Organellar destabilization 1 Mitochondrial dysfunction

The mitochondrial network plays a vital role in the supply of cellular energy currency in the form of adenosine triphosphate (ATP) to ensure the proper functioning of a variety of metabolic processes within a cell. Simpler molecules resulting from the cellular cyclic processing of macromolecular nutrients transfer electrons to carrier proteins such as nicotinamide and flavin adenine dinucleotides (NAD+ _{and FAD}+_{) producing NADH and} FADH2, which transfer the electrons to the electron transport chain (ETC) localized at the inner mitochondrial membrane (Saraste, 1999). Due to the constitutive cyclic fluctuation of the redox status between ETC enzymatic protein complexes with consequent high consumption of cellular oxygen in the oxidative phosphorylation process, mitochondria are assumed to be the main cellular producers of ROS (Orrenius et al., 2007). Escaping

26 electrons from the ETC can potentially reduce oxygen to form the highly reactive O2•-_, which can undergo further Fenton reaction to generate hydroxyl radical (OH•) and hydrogen peroxide (H2O2) which similarly can cause detrimental cellular damages (Boveris et al., 1972).

As a result of this pivotal physiological function of mitochondria, which if not properly managed can have adverse effects on cell survival, mitochondrial functionality has been proposed to be a crucial regulator and indicator of cellular homeostasis. Indeed, decline in mitochondrial functionality has been closely linked to increasing age of mammalians. This age-correlated respiratory chain deficiency is especially prevalent in only a subset of mammalian tissues, such as heart, skeletal muscle, colonic crypts and neurons (Dufour et al., 2008). A recent study by Dufour et al., 2008 demonstrated that the co-existence of functional respiratory chain-deprived and normal neurons accelerated the neurodegenerative process of the adjacent normal cells through a trans-neuronal signaling mechanism.

1.3.6.2Lysosomal rupture

Calpain activation has been reported to associate with lysosomal rupture leading to the death of post-ischemic CA1 neurons (Yamashima et al., 2003). A ―calpain-cathepsin hypothesis‖ was formulated by Yamashima et al. (1998) on the basis of the experimental paradigm of global brain ischemia in primates. The calpain-cathepsin cascade mechanism of cell death involves Ca2+ mobilization through the uptake of extracellular Ca2+ and/or the release from internal Ca2+ _{stores. Ca}2+ _{mobilization can lead to the activation of}

27 calpains which induces lysosomal rupture, possibly aided by ROS. The released lysosomal proteases, mainly the cathepsins, will then degrade the cell constituent proteins, ultimately leading to cell death. Cytoplasmic activation of cathepsin B (CTSB) upon lysosomal rupture mediates activation of pro-inflammatory caspase-1 and -11 upon focal cerebral ischemic induction and Aß42-induced neurotoxicity (Benchoua et al., 2004; Gan et al., 2004). However, recent finding by (Mueller-Steiner et al., 2006) suggested CTSB by its endogenous proteolytic activity reduced amyloid plaque accumulation through increased protein turnover.

1.3.6.3Endoplasmic reticulum (ER) stress

ER, with a pivotal pleiotropic physiological role in cellular biogenesis, metabolism, signaling and survival, is also a vital homeostatic organellar regulator of cellular stress (Travers et al., 2000). It is the site for the proper synthesis, folding and post-translational modification of cellular proteins (Ron and Walter, 2007) as well as production of steroids, cholesterol and other lipids (Chang et al., 2006). It also serves as a major intracellular Ca2+ _{ion store (Verkhratsky, 2005).}

Presence of ER stress has been reported in AD (Hoozemans et al., 2005), PD (DeGracia and Montie, 2004) and ischemic stroke (Kitao et al., 2007). ER stress, characterized by the accumulation of unfolded proteins in the ER lumen, is frequently manifested upon presence of oxidative stress. This stress induction can occur upon perturbation of any of ER cellular functions, i.e. via protein oxidation, disturbance of Ca2+ signaling, and alteration of the homeostatic redox balance (Chakravarthi et al., 2006; Gorlach et al.,

28 2006). An intimate communicative, functional coupling relationship between ER and mitochondria has also been established on the basis of these cellular functions. One instance would be the maintenance of Ca2+ _{equilibrium, crucial for the proper functioning} of both organelles (Csordas et al., 2006). Mitochondria act as an emergency Ca2+ _store upon sudden transient surge in cytosolic Ca2+ level, to buffer the ER against any functional disruption. Furthermore, several members of the B-cell lymphoma 2 (BCL2) family prominent for their roles in regulation of mitochondrial-mediated apoptosis, also seem to participate in ER-induced cell death and Ca2+ _{signaling between the ER and} mitochondria (Breckenridge et al., 2003; Gorlach et al., 2006; Rao et al., 2004; Szegezdi et al., 2006; Wu and Kaufman, 2006). Initiation of ER stress has been demonstrated to occur upon mitochondrial energy deficits (Flores-Diaz et al., 2004; Xu et al., 2004).

Extensive ER damage can trigger cell death via the production of unfolded proteins, the release of Ca2+ _{into the cytoplasm or altered redox homeostasis (Breckenridge et al.,} 2003) resulting in either classical programmed cell death (PCD) or other mitochondrial cell death pathways (Jimbo et al., 2003). As such, dysfunctional Ca2+ _{regulation arising} from ER stress and increased molecular oxidative damage further potentiates activation of programmed necrotic pathway involving calpains, forming a positive feedback regulatory loop (Crocker et al., 2003; Nakagawa and Yuan, 2000).

29 Figure 1.1 A simplified diagram summarizing the major biological processes implicated during neuronal excitotoxicty.

1.4 Ischemia

Stroke, a cerebro-vascular disease/accident, occurs when blood supply to the brain is disrupted in the event of occlusion or rupture of blood vessels, resulting in the loss of neurological function. As such, stroke can be subdivided into two types: ischemic stroke (lack of blood flow due to thrombosis or arterial embolism) and hemorrhagic stroke

30 (vascular leakage). It has been shown that majority of stroke cases (accounting for ~85%) is attributed to acute ischemic cause with the rest categorized as hemorrhage (Lakhan et al., 2009). Ischemic stroke is a general term with reference to a heterogenous group of etiologies e.g. embolism, relative hypoperfusion and thrombosis. Nevertheless, ischemic stroke is ubiquitously caused by atherothrombosis of large cervical and intracranial arteries and embolism from the heart.