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