**4.2 Pathophysiology of ROS**

*Amyloid Diseases*

**4. Oxidative stress**

(O2

proline containing peptides from cis to trans conformational isomers thereby facilitating their folding process [47]. Some inherently amyloidogenic proteins such as PrP, hTTR and BRICHOS-domain containing proteins can paradoxically also inhibit aggregation of other amyloids. BRICHOS-domain containing protein ITM2B (Bri2) is a CNS membrane protein which shows chaperone functions for amyloids by binding to tyrosine and other charged residues [43]. Tetrameric form of Transthyretin (TTR) can also prevent aggregation of amyloid oligomers [44]. In recent years, some small molecules have also been put forward as possible inhibitors of amyloid aggregation—such as polyphenols, e.g., epigallocatechin gallate (EGCG), curcumin, resveratrol, etc. [7, 12]. Understanding inhibition mechanisms of non-proteolytic endogenous chaperone proteins and other molecules is very crucial to develop therapies targeting amyloid aggregates in AD (**Table 2**).

Oxidative stress in human body is mainly mediated by reactive oxygen species (ROS) including hydrogen peroxide (H2O2), hydroxyl free radical (•OH), superoxide

•<sup>−</sup>), peroxyl (•OOR), hypochlorous acid (HOCl), nitric oxide (NO), peroxynitrite (ONOO<sup>−</sup>) and other reactive nitrogen species. Increase in concentration of these reactive species can trigger downregulation of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase, catalase, thioredoxin and small molecules such as melatonin and coumarin [81]. Endogenous sources of ROS include components in mitochondrial electron transport chain and NADPH oxidase, which can trigger pathological responses leading to lipid peroxidation, DNA damage and cell death mediated by toxic products. Amyloid plaques have been shown to contain multi-fold concentrations of metals like iron (Fe), copper (Cu) and zinc (Zn) [81]. Intracellular Aβ can also trigger production of highly toxic molecules such as 4-hydroxyl-2,3-nonenal (HNE) and malandialdehyde through interactions with Fe or Cu which can catalyze Fenton reactions to generate more OH<sup>−</sup> ions. Hydroxyl radicals can also be generated from other brain ROS through Haber-Weiss reaction, particularly within mitochondria which are most prone to such oxidative damage [82]. Such ROS are generated as by-products of amyloid aggregation and are involved with metallobiology of dementia. High concentrations of Fe in brain have drastic effects in cases of dementia leading to cognitive decline through interactions with brain amyloids [83]. Metal ions are crucial for many brain functions and Fe and Cu even possess active sites for binding antioxidant SOD. However, increase in concentrations of some of these metals can cause high levels of toxicity. Twenty percent of cases in familial ALS is affected by mutation in a single enzyme SOD1 functionally affected by Cu/Zn [83]. Metal ions have propensity to bind high affinity, well-protected and redox-shielded binding sites of proteins, and in higher concentrations they can bind to other putative active sites in proteins involved in pathology of various degenerative diseases [84]. While zinc compounds have been tested for their probable therapeutic role in overcoming cognitive degeneration, Zn2+ ion has also been implicated as a contributor to formation of amyloid plaques which act as metal sinks [84]. Such mitochondrial damage and mutations have been associated with age related late-onset, non-autosomal dominant AD pathology [85].

Aβ binds and reduces Fe3+ and Cu2+ in presence of endogenous reducing agents

to generate H2O2 further producing other partially ROS [86, 87]. Studies have revealed the role of metal ions such as Fe, Cu and Zn in inducing Aβ aggregation

**198**

**4.1 Amyloid-β and oxidative stress**

Excessive free radical production as a result of oxidative stress at cellular levels causes protein oxidation and lipid peroxidation [91]. Lipid peroxidation leads to break down of unsaturated fatty acids among other components of membrane phospholipids, leading to accumulation of isoprostanes, acrolein, thiobarbituratereactive substances, etc. [84, 92]. Glutamate receptors overstimulation can trigger downstream cell death cascades through increased calcium influx and generation of nitric oxide species [48]. 8-hydroxy-2′-deoxyguanosine (OHDG) is one of the oxidative markers for DNA, found in PD patients [36]. These products impair glucose transport and glutamate uptake, hence contributing to cell apoptosis. ROS cause imbalance in metal and ion homeostasis, for example Ca2+, which can trigger imbalance in downstream signaling cascades. Oxidative damage can lead to hydroxylation of nucleic acids and carbonylation of proteins. Carbonyls are markers of protein oxidation and have been found concentrated in frontal brain regions of AD patients [92]. Free radicals generated as result of amyloid oligomerization or aggregation can directly mediate mitochondrial damage which triggers neuronal death through downstream pathways, one of them being cytochrome C reduction [48]. OS in PD cases may be a result of deregulation of dopamine-iron redox pathway, since αS can alter expression of enzymes indirectly regulating dopamine synthesis [84]. αS is also known to directly interact with metal ions causing protein aggregation. ALS is mainly characterized by loss of motor neurons, which combined with SOD mutations diminishing its free radical scavenging abilities can exacerbate the impacts to oxidative injury [84]. Oxidative markers localized in plaques and NFTs are toxic products such as 3-nitrotyrosine, HNE, pyrraline and pentosidine, while metal enriched protein carbonyls including ferritin, catalyst, Cu/Zn-SOD and Mn-SOD [93].

#### **4.3 Protective mechanisms from ROS**

Chaperones can bind ROS generated as by-product of amyloids and thus prevent triggering breakdown of homeostasis. α2-Macroglobulin can directly bind Aβ and potentially act as a chaperone in addition to its zinc-binding capabilities which can help mitigate redox activity of Aβ [94]. Zn2+ is redox-inert and may be helpful in mitigating metal mediated Aβ redox activity. ApoE can mediate Aβ clearance as a chaperone depending on specific isoform interactions; ε4 may potentially increase

Aβ pathology [95]. ApoE interaction with Aβ is modulated by metals [93]. There are two key requirements for metal interactions in dementia-either supplementation or chelation [83]. Metal chelators can help solubilization of Aβ plaques. Effects of chaperones on metal ions need to be carefully curated to maintain metal homeostasis in brain and other organs of nervous system. Several endogenous brain proteins show potential protective response against ROS in diseased brain, including β-trace protein, which is the second most abundant protein in CSF. Competitive binding of metal ions by chaperone proteins may be advantageous in decreasing generation of ROS by-products. Overexpression of metal-affinity proteins have been shown, as example of iron-binding ferritin. Antioxidant alpha-tocopherol has been reported to potentially slow AD progression in addition to action of metal chelators such as clioquinol and desferrioxamine [92]. L-PGDS scavenges ROS and in process protects against neuronal cell death with its ligand binding function intact [96]. It also reduces cytotoxicity mediated by oxidation of heme metabolites such as bilirubin [97]. We have found that L-PGDS can directly interact with Aβ-heme complex and lower its peroxidase activity (to be published). Other non-proteolytic chaperones such as α2M, clusterin and haptoglobin, also show neuroprotective potential in similar manner as L-PGDS and are good candidates for more comprehensive oxidative stress related studies.
