Файл: Extracellular and Intracellular Signaling (книга).pdf

mitochondrial Ca21 overload, decrease in mitochondrial complex I activity and an increase of levels of ROS production.54 PINK1 deficient cells are more susceptible to die after exposure to MPP1 or Rotenone.55 A collaborative function for PINK1 with parkin and DJ1 in protecting from oxidants has been suggested; this is supported by the susceptibility of PINK1 deficient cells to die, which can be rescued by over-expressing parkin, implicating that PINK1 is upstream on a linear pathway.56 This also indicates that DJ1 stabilizes PINK1 allowing for their binding to have a synergistic action protecting from MPP1 exposure.57

In PD etiology, genetic deficiencies or environmental insults generate toxic cell conditions that a ect the balance between production of reactive species and antioxidant defenses giving rise to oxidative and nitrosative stress. ROS and RNS react rapidly with cell components and are responsible for nitration and oxidation to proteins, lipids and/or nucleic acids. Protein nitration, oxidation and carbonylation are increased in PD.58–60 Specifically, PD-related proteins are oxidatively damaged, including a-synuclein oxidation and nitration and parkin nitrosilation. Additionally, in PD more ubiquitous enzymes are found to be oxidatively modified a ecting central cell pathways. Aconitase, an iron-sulfur enzyme, is inactivated by superoxide-induced loss of the labile Fe21 atom, which a ects the tricarboxylic acid cycle and consequently cellular energy metabolism (Figure 13.1).61 Lipid peroxidation is increased in PD as

high levels of lipid hydroperoxides and 4-hydroxynonenal (HNE) are found in the SN.62,63 HNE is found as part of LBs and also forming adducts

with nucleophilic groups on proteins, such as dopamine transporter.64 Lipid peroxidation in PD has been associated with apoptosis, PARP-induced cleavage, decreased GSH levels and inhibited mitochondrial complexes I and II.62 Lastly, oxidative stress in PD produces several oxidative lesions to genomic and mitochondrial DNA. Mitochondrial DNA (mDNA) is particularly susceptible given its proximity to the mitochondria, an important ROS production site. A higher number of deletions in mDNA are found in PD patients as a result of oxidative damage48 and missense mutations a ect respiratory chain genes65 (Figure 13.1). Additionally, loss of function of PINK1 has resulted in decreased mDNA synthesis.66 Levels of 8-hydroxyguanine and 8-hydroxy-2- deoxyguanosine are increased in PD,67 whereas exposure to 6-OHDA induces an increase of double strand breaks.68 Severely damaged nucleic acids could a ect gene expression and lead to apoptotic cell death in PD. Given that neuronal cells are post-mitotic and DNA does not benefit from turnover, DNA repair systems are critical to defend against oxidative lesions in neuronal cells. The e ciency of DNA repair systems may influence aging in response to oxidative stress.69

Damage to macromolecules due to oxidative and nitrosative stress results in cell components dysfunction. Two of the most a ected organelles in PD are mitochondria and endoplasmic reticulum (ER). Mitochondrial dysfunction in PD is likely the result of complex I inhibition, increased ROS production and Ca21 overload.70 Under these conditions, mitochondria lose their control over Ca21 levels, mitochondrial membrane potential (MMP) and generation of NADH and ATP. Mitochondrial Ca21 overload stimulates mitochondrial

permeability transition pore (mPTP) opening, which results in mitochondrial membrane depolarization and mitochondrial swelling. As a result, there is loss of GSH and NAD(P)H from the mitochondria and release of cytochrome c to the cytosol.71 Under physiological conditions, mitochondrial Ca21 stimulates tricarboxylic acid cycle and oxidative phosphorylation, however, it is uncertain how this stimulation changes under Ca21 overload conditions.71 Decreased MMP a ects normal mitochondrial processes such as fusion and fission72 and mitochondrial tra cking.73 By nature, the main function of mitochondria is to supply energy to maintain physiological cell processes, and impaired ATP production directly a ects the function of ATP-dependent processes such as maintenance of plasma membrane potential, glucose transport into the mitochondria, ubiquitination and the proteasome function. Additionally, master cell energy regulators, such as AMP-activated protein kinase (AMPK), respond to changes in AMP/ATP ratio, Ca21 levels and ROS initiating the activation of an intracellular signaling pathway that prevents cell death.74

PD-related proteins that are localized in the mitochondria, such as DJ1, PINK1 and parkin, can show di erences in their functions under mitochondrial dysfunction.75 Parkin redistribution from the mitochondria into the cytosol occurs in response to inhibitors of respiratory chain activity and cell cycle blockers.48 Oxidized DJ1 changes its distribution from being di used throughout the cytoplasm to the proximity of the mitochondria. Additionally, mitochondrial dysfunction could induce ER stress due to their adjacency in the cytoplasm. ER stress is found in PD and it is accompanied by the unfolded protein response (UPR) and protein aggregation (discussed in the next section). It has been proposed that a cross-talk between the mitochondria and ER could initiate a signaling pathway resulting in apoptosis in dopaminergic cells.76

Although believed to be only damaging, a new role for ROS in normal intracellular signaling has emerged. Such function in signaling has especially been applied to hydrogen peroxide since its half-life and specificity allows it to fit criteria for being a second messenger.77 Thiols in proteins can be oxidized to sulfenic acid residues, glutathionylated residues or to the formation of intramolecular disulfide bonds in the presence of hydrogen peroxide. These protein modifications determine the origin of cell messages. In the 6-OHDA model, oxidation of thioredoxins (Trx), such as Trx1, allows apoptosis signalregulated kinase1 (ASK1) to dimerize and activate the apoptosis-inducing pathways, p38 and JNK intracellular pathway.78,79 Using a similar mechanism, in cells treated with hydrogen peroxide, oxidized DJ1 converts Cys-106 to cysteine sulfinic acid (Cys-SO2H), and binds to ASK1 inducing cytoprotection.80 The activation of another cell survival intracellular signaling cascade, the Akt pathway, has been found to be facilitated by DJ1.81 Additionally, the ‘‘floodgate hypothesis’’ proposes that, under oxidative stress conditions, hydrogen peroxide oxidizes peroxydases, thus making them inactive.77 Peroxidase inactivation, specifically Prx1, has been described to induce cell-cycle arrest mediated by the activation of p38 and caspase-3.82 Some promising examples of hydrogen peroxide-mediated signaling include oxidative modifications in AP-183 and PTEN.84

13.3.1.2Response to Altered Proteostasis

Proteostasis refers to a complex regulation of protein synthesis, folding, quality control, transport and degradation.85 This regulation is important in order to keep the normal protein structure and function and to prevent abnormal protein accumulation. The identification of LBs as hallmarks of PD has raised the interest in the role of alterations of proteostasis in PD. LBs, found in most degenerating dopaminergic neurons in PD patients, are mainly proteinaceous inclusions formed by overlapping steps of protein aggregation, protein posttranslational modifications and proteolysis.86 a-Synuclein is the main filamentous component of LBs;87 it is natively unfolded and has a structural trend to aggregate due to its hydrophobic non-amyloid beta domain.25 In the formation of LBs, a-Synuclein monomers aggregate to form fibrils, which have been shown as being favored by oxidative modifications.88 The role of a-Synuclein as a causal factor of PD is supported by the findings that mutations and gene variations of PARK 1 increase the aggregation propensity of a-Synuclein,89,90 and that a-Synuclein over-expression is a causative factor in familial cases of PD.26 Therefore, the pathogenic mechanisms of a-Synuclein in PD include self-aggregation and decreased ability of cells to eliminate a-synuclein before it reaches critical intracellular concentrations to aggregate.91 Ubiquitinated proteins, parkin and chaperones Hsp70/Hsp90, as well as mitochondrial proteins such as cytochrome c, also colocalize in LBs.92 Some other of the familial PD-associated mutations and gene variations have been related to changes in protein structure that a ect hydrophobicity, protein-protein interaction and degradation, such as mutations in DJ1 that make its protein product easily degraded.93

Di erent cellular systems participate in proteostasis, including the ER, the ubiquitin-proteasome system (UPS), ubiquitin-independent proteases and autophagy. The ER has a function in folding and post-translational modification of proteins after they are synthesized. Abnormal proteins, such as truncated proteins, are not folded properly and are not transported out of the ER, which favors their subsequent accumulation. Protein accumulation into the ER interferes with protein tra c from ER to the Golgi and induces ER stress.94 ER stress induces a compensatory response cascade, the unfolded protein response (UPR), which involves an increased expression of ER chaperones and ER-associated degradation (ERAD)-associated molecules to increase the cellular folding capacity and the translocation of unfolded proteins from the ER into the cytosol. Some of these ER chaperones include Hsc/Hsp70 and Hsp40, which re-fold unfolded or misfolded proteins. In the cytosol, the ERAD directs unfolded proteins to be degradated by the proteasome. ER stress also induces an ER Ca21 imbalance and persistent protein accumulation resulting in caspase-12 and ER-specific apoptosis activation.95

If proteins are normally synthesized and folded, molecular crowding can account for an abnormal protein aggregation. The neuronal cytoplasm is a crowded environment, mostly occupied by macromolecules, that o ers a limited access to proteins. Molecular crowding is non-specific and can fluctuate as cell

volume changes with age or disease. Under the molecular crowding scenario, protein folding and protein-protein interaction equilibrium is driven towards the lower volume (globular/oligomeric) species as well as an entropic driving force that compensates the decreased entropy of fibril-forming protein for the high entropy of other proteins.96 The failure to properly dispose proteins and the over-expression of PD-related proteins lead to excessive cytoplasmatic protein content, molecular crowding and spontaneous oligomerization.91

The UPS is the major system responsible for degradation of cytosolic abnormal proteins. UPS involves degradation of damaged proteins as part of the physiological protein turn-over through a sequential ATP-dependent processing by the ubiquitin-activating (E1), -conjugating (E2) and -ligating (E3) enzymes. These enzymes function in degradation of proteins by adding a ubiquitin tag to the substrate protein to be recognized by the proteasome.97 E1 activates ubiquitin in its glycine 76, E2 transfers the activated ubiquitin from E1 to E3, which binds covalently to the substrate protein in a substrate-specific manner. Following this first ubiquitination, a polyubiquitin chain is added to the substrate protein making it a target for degradation by the 26S proteasome. The 26S proteasome is assembled by a catalytic core 20S proteasome and two regulatory subunits, 19S and 11S. The 20S proteasome hydrolyzes peptide bonds by proteolysis.98 The UPS has been shown to be a ected in PD99 as well as proteasome function has been shown to be inhibited leading to LBs-

like intraneuronal inclusions and selective degeneration of dopaminergic neu- rons100–102 (Figure 13.2). The pathogenic processes responsible for UPS

and proteasome dysfunction may involve a-Synuclein and parkin. Specifically, an a-Synuclein alternatively spliced form, the 112-aa form, has been shown to induce proteasome disfunction14 whereas E3-ubiquitin ligase function of parkin may be a ected by mutations in PARK2 and S-nytrosylation.103 Parkin-associated endothelin-receptor-like receptor (Pael-R) is a substrate for parkin and has been shown to form insoluble aggregates in PD patients carrying mutations in PARK2.15 Accumulation of Pael-R results in ER stress and neuronal death, while PARK2 over-expression protects against ER stress induced by unfolded proteins15 (Figure 13.2). An alternative proteolytic processing for unfolded Pael-R requires its binding to Hsp70 and ubiquitination by an E4 reaction.104 Programmed cell death-2 (PDCD2) protein and CDCrel-1 are potential parkin substrates. PDCD2 is highly homologous to Rp-8, a protein associated with apoptosis, inflammation and cell proliferation in rodent brain.28 CDCrel-1 is a synaptic protein predominantly expressed in the nervous system and involved in cytokinesis.105 In addition, a mutation in ubiquitin carboxy terminal hydrolase L1 gene (UCH-L1), involved in autosomal dominant PD cases, can be related to altered UPS function since it has been shown to have a function in cleaving polymeric ubiquitin.106 The impact of abnormal function in

these PD-related genes on UPS and proteasome function have been involved in the resulting ER stress, UPR and apoptosis in PD.107

When the action of chaperones is overcome, other mechanisms are used by the cellular quality control machinery to control protein accumulation.108 Ubiquitin-independent protein degradation constitutes a mitochondrial system

of ATP-dependent proteases to monitor protein quality in the mitochondrial matrix. HtrA2 is a mitochondrial serine protease that can interact with and is regulated by PINK1 (Figure 13.1).109 HtrA2 has a dual role in neuronal death: it activates proapoptotic proteins upon release into the cytosol and induces apoptotic death upon its inactivation.110 Autophagy is another protein quality control in cells and it is considered a last line of defense against damage. Autophagy is initiated by the UPR to compensate UPS dysfunction and high protein accumulation states. Autophagy involves the removal of damaged or redundant cell components utilizing the lysosomes. MPP1, mutations in the

PD-related gene leucine rich repeat kinase 2 (LRRK2), neurotoxic doses of dopamine, as well as 6-OHDA induce autophagy.111–114

Although the link between oxidative stress and alteration of proteostasis is not completely clear, proteasome dysfunction is likely a result of an overload of unprocessed oxidized proteins and the limited availability of ATP for ATP-dependent peptidases required for proteasome function.115 In some cases, oxidatively damaged proteins a ect proteostasis. a-Synuclein oxidation and

nitration cause its misfolding and decreased ubiquitination.88 Oxidative modifications in parkin a ect its E3 function.103,116 Paraquat toxicity, which is

associated with oxidative stress, upregulates and induces aggregation of a-Synuclein.117 DJ1, which may have antioxidant properties, reduces aggre-

gation of a-Synuclein and Pael-R preventing the apoptotic cell death induced by their toxic accumulation.118,119 Some of the PD-related proteins that are

involved in oxidative stress such as PINK1 and UCH-L1, as well as oxidized proteins and HNE, have been found colocalized to aggrosomes and LBs.120 The e ect of oxidative stress on proteostasis may be mediated by di erent mechanisms depending upon the initiator stimulus. A comparison between the response to 6-OHDA and MPP1 shows that 6-OHDA activates the expression of genes involved in UPR and translation, whereas MPP1 activation is restricted to genes involved in translation.121 Both 6-OHDA and MPP1 induce CHOP,121 a stress-induced transcription factor involved in apoptosis (Figure 13.3).122

13.3.1.3Response to Glutamate

Some dopaminergic neurons in the SN pars compacta receive moderate excitatory glutamatergic input from the subthalamic nucleus in the striatum. As the cell density progressively decreases in the SN in PD, dopamine levels in the striatum are progressively decreased causing reduction in the inhibition and over-activity of the internal globus pallidus and subthalamic nucleus resulting in overstimulation of dopaminergic nigral cells by glutamate. This excessive stimulation by glutamate causes an increased influx of Ca21 into the cell through NMDA receptors (Figure 13.2).123 Their characteristic NMDA NR2 subunit contents may make these neurons more susceptible to damage due to

their direct interaction with specific small GTP-binding proteins, such as Ras and Rap.124,125 As it will be discussed below, activation of small GTP-binding

could be contributing factors to Ca21 imbalance in dopaminergic neurons. Cybrids containing dysfunctional mitochondria from PD patients have been shown to increase intracellular Ca21 levels correlating with increased Calpain activation (Figure 13.2).134 Intracellular Ca21 levels are selectively increased by exposure to MPP1.135 Supporting the role of increased intracellular Ca21 in PD, midbrain cells containing calbindin, a Ca21 binding protein, are spared from degeneration in PD patients and MPTP-induced parkinsonism.136 The resulting Ca21 imbalance is associated with altered membrane permeability, abnormal microtubule function and activation of Ca21-dependent enzymes.

In the mitochondria, loss of PINK1 function a ects Ca21 e ux resulting in increased mitochondrial Ca21 levels and ROS production (Figure 13.1).137 Elevated intracellular Ca21 levels arrest microtubule-based mitochondrial movement seemingly by a ecting formation of kinesin-Miro-Milton complex.72 As an intracellular signaling messenger, Ca21 can activate calmodulin/ Ca21-calmodulin-dependent protein kinase (CaM/CaMKII), calpain and protein kinase C (PKC) in the cytosol (Figure 13.2). Upon CaM activation by Ca21, CaMKII is activated. CaMKII activation is associated with increased mitochondrial ROS production either by activation of Ca21-dependent dehydrogenases that stimulate oxidative phosphorylation or via activation of downstream targets such as MAO. Also, CaM functions as a co-activator of nNOS. nNOS is involved in production of NO from L-arginine. NO is an unconventional neurotransmitter and is important in messenger functions during inflammatory responses in the SN and in signaling pathways in striatal neurons. Elevated nNOS activity could contribute to dopaminergic cell death in SN by increasing NO available for peroxynitrite production.

Calpains are cystein proteases. Calpains have been found to cleave several substrates including cytoskeletal components, proteases and cell signaling

molecules. Calpain activity is increased in PD patients as well as in the MPTP and Rotenone rodent model.138,139 Calpain has been involved in the induction

of apoptosis through conversion of p35 to p25 (Figures 13.2 and 13.3).140 p25 causes cdk5 to be constitutively activated and mislocated. Increased p25/cdk5 activity cause degeneration and apoptotic cell death, suggestively mediated by phosphorylation of Prx2, a Prx located in the cytoplasm of neurons.141 Calpain has shown to cleave a-synuclein in vitro, which di erentially a ects a-synuclein aggregation because calpain cleavage of a-synuclein monomers prevents

further oligomerization, while cleavage of a-synuclein fibrils promotes further co-assembly.142

PKC is an important initiator kinase in neurodegeneration. PKC is a family of serine/threonine kinases divided into four groups: the conventional, the novel PKCs, the atypical PKC and a PKN subfamily. The conventional PKCs comprise PKCa, PKCb and PKCg, which are activated by a combination of diacylglycerol (DAG) and phospholipid, and Ca21-dependent phospholipid binding (Figures 13.2 and 13.3). The novel PKCs include PKCd, PKCe, PKCy and PKCZ and di er in that they do not respond to Ca21. The atypical PKC and the PKN subfamily do not depend on Ca21 or DAG for activation but are allosterically activated.143 Given their activation under elevated Ca21 levels,