Keywords

1 Importance of Dementia in Ageing Society

In the modern countries, increasing number of aged people induces serious social, medical, and economical problems. Among them, the increase of demented people according to ageing is one of the biggest problems. It is known that the incidence of dementia doubles in the people of 5 years elder, that means the risk of dementia is 10 % in the people over 65 years old, and 25 % in over 85 years old. The three major causes of dementia are Alzheimer disease (AD), Lewy body dementia (LBD), and vascular dementia (VD). In addition, age-related decline of mental activity is closely related to the occurrence of dementia and sometimes it is mixed and impossible to determine the distinct contribution of the diseases and ageing in demented people in individual. To clarify the mechanism of brain ageing is essential to understanding the etiology of the dementia.

2 Implication of Oxidized Polyunsaturated Fatty Acids, Especially DHA to Brain Ageing and Neurodegeneration

Oxidative stress is suggested to be involved in the etiology of both brain ageing and neurodegeneration such as AD and LBD. Because that oxygen consumption is high in the brain neurons where oxidizable unsaturated fatty acid is rich, the level of lipid peroxidation should be continuously high and might injure the cells. Among unsaturated fatty acids, docosahexanoic acid (DHA; 22:6 n-3) is the most abundant omega-3 polyunsaturated fatty acid (PUFA) in the brain. DHA is a component of membrane lipid bilayer and is implicated in the various neuronal functions, such as neuronal plasticity, neural transmission, and signal transduction (Eady et al. 2012; Wurtman et al. 2010). In addition, DHA is known as a potent antioxidant, and in vitro and in vivo studies proved that application of DHA prevented neuronal damage by oxidative stress (Bazan et al. 2011; Berman et al. 2009; Glozman et al. 1998). However, lipophilic antioxidants such as DHA and beta-carotene are pro-oxidants and when they are oxidized, they become toxic lipid-peroxidation products to make chain reaction of peroxidation (Yavin et al. 2001). Lipid peroxide is the early product of lipid peroxidation compared with the aldehyde (Kato and Osawa 2010) (Fig. 11.1). The distinct chemical reaction of DHA and intermediates are unknown, although it was found that human neuroblastoma SH-SY5Y cells, DHA is metabolized into intermediates, 17-, 14-, 7- or 4-hydoxydocosahexaenoic acid (HDHA) and toxic 17-hydroperoxydocosa-hexaenoic acid (17-HpDHA) (Gleissman et al. 2010). These hydroxyl or cyclic intermediates may be associated with the formation of propanoyl and succinyl to form adduct with lysine residue in the proteins, which is named propanoyl-lysine (PRL) and succinyl-lysine (SUL) respectively. In the same way, oxidation of omega-6 PUFA, arachidonic acid forms hexanoyl-lysine (HEL) and glutaroyl-lysine (GLL) (Fig. 11.1). PRL- and SUL-positive proteins were detected in albumin incubated with oxidized DHA. Furthermore, by animal experiments, the increase of SUL-modified proteins was identified in the liver of mice fed with the diet containing high level of DHA under oxidative stress induced by carbon tetrachloride but not in the mice with high DHA diet only (Kawai et al. 2006). In human, PRL-adduct proteins were found to increase in the urine of diabetic patients (Hisaka et al. 2009). However, little is known about the existence and the role of lipid peroxide derived from DHA in the brain. The study to clarify the involvement of PRL- or SUL-adduct proteins in brain ageing and neurodegeneration has been waited for.

Fig. 11.1
figure 1

Hypothetic pathway of oxidation pathway of DHA

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3 LBD and Lipid Peroxidation

One of the common pathological feature of neurodegeneration is accumulation and aggregation of abnormal proteins such as amyloid β protein to make senile plaque in AD and α-synuclein as a main component of Lewy body in LBD. Among neurodegenerative disorders in the aged, increased level of oxidative stress is one of the most important pathogenetic factors of Parkinson disease (PD), the most common phenotype of LBD. In the dopamine neurons of the substantia nigra, neurotransmitter dopamine is easily auto-oxidized to make quinone and produces superoxide (O2 −∙) simultaneously. The half-life of superoxide is very short and it reacts with H2O to produce hydrogen peroxide (H2O2). Dopamine is oxidized enzymatically by monoamine oxidase (MAO) and produces H2O2 also. In the presence of iron, which is rich in dopamine neurons as a co-factor of tyrosine hydroxylase, H2O2 produces reactive and deleterious hydroxyl radical (OH∙) in the neurons. These reactions are summarized in following lines.

$$ \begin{array}{l}{{\mathrm{O}}_2}^{-\cdotp }+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{O}}_2+{\mathrm{O}\mathrm{H}}^{-}+{\mathrm{O}\mathrm{H}}^{\cdotp}\kern1em \left(\mathrm{Haber}\hbox{-} \mathrm{Weiss}\kern0.5em \mathrm{reaction}\right)\\ {}{\mathrm{Fe}}^{2+}+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{Fe}}^{3+}+{\mathrm{O}\mathrm{H}}^{-}+{\mathrm{O}\mathrm{H}}^{\cdotp}\kern1.25em \left(\mathrm{Fenton}\kern0.5em \mathrm{reaction}\right)\end{array} $$

High level of ROS derived from dopamine metabolism is one of the reason why dopamine neurons are vulnerable in ageing (Carlsson 1978).

Another source of reactive oxygen species (ROS) in the cells is mitochondria. From mitochondrial respiratory chain, ROS is continuously generated and about 1 % of ROS is failed to be quenched by anti-oxidants and anti-oxidizing enzymes to injure the bioactive molecules. Because half-life of ROS is generally short, the most potential target of ROS should be mitochondrial membrane and membrane-bounded proteins. Indeed, we found that in cybrid cells, made by the fusion of the mitochondria-negative cells containing nucleus and another cells containing nucleus without mitochondria, the cells containing mutated mitochondrial genome (mitochondrial encephalopathy with lactic acidosis, MELAS type of mutation) which was established as reported previously (Fujita et al. 2007) was stained by HEL and 4-hydroxy-2-nonenal (HNE) antibodies. By immuno-histochemical observation was done according to Shamoto-Nagai et al. (2007). HEL and HNE positive staining was accumulated in the mitochondria as detected by immunohistochemistry (Fig. 11.2). The presence of modified proteins in mitochondrial fraction was confirmed by Western blotting (Fig. 11.3). Mutation and deletion of mitochondrial genome is causable for mitochondrial cytopathy, in which brain and muscle are commonly disturbed (Morgan-Hughes et al. 1982). Accumulation of functionally impaired mitochondria due to dysfunction of quality control system of mitochondria is now gathering attention as a pathogenesis of some familial type of PD (See Exner et al. (2012) as a review). PD and brain ageing might share the common death pathway such as oxidative stress, mitochondrial dysfunction and lipid peroxidation-derived membrane injury.

Fig.     11.2
figure 2

HEL-modification was accumulated in the mutated mitochondrion. (a). Immunocytochemical detection of HEL moieties in independent cybrid cell lines. Immunostaining was performed with biotynylated anti-rabbit IgG and peroxidase-conjugated streptavidin and 3, 3’-diaminobenzidine tetrahydrochloride as chromogen. Increased immunoreactivity was detected in cybrid cells fused with mutated mitochondrial DNA (2SA, 2SD, 59A) but not in the cells fused with normal mitochondrial DNA (data not shown). (b). 59A cybrid cells were double-stained with CMTMRos, a mitochondrial marker with red fluorescence (upper) and also immunohistochemically using anti-HEL antibody and FITC green fluorescence dye (lower)

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Fig. 11.3
figure 3

(I) Immunochemical detection of HEL-modified proteins. (a). Total protein (10 mg) derived from various cell lines (2SA, 2SD, 59A) was lysed and separated by SDS-PAGE (12 % polyacrylamide) then probed using polyclonal antibody against HEL-conjugated proteins. (b). Proteins derived from various cell lines was separated into cytosolic, mitochondrial, microsomal, and nuclear fractions. Each fractions were separated and probed by anti-HEL antibody. HEL-positive proteins were detected in cytosolic and mitochondrial fractions but not in microsomal or nuclear fraction (data not shown). In mitochondrial fraction, two major bands at 51 and 65 kDa were detected. (II) Immunochemical detection of HNE-modified proteins. (a). Total protein (10 mg) derived from various cell lines (2SA, 2SD, 59A) was lysed and separated by SDS-PAGE (12 % polyacrylamide) then probed using polyclonal antibody against HNE-conjugated proteins. (b). Proteins derived from various cell lines was separated into cytosolic, mitochondrial, microsomal, and nuclear fractions. Each fractions were separated and probed by anti-HNE antibody. HNE-positive proteins were detected in cytosolic and mitochondrial fractions but not in microsomal or nuclear fraction (data not shown). In mitochondrial fraction, two major bands at 45 and 53 kDa were detected

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4-HNE and acrolein (ACR) are lipid-derived aldehyde and they are reported to increase in the dopamine neurons in the substantia nigra of PD brain (Dalfo et al. 2005; Shamoto-Nagai et al. 2007; Yoritaka et al. 1996) the increased ACR-modified proteins was confirmed by our group also (Fig. 11.4). In vitro study revealed that 4-HNE and ACR can make direct adduct with αSyn and produces abnormal oligomer with β-sheet structure or aggregates. Such aldehyde-adduct αSyn shows increased the toxicity to primary cultured neurons and inhibits the proteasome activity in vitro and in the cultured neuronal cells (Qin et al. 2007; Shamoto-Nagai et al. 2007). Recently, we investigated the involvement of lipid peroxide such as PRL and SUL on the modification of αSyn in vitro and in the cellular model of PD. DHA enhanced oligomerization and aggregation of αSyn and in the cells treated with oxidative stress, lipid-peroxide modified aggregates similar to Lewy body was detected (Shamoto-Nagai et al. in preparation). It is consistent with the in vivo report that DHA administration enhanced oligomerization of αSyn in the brain of transgenic mice (Yakunin et al. 2012).

Fig. 11.4
figure 4

Immunohistochemical detection of HNE-positive moieties using anti-HNE antibody. Control and Parkinsonian substantia nigra was stained with anti-HNE antibody as previously reported (Shamoto-Nagai et al. 2007). The increase of HNE-reactivity in melanine-containing dopaminergic neurons are apparent

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4 Can Oxidatively Modified Proteins Be Applicable as a Marker of LBD?

In LBD, not only central nervous system but also peripheral sympathetic and parasympathetic neurons are degenerated with the existence of Lewy body (Orimo et al. 2002). It is indicated that continuous oxidative stress and lipid peroxidation should occur in the degenerating peripheral neurons and there may be oxidatively modified molecules which can be applied as a marker to diagnose or evaluate the progression of the disease.

Recently, neuroprotective or neurorescue therapy is coming to be realistic issue. Several species of natural compounds and drugs have been proposed to be effective to prevent neuronal death in cellular and animal models of PD. Our group reported that the inhibitors of MAO type B (MAO-B), rasagiline and (−) deprenyl protect neuronal cell death induced by various insults (Naoi and Maruyama 2009, 2010). The mechanism of the neuroprotective action is under the investigation. It was found that MAO-B inhibitors and some kind of polyphenols increase the expression of mRNA and protein levels of neuroprotective molecules such as Mn- and Cu/Zn- superoxide oxidase (SOD), brain-derived neurotrophic factor (BDNF) and glial cell line-neurotrophic factors (GDNF) in the cells, rodents, and primates (Carrillo et al. 2000; Maruyama et al. 2002, 2004; Maruyama and Naoi 2013). In addition, MAO-B inhibitor (−)deprenyl, polyphenol tetrahydrocurcumin and resveratrol prolonged the lifespan of rodents and other animals (Baur 2010; Kitani et al. 2004; Knoll 1988; Rubinsztein et al. 2011; Xiang et al. 2011). Decreased harmful oxidative stress and increased growth factors might be ascribed to the longevity effect of these candidates for neuroprotective drugs (Mattson and Magnus 2006). In neurodegenerative disorders, the symptom and progression of the disease is heterogenous and even though the development of new brain imaging, it is not easy to evaluate the neuroprotective effects of the drugs. Oxidatively modified proteins and humoral factors such as BDNF and GDNF might be applicable to evaluate the efficiency of neuroprotective compounds in human.

5 Conclusion

Lipid peroxidation is involved in the neurodegenerative disorders and brain ageing. Especially, membrane composing PUFA, such as DHA is easily oxidized and produces lipid peroxides and aldehydes which make adducts to proteins with abnormal conformation and are toxic. Lipid peroxide is an early product of lipid peroxidation compared to aldehyde and so, the investigation of lipid peroxide adduct proteins may open a new field of science which clarify the mechanism of neuronal dysfunction and death in the aged people.