 of α-Syn can be phosphorylated by G protein-coupled receptor kinases (GRK1, GRK2, GRK5, and GRK6) [61–63], casein kinases 1 and 2 (CK1 and CK2) [26, 64–69], and the polo-like kinases (PLKs) [70]. Current studies have shown that, GRKs may also phosphorylate non-receptor substrates, comprising the four members of the Syn family (α-,β-,γ-Syn, and synoretin) in addition to phosphorylating agonist-occupied G protein-coupled receptors (GPCRs) [62]. Overexpression of GRK2 or GRK5 in COS-1 cells, showed that these kinases phosphorylate α-Syn at serine129 [62]. Endogenous GRK-induced phosphorylation of α-Syn at serine129 was demonstrated *in vitro* in HEK293 cells, and GRK3 and GRK6 were seen to be playing the main roles in this modification [63]. Post-mortem analysis showed that GRK5 co-localized with α-Syn in the LBs of the SNpc of PD patients, but was not detected in cortical LBs of Dementia with lewy bodies (DLB), or in the glial cytoplasmic inclusions of MSA [61]. Overexpression of α-Syn in SH-SY5Y cells and human α-Syn expressing transgenic mice also showed an increase in GRK5 protein [71]. A genetic association study revealed a haplotype association of the GRK5 gene with susceptibility to sPD in the Japanese population [61]; however, GRK5 polymorphisms in southern Italy failed to correlate with sPD [72]. The knockdown of endogenous GRK5 in SH-SY5Y cells fails to suppress the phosphorylation of α-Syn completely [71], confirming the involvement of other kinases in this phosphorylation.

The other group of kinases that phosphorylates α-Syn at serine129 is CK1 and CK2. This has been demonstrated in the yeast model [69], in mammalian cells [26, 68] and in rat primary cortical neurons [64]. It has been suggested that phosphorylation of serine129 in α-Syn by CK2 may promote *in vitro* fibrillation [59] and *in situ* inclusion formation [73]. Phosphorylation by CK2 and dephosphorylation by PP2C in *in vitro* model indicate that these may be important enzymes that regulate the phosphorylation of α-Syn [74]. Furthermore, surplus α-Syn can form inclusions that sequester CK1, diminishing CK1 activity and aggravating synaptic defects, generating a vicious toxic cycle. CK1 has been found to co-localize with pS87 in transgenic mice and in LB-like structures in LBD/PD-diseased brains [75, 76], and phosphorylates α-Syn at serine87 as well [26]. Oxidative stress induced by iron is reported to upregulate CK2 that leads to increased phosphorylated α-Syn serine129 with an associated increase in oligomerization and inclusion formation [65]. Smith et al. [66] have shown in SH-SY5Ycells that the increase in α-Syn phosphorylation under oxidative stress is mediated by CK2 and correlates with enhancement of inclusion formation.

synucleinopathy lesions has also confirmed that the protein is phosphorylated on Ser129 [49, 50].  Moreover, the phosphorylated α-Syn in LBs is usually ubiquitinated [50, 51, 55]. The fact that most of the α-Syn is not phosphorylated under physiological conditions *in vivo* suggests that α-Syn phosphorylation

The central processing unit of the human body is its CNS consisting of specialized cells called neurons relaying electrical and chemical signals to all parts of our body [58]. However, the most abundant cell type in the CNS is the glial cells comprising of astrocytes, oligodendrocytes and microglia [59, 60]. In addition, it is interspersed with microvasculature that provides the nutrients and support to these CNS cells. The central theme in CNS function is equilibrium among these various cell types to maintain optimal synaptic strengths, neuronal firing rates, and neurotransmitter release. The regulation of these functions can be either through inside-out or outside-in stimuli and are strongly associated with several signaling pathways within these cells. Virtually every class of neuronal protein is regulated by phosphorylation and most types of extracellular signals, including neurotransmitters, hormones, light, electrical potential, extracellular matrix, neurotrophic factors, and cytokines, can produce diverse physiological effects by regulating the phosphorylation of specific phosphor-proteins in their target cells. These extracellular signals modify the activity of protein kinases and/or phosphatases either directly (e.g. receptors with kinase activity) or via cascades of enzymatic reactions

at serine129 contributes to the pathology of the disease [49, 56, 57].

(e.g. receptor → G protein → enzyme ~ second messenger ~ protein kinase).

α-Syn phosphorylation can be induced by several kinases. Serine129 of α-Syn can be phosphorylated by G protein-coupled receptor kinases (GRK1, GRK2, GRK5, and GRK6) [61–63], casein kinases 1 and 2 (CK1 and CK2) [26, 64–69], and the polo-like kinases (PLKs) [70]. Current studies have shown that, GRKs may also phosphorylate non-receptor substrates, comprising the four members of the Syn family (α-,β-,γ-Syn, and synoretin) in addition to phosphorylating agonist-occupied G protein-coupled receptors (GPCRs) [62]. Overexpression of GRK2 or GRK5 in COS-1 cells, showed that these kinases phosphorylate α-Syn at serine129 [62]. Endogenous GRK-induced phosphorylation of α-Syn at serine129 was demonstrated *in vitro* in HEK293 cells, and GRK3 and GRK6 were seen to be playing the main roles in this modification [63]. Post-mortem analysis showed that GRK5 co-localized with α-Syn in the LBs of the SNpc of PD patients, but was not detected in cortical LBs of Dementia with lewy bodies (DLB), or in the glial cytoplasmic inclusions of MSA [61]. Overexpression of α-Syn in SH-SY5Y cells and human α-Syn expressing transgenic mice also showed an increase in GRK5 protein [71]. A genetic association study revealed a haplotype association of the GRK5 gene with susceptibility to sPD in the Japanese population [61]; however, GRK5 polymorphisms in southern Italy failed to correlate with sPD [72]. The knockdown of endogenous GRK5 in SH-SY5Y cells fails to suppress the phosphorylation of α-Syn completely [71], confirming the involvement

**2.3. Kinases involved in α-synuclein phosphorylation**

of other kinases in this phosphorylation.

**2.2. Phosphorylation and CNS**

120 Protein Phosphorylation

*In vitro* studies employing kinase assays showed involvement of another family of cellular kinases in the phosphorylation of α-Syn at serine129: PLK1, PLK2, and PLK3 [70, 77]. The PLKs comprise a family of conserved Ser/Thr protein kinases that are known to play a role in cellcycle regulation and cellular response to stress and carcinogenesis [78]. PLK2 directly phosphorylated α-Syn at serine129 in an *in vitro* biochemical assay [70]. Inhibitors of PLK kinases inhibited α-Syn phosphorylation both in primary cortical cell cultures and in mouse brain *in vivo*. Further, using PLK2 KO mouse, Buck et al. [79] too have shown that PLK2 plays a key role in Ser129 α-Syn phosphorylation in mouse brain. Aubele et al. [80] have shown in an *in vivo* model that brain-permeable Plk-2 inhibitors reduce α-Syn phosphorylation in rat brain. In response to synaptic activation, PLK2 and PLK3 expression is reported that is associated with synaptic plasticity, remodeling, and homeostasis [81, 82], thus suggesting that these kinases could play an important role in modulating the normal physiology of α-Syn. Leucine-rich repeat kinase 2 (LRRK2) is also known to pSyn [83], but this remains debatable as there are no other studies confirming this, despite the existence of a clear interaction between the two proteins [83, 84].

#### **2.4. Phosphorylation at serine129 modulates α-synuclein protein-protein interaction**

The C-terminal domain of α-synuclein (residues 96–140) is an acidic tail of 43AA residues, containing 10 Glu and 5 Asp residues. C-terminal truncations of α-Syn induce aggregation, suggesting that C-terminal modifications might be involved in the pathology of α-Syn [85]. An interaction between the C-terminal domain and the NAC region of α-Syn is postulated to be responsible for the inhibition of α-Syn aggregation. Moreover, there are several studies on the interaction of α-Syn C-terminal tail with different proteins [86–91]. McFarland et al. [92] were the pioneers to address this using targeted functional proteomics [51]. The authors showed that the non-phosphorylated α-Syn peptide primarily interacts with proteins related to the mitochondrial electron transport chain (ETC) (complex I, III, and IV proteins of the ETC) [51]. It was hypothesized that changes in α-Syn phosphorylation could represent a response to biochemical events associated with PD pathogenesis. Among these, mitochondrial complex I dysfunction, oxidative stress, and proteasome dysfunction are processes that are known to be involved in synucleinopathies [93, 94]. The low levels of pSyn under physiological conditions as well as the absence of other phosphorylated residues such as pY39, pS87 and pY125 [26, 49, 50] suggest a faster degradation of this form under normal conditions. In fact, the phosphorylation status of α-Syn was recently correlated with clearance mechanisms [95, 96]. Another group Chau et al. [97] too reported that α-Syn phosphorylation at serine129 is toxic to DA cells and both the levels of serine129 phosphorylated protein as well as its toxicity are increased with proteosomal inhibition, emphasizing the interdependence of these pathways in PD pathogenesis.

However, the pSyn has a greater affinity for certain cytoskeletal and presynaptic proteins associated with synaptic transmission and vesicle trafficking [51]. Yin et al. [98] showed that α-Syn interacts with the switch region of Rab8a, a small guanine nucleotide-binding protein, in a serine129 phosphorylation-dependent manner; thus implicating its role in coordinating vesicle trafficking. Hara et al. [99] reported that serine129 phosphorylation of membraneassociated α-Syn modulates dopamine transporter function in a G protein-coupled receptor kinase-dependent manner. These observations suggest that pSyn could serve as a molecular switch to control α-Syn interaction with different protein partners and therefore may modulate the function of DA neurons. However, further investigations are required to assess the impact and the physiological consequences of serine129 phosphorylation on α-Syn interaction with other proteins, such as SNARE proteins [35, 100], cytoskeletal proteins (i.e. tubulin) [55, 101] and other amyloidogenic proteins (i.e. tau) [19, 102]. Jensen et al. [103] have hypothesized that an interaction between α-Syn and tau could link synaptic vesicles with microtubules. Tau has been shown to co-localize and interact directly with the Src PTK family member, Fyn [104]. It is hypothesized that tau could bring Src PTK family members such as Fyn into closer proximity to α-Syn, thereby enhancing the activity of these kinases for α-Syn. Samuel et al. [105] showed that the membrane binding of α-Syn monomers was differentially affected by phosphorylation depending on the PD-linked mutation. WT α-Syn binding to presynaptic membranes was not affected by phosphorylation, while A30P α-Syn binding was greatly increased and A53T α-Syn was marginally lower, implicating the distal effects of the carboxyl terminal on amino-terminal membrane binding. The un-phosphorylated form of serine129 associates mainly with mitochondrial electron transport proteins, while the phosphorylated form associates with cytoskeletal, vesicular trafficking proteins and enzymes involved in protein serine phosphorylation [92]. Further work by Sugeno et al. [106] using α-Syn-over expressing cells exposed to a low dose of rotenone as an environmental toxin, showed that phosphorylation of α-Syn at serine129 promoted intracellular aggregate-formation and induced ER stress that was followed by mitochondrial damage and apoptosis.

Phosphorylation also seems to play an important role in the regulation of α-Syn axonal transport as the serine129D mutation significantly decreases its rate of transport in neurons, probably due to the modulation of α-Syn interaction with motor and/or accessory proteins involved in this process [107]. Moreover, the interplay between the different phosphorylated residues could increase the diversity in the possible protein interactors. Several differences were observed in the set of proteins that were found to interact with serine129 and Y125 phosphorylated forms of α-Syn [92]. S129 and Y125 residues both residing in the C-terminal region of α-Syn have been implicated in the majority of α-Syn interactions with proteins [103, 108, 109], reinforcing the significance of phosphorylation in these residues in modulating the biological role of α-Syn. All these findings together suggest that phosphorylation of α-Syn at serine129 has a widespread effect on protein-protein interaction of α-Syn.

Phosphorylation also seems to alter the subcellular localization of α-Syn. pSyn was found to be preferentially localized in the nuclei of DA neurons in rat and mouse models of synucleinopathy [67, 100]. In studies using PD rat models, the phospho-resistant S129A was found to be localized in the nucleus at higher levels than the S129D form, and was found to correlate with enhanced toxicity [110, 111]. Our group too demonstrated the nuclear localization of pSyn in SH-SY5Y cells under 6-hydroxydopamine toxicity [112]. Gonçalves and Outeiro [113] showed that S129 phosphorylation modulates the shuttling of α-Syn between nucleus and cytoplasm in human neuroglioma cells, using photo-activatable green fluorescent protein as a reporter. Moreover, the study showed that co-expression of α-Syn with different kinases altered the translocation dynamics of the protein. While G protein-coupled receptor kinase 5 (GRK5) promotes the nuclear localization of α-Syn, PLK2 and three modulate the shuttling of the protein between the nucleus and cytoplasm [114]. This difference reflects different α-Syn phosphorylation patterns in serine129 and/or other residues. Although the function of α-Syn in the nucleus is still unclear, it appears to be related to pathological insults. In particular, nuclear localization of α-Syn increases under oxidative stress conditions [112, 114, 115]. Nuclear α-Syn interacts with histones, inhibits acetylation, and promotes neurotoxicity [116, 117]. Furthermore, α-Syn may act as a transcriptional regulator, binding promoters such as PGC1-α, a master regulator of mitochondrial gene expression [106]. The significance of pSyn in regulating nuclear proteins still needs to be unraveled.

#### **2.5. Oxidative stress and α-Syn phosphorylation**

be involved in synucleinopathies [93, 94]. The low levels of pSyn under physiological conditions as well as the absence of other phosphorylated residues such as pY39, pS87 and pY125 [26, 49, 50] suggest a faster degradation of this form under normal conditions. In fact, the phosphorylation status of α-Syn was recently correlated with clearance mechanisms [95, 96]. Another group Chau et al. [97] too reported that α-Syn phosphorylation at serine129 is toxic to DA cells and both the levels of serine129 phosphorylated protein as well as its toxicity are increased with proteosomal inhibition, emphasizing the interdependence of these pathways

However, the pSyn has a greater affinity for certain cytoskeletal and presynaptic proteins associated with synaptic transmission and vesicle trafficking [51]. Yin et al. [98] showed that α-Syn interacts with the switch region of Rab8a, a small guanine nucleotide-binding protein, in a serine129 phosphorylation-dependent manner; thus implicating its role in coordinating vesicle trafficking. Hara et al. [99] reported that serine129 phosphorylation of membraneassociated α-Syn modulates dopamine transporter function in a G protein-coupled receptor kinase-dependent manner. These observations suggest that pSyn could serve as a molecular switch to control α-Syn interaction with different protein partners and therefore may modulate the function of DA neurons. However, further investigations are required to assess the impact and the physiological consequences of serine129 phosphorylation on α-Syn interaction with other proteins, such as SNARE proteins [35, 100], cytoskeletal proteins (i.e. tubulin) [55, 101] and other amyloidogenic proteins (i.e. tau) [19, 102]. Jensen et al. [103] have hypothesized that an interaction between α-Syn and tau could link synaptic vesicles with microtubules. Tau has been shown to co-localize and interact directly with the Src PTK family member, Fyn [104]. It is hypothesized that tau could bring Src PTK family members such as Fyn into closer proximity to α-Syn, thereby enhancing the activity of these kinases for α-Syn. Samuel et al. [105] showed that the membrane binding of α-Syn monomers was differentially affected by phosphorylation depending on the PD-linked mutation. WT α-Syn binding to presynaptic membranes was not affected by phosphorylation, while A30P α-Syn binding was greatly increased and A53T α-Syn was marginally lower, implicating the distal effects of the carboxyl terminal on amino-terminal membrane binding. The un-phosphorylated form of serine129 associates mainly with mitochondrial electron transport proteins, while the phosphorylated form associates with cytoskeletal, vesicular trafficking proteins and enzymes involved in protein serine phosphorylation [92]. Further work by Sugeno et al. [106] using α-Syn-over expressing cells exposed to a low dose of rotenone as an environmental toxin, showed that phosphorylation of α-Syn at serine129 promoted intracellular aggregate-formation and induced ER stress that

Phosphorylation also seems to play an important role in the regulation of α-Syn axonal transport as the serine129D mutation significantly decreases its rate of transport in neurons, probably due to the modulation of α-Syn interaction with motor and/or accessory proteins involved in this process [107]. Moreover, the interplay between the different phosphorylated residues could increase the diversity in the possible protein interactors. Several differences were observed in the set of proteins that were found to interact with serine129 and Y125 phosphorylated forms of α-Syn [92]. S129 and Y125 residues both residing in the C-terminal region of α-Syn have been implicated in the majority of α-Syn interactions with proteins

in PD pathogenesis.

122 Protein Phosphorylation

was followed by mitochondrial damage and apoptosis.

Oxidative stress can increase α-Syn phosphorylation [97]. Perfeito et al. [118] showed through an *in vitro* model that exposure to ferrous iron and rotenone resulted in increase in pSyn. A similar increase was reported by Ganapathy et al. [112] in the presence of the endogenous toxin 6-hydroxydopamine. Proteosomal inhibition by epoxomycin and increased oxidative stress by paraquat treatment has led to increases in pSyn [97]. This may reflect either an increased activity of the kinase responsible or a decrease of phosphatase activity. Under physiological conditions, α-Syn is degraded by chaperone-mediated autophagy [119], and studies suggest that this gets reduced upon oxidation or nitration [12, 106]. α-Syn may also be degraded by proteasomes [120]. The increase in pSyn levels in LBs suggests a change in the turnover or degradation of the phosphorylated protein (**Figure 3**). It is possible that at elevated levels, this phosphorylated species may be toxic.

#### **2.6. α-Syn phosphorylation at serine129 and cellular events**

The role of α-Syn phosphorylation in the cellular pathogenesis of PD remains debated [92, 110, 111, 121, 122]. This apparent controversy is due to the fact that phosphomimics (S129D/E) do not reproduce the exact properties of the endogenous authentically phosphorylated α-Syn [122–124]. The expression of a variant showing prevention of phosphorylation by sitedirected mutagenesis of serine129 to alanine (S129A) caused an increase in α-Syn inclusions

**Figure 3.** Schematic representation of suggested pathological role of oxidative stress in the α-Syn phosphorylation and aggregation. Normally genetic mutation, neurotoxins, dopamine auto oxidation in the presence of excess iron and the release of reactive oxygen species (ROS) leads to mitochondrial dysfunction and oxidative stress in turn leading to cell death. However, increase in the oxidative stress and direct effect of neurotoxins can lead to phosphorylation of α-Syn and their aggregation which inturn inhibits the lysosomal autophagy and impaired proteosomal degradation resulting in LB formation and cell death.

and toxicity [110] On the other hand, several investigators have reported that the expression of S129A mutant protein led to fewer inclusions [66, 94, 110]. It has also been reported that the expression of phosphorylation mimicking serine129 to the aspartate (S129D) variant does not show DA deficits [92, 110, 111, 121, 123]. Hence, several groups have sought to address the issue by overexpressing α-Syn and using siRNA for its natural kinases [99, 118, 124]. As discussed in the earlier section, among the kinases primarily responsible for the α-Syn phosphorylation at serine129 are CKs, GRKs, LRRK2, and PLKs. The modulation of phosphorylation of α-Syn has also been reported by targeting the kinases in A53T mutant α-Syn expressing cells [99, 118, 124–126]. siRNA studies targeting kinases such as PLK, GRK2, and CK2 have been used to study the effect of phosphorylated α-synuclein on WT and A53T mutant α-synuclein expressing cells [99, 118, 124, 125, 127]. These studies have shown the effect of α-Syn phosphorylation with respect to ROS generation, mitochondrial alterations, proteasomal changes, and dopamine transport. The work of Perfeito et al. [118] suggest that stimuli that promote ROS formation and mitochondrial alterations highly correlate with mutant α-Syn phosphorylation at serine129, which may precede cell degeneration in PD. Similarly, we have shown in our recent work that at sub-lethal 6-hydroxydopamine concentrations, the decrease in resting vesicles (VMAT2) and vesicular dopamine release are not attributable to apoptotic cell death and occur concomitantly with the phosphorylation of α-Syn [112].

#### **2.7. α-Syn phosphorylation at serine129 during PD pathogenesis: an early or late event?**

The evidence of pSyn accumulation in the brain has been collected largely from post mortem analysis and it fails to answer if this accumulation occurs during the early or late stages of synucleinopathies. In a recent work, Walker et al. [127] investigated how pSyn levels and solubility change in cingulate and temporal cortex of DLB patients, at different stages of the disease. The authors reported a progressive accumulation of pSyn-immunoreactive species in diseased brains compared to healthy controls, as well as a positive correlation between pSyn levels and the severity of disease symptoms. A similar study using brain samples from PD patients also reported a drastic accumulation of pSyn-positive inclusions in different brain regions at the late stages of the disease [128]. Together, these results suggest that the occurrence of pSyn is linked to the severity of disease progression.

In our recent work, we demonstrated using biophysical and biochemical analysis in an *in vitro* model that under sub-lethal concentrations of 6-hydroxydopamine, phosphorylation of α-Syn precedes apoptosis and occurs concomitantly with the decrease in vesicular dopamine release [21]. This study provides a new perspective on the occurrence of pSyn even in early stages of the disease that may contribute to the impairment of neuronal function. Another recent work by Takahashi et al. [53] demonstrated increased accumulation of pSyn in the motor cortex of normal aging mice along with early onset motor impairment, which was further ameliorated by coenzyme Q.

#### **2.8. pSyn in human fluids and PNS of synucleinopathies**

and toxicity [110] On the other hand, several investigators have reported that the expression of S129A mutant protein led to fewer inclusions [66, 94, 110]. It has also been reported that the expression of phosphorylation mimicking serine129 to the aspartate (S129D) variant does not show DA deficits [92, 110, 111, 121, 123]. Hence, several groups have sought to address the issue by overexpressing α-Syn and using siRNA for its natural kinases [99, 118, 124]. As discussed in the earlier section, among the kinases primarily responsible for the α-Syn phosphorylation at serine129 are CKs, GRKs, LRRK2, and PLKs. The modulation of phosphorylation of α-Syn has also been reported by targeting the kinases in A53T mutant α-Syn expressing cells [99, 118, 124–126]. siRNA studies targeting kinases such as PLK, GRK2, and CK2 have been used to study the effect of phosphorylated α-synuclein on WT and A53T mutant α-synuclein expressing cells [99, 118, 124, 125, 127]. These studies have shown the effect of α-Syn phosphorylation with respect to ROS generation, mitochondrial alterations, proteasomal changes, and dopamine transport. The work of Perfeito et al. [118] suggest that stimuli that promote ROS formation and mitochondrial alterations highly correlate with mutant α-Syn phosphorylation at serine129, which may precede cell degeneration in PD. Similarly, we have shown in our recent work that at sub-lethal 6-hydroxydopamine concentrations, the decrease in resting vesicles (VMAT2) and vesicular dopamine release are not attributable to apoptotic cell death

**Figure 3.** Schematic representation of suggested pathological role of oxidative stress in the α-Syn phosphorylation and aggregation. Normally genetic mutation, neurotoxins, dopamine auto oxidation in the presence of excess iron and the release of reactive oxygen species (ROS) leads to mitochondrial dysfunction and oxidative stress in turn leading to cell death. However, increase in the oxidative stress and direct effect of neurotoxins can lead to phosphorylation of α-Syn and their aggregation which inturn inhibits the lysosomal autophagy and impaired proteosomal degradation resulting

and occur concomitantly with the phosphorylation of α-Syn [112].

in LB formation and cell death.

124 Protein Phosphorylation

Several studies in the field of PD and diagnosis of Parkinsonism are based on the fundamental molecular events associated with or without LBs. Total α-Syn quantification in the CSF of PD, DLB, and MSA patients in comparison to healthy controls has been proposed as a biomarker for α-Syn-related disorders [129–131]. However, an ideal biomarker for a particular disease must be easy to detect, and also reflect disease onset and progression with associated primary changes. A longitudinal study conducted by Foulds et al. [132] in the blood plasma of patients suffering from PD showed that total α-Syn in blood plasma of PD patients remained similar to that in normal individuals, but the level of α-Syn phosphorylated at serine129 was significantly higher in PD patients. Statistical analysis confirmed the usefulness of plasma levels of pSyn in discriminating patients with PD from healthy controls. In addition, pSyn inclusions were also detected in the PNS which might also serve as a useful diagnostic test for PD and related synucleinopathies. Work from two independent groups on skin biopsies showed that the majority of PD patients had accumulation of pSyn in small and large nerve fibers, while no signal was detected in healthy controls and in MSA or essential tremor control subjects [133, 134]. This cutaneous pathology was correlated with the progression of disease symptoms suggesting the use of this peripheral marker as a biomarker for the disease [135]. Presence of pSyn immunoreactivity was detected in gastric, duodenal and colonic biopsies [136, 137]. Hilton et al. [137] further reports that this accumulation of pSyn in the bowels of patients is detected in the pre-clinical phase of PD. Taken together, these reports suggest that accumulation of pSyn might provide a more reliable biomarker to detect PD at early stages and further discriminate between synucleinopathies, compared to total α-Syn.

#### **3. Conclusion**

In comparison to the relatively small number of genes, the large diversity of the proteome is achieved mainly by post-translational protein modifications, phosphorylation being the most widespread. Since the C-terminal region of α-Syn is involved in interaction with proteins [103, 104, 109] and metal ions [138–140], any phosphorylation in this site can alter its interaction capability [77]. The significance of α-Syn phosphorylation at serine129 has gained importance in PD because its accumulation is distinctly enhanced in the diseased condition. The revelation of the involvement of pSyn on α-Syn aggregation, LB formation, and neurotoxicity is crucial to understanding the pathogenesis and progression of PD and related disorders. Since some *in vitro* and *in vivo* studies have indicated that the pSyn is an early event preceding apoptosis, some important questions now needs to be explored in reference to the physiological functions regulated by phosphorylation such as dopamine synthesis, vesicle mobilization, and regulation of synaptic proteins, synaptic plasticity, and its subcellular localization. A systemic investigation of the role of each kinase on the phosphorylation of α-Syn and α-Syn clearance also needs to be carried out to identify viable targets for development of new therapeutics. A further *in vivo* study by Hirai et al. [57] elevates the significance of pSyn as a region-specific phenomenon, and the level of pSyn in response to physiological stimuli is selectively higher in the striatum region in comparison to cortex and hippocampus [57]. The relative sensitivity of this phenomenon in the striatum in reference to PD pathology needs to be assessed to address the increased susceptibility of some brain regions to α-Syn phosphorylation and pathology. In addition, the synergistic association of PD-linked mutations and pSyn in the regulation of α-Syn toxicity needs to be evaluated. Investigation of the possible implications of phosphorylation of α-Syn on cell-to-cell transmission and its pathological propagation in PD-diseased brains is also pending. Thus, the research evidence clearly suggests that the phosphorylation of α-Syn plays a significant and possibly pivotal role both in PD pathogenesis and progression, and that answers to the questions above are crucial for the identification of novel, disease-modifying, therapeutic targets for the treatment of PD and related synucleinopathies.

#### **Author details**

Indrani Datta\* and Kavina Ganapathy

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Biophysics, National Institute of Mental Health and Neurosciences, Bengaluru, India

#### **References**

[1] De Lau LM, Breteler MM. Epidemiology of Parkinson's disease. The Lancet Neurology. 2006;**5**(6):525-535

[2] Abeliovich A, Schmitz Y, Farinas I, Choi-Lundberg D, Ho WH, et al. Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron. 2000;**25**:239-252

**3. Conclusion**

126 Protein Phosphorylation

**Author details**

India

**References**

2006;**5**(6):525-535

Indrani Datta\* and Kavina Ganapathy

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Biophysics, National Institute of Mental Health and Neurosciences, Bengaluru,

[1] De Lau LM, Breteler MM. Epidemiology of Parkinson's disease. The Lancet Neurology.