Insights Into The Pathogenesis Of Neurodegenerative Diseases: Focus On Mitochondrial Dysfunction And Oxidative Stress Part 1

Abstract: As the population ages, the incidence of neurodegenerative diseases increases. Due to intensive research, important steps in the elucidation of pathogenetic cascades have been made and significantly implicated mitochondrial dysfunction and oxidative stress. 

With the continuous improvement for modern medicine and living standards, the average life expectancy of human beings is getting longer and longer, which also means that the aging of the population is becoming more and more serioua s. Although the elderly have to face many health and life problems, memory loss is not necessarily one of these problems.

In fact, there are many elderly people with excellent memory. They can not only maintain their daily lives, but also use their life ex,perience to solve problems. Regarding the decline in memory, many people think that this is due to age, but in fact this is just a misconception.

Studies have sh is no direct connection beprovides loss and population aging. Whether young or old, our brains have the ability to repair themselves, as long as we train regularly, we can improve our memory. The decline in memory of the elderly is more because their brains are not fully exercised, leading to brain decline.

We can take some measures to strengthen our memory, such as exercising our brains through various activities such as reading, writing, painting, traveling and socializing. In addition, we can also pay attention to diet and lifestyle habits, such as maintaining good sleep quality, a balanced diet, and avoiding unhealthy behaviors such as smoking or drinking.

Therefore, we should not emphasize the decline in memory because of the aging of the population, but should be more active in exercising and maintaining the brain. By improving our memory, we can protect our health and free life. It can be seen that we need to improve memory, and Cistanche can significantly improve memory, because Cistanche is a traditional Chinese medicinal material with many unique effects, one of which is to improve memory. The effect of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in various ways.

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However, the available treatment in Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis is mainly symptomatic, providing minor benefits and, at most, slowing down the progression of the disease. 

Although in preclinical setting, drugs targeting mitochondrial dysfunction and oxidative stress yielded encouraging results, clinical trials failed or had inconclusive results. It is likely that by the time of clinical diagnosis, the pathogenetic cascades are full-blown and significant numbers of neurons have already degenerated, making it impossible for mitochondria-targeted or antioxidant molecules to stop or reverse the process. 

Until further research will provide more efficient molecules, a healthy lifestyle, with plenty of dietary antioxidants and avoidance of exogenous oxidants may postpone the onset of neurodegeneration, while familial cases may benefit from genetic testing and aggressive therapy started in the preclinical stage.      

Keywords: mitochondrial dysfunction; oxidative stress; antioxidants; Alzheimer's disease; Parkinson's disease; amyotrophic lateral sclerosis.

1. Introduction

Aging associates a series of physiologic deficits and a variable degree of cognitive impairment, being also a major risk factor for neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS) [1]. 

From the enormous amount of research aimed at unraveling the mechanisms of aging and neurodegeneration several pieces of the puzzle emerged, but we have still a long way to go to grasp the whole picture. However, it appears that oxidative damage induced by free radicals and mitochondrial dysfunction play a major role in both processes.                                   

2. Normal Aging

Aging of the brain occurs at molecular, cellular, and histological levels [2]. It associates lower levels of neuronal metabolic activity, subtle alterations in neuronal structure in several neuronal circuits, as well as synaptic atrophy, cytoskeletal abnormalities, accumulation of fluorescent pigments, and reactive astrocytes and microglia [3,4]. 

Research points toward the hypothalamus as initiating and controlling the gradual decline of energy metabolism of the entire body [5,6]. Through the secretion of neurohormones, the connections with the endocrine system, and projections of the orexinergic nucleus to the reticular activating system [5], the hypothalamus regulates the stress levels, metabolism, sleep, and influences the subjectively perceived quality of life and establishing of social relationships [7–9]. 

Degeneration of the suprachiasmatic nucleus may additionally contribute to circadian rhythm disorders and impaired sleep [10–12]. Limiting sleep may cause neuronal toxic waste products to accumulate and limit neurogenesis in the aging brain, igniting a vicious cycle which augments the neurodegenerative process [13].               

Additional factors contribute to the declining metabolism of the brain cells. Cerebral metabolism relies mainly on a constant supply of glucose and oxygen through the blood flow, and, to a limited extent, on lactate [14]. 

Neuronal glucose uptake is mediated by glucose transporters (GLUTs), after which it is converted into glucose-6-phosphate (G6P) by hexokinase. As such, the availability of ATP, depending on oxygen supply and mitochondrial oxidative phosphorylation (OXPHOS), interferes with glucose uptake [5,15]. OXPHOS produces considerable higher amounts of ATP as compared to glycolysis [16,17]. 

In aging, reduced metabolism can be caused by mitochondrial dysfunction and reduced ATP synthesis, as well as by vascular changes which lead to limited oxygen supply. 

The high cellular energy expenditure in the nervous system, used for synaptic transmission (around 80% of the energy consumption of the brain) [18,19], synaptogenesis, and synaptic pruning [18,20] associates a high rate of generation of reactive oxygen species mainly by electrons leaked from the mitochondrial electron transport chain (ETC). 

The excessive generation of free radicals cannot be neutralized by the antioxidant defenses and leads to oxidative stress, implicated in aging since the 1950s, when Harman suggested that free radical-induced damage of biomolecules, such as proteins, lipids, and DNA, causes a reduction of their biochemical and physiological function in aging [21]. 

Indeed, research has demonstrated altered composition of phospholipids in the brains of aged humans and animals together with increased malondialdehyde (a marker of lipid peroxidation) generation, which forms deposits connected with intraneuronal lipofuscin [22], and elevated carbonyl residues (a marker of protein oxidation) [23]. 

In addition, aging decreases the antioxidant defense systems, such as the astrocytic glutathione system [24], which further potentiates oxidative stress [2]. Mitochondria undergo a series of age-related changes such as fragmentation or enlargement [25], increased mitochondrial DNA (mtDNA) oxidative damage [26], exhibit dysfunctions of the respiratory chain [27] and of calcium homeostasis [28]. 

These changes associate a reduction of the intracellular NAD+ levels which impairs the function of NA dependent enzymes such as sirtuins (SIRT) and histone deacetylases [29,30]. Sirtuins are highly evolutionary conserved enzymes involved in the regulation of lifespan and aging in different organisms, from yeast to mammals [31]. 

In mammals, SIRT 3, 4, and 5 are located in the mitochondria, SIRT 2 in the cytosol, and SIRT 1, 6, and 7 are located in the nucleus [32]. Research has shown that SIRT 1, mainly in the hypothalamus, is a key player in controlling aging and longevity in mammalian organisms [33,34]. 

Shortening of the telomeres, which promote chromosomal stability during cell replication [35], was initially disregarded as having important influence in the brain since neurons are essentially postmitotic cells which no longer replicate. 

However, this view has been challenged by demonstrating cell cycle activity in 10–20% of neurons in the cortex of healthy aging brains and in AD [36,37], as well as by the presence of neural stem cells in the subventricular and subgranular zones, in the choroid plexuses and meninges [38]. In addition, glial cells (especially microglia) do replicate actively, with telomeres gradually shortening after each cellular replication [39]. 

Aging induces also an inflammatory phenotype during which, in response to mutations and DNA damage, nuclear factor-κB (NF-κB) initiates the transcription of tumor necrosis factor-α (TNF-α) and various inflammatory interleukins (IL-1β, IL-6, IL-8) [40]. 

ROS have a crucial role in this process, since they phosphorylate and degrade IκB, which binds to and inactivates NF-κB [41]. Low amounts of ROS initiate pro-survival signaling cascades, the activated NF-κB suppressing c-Jun N-terminal kinases (JNKs) and apoptosis and upregulating antioxidant and anti-apoptotic genes such as manganese superoxide dismutase (MnSOD) [42]. 

However, high ROS concentrations activate NF-κB through protein kinases and initiate cellular stress signaling pathways. Damaged neurons alter the ionic balance in the interstitial space and lead to cytokine release, thereby activating microglia [43,44]. 

Although TNF-α has important roles in learning and synaptic plasticity [45], excessive microglial activation will cause degeneration of synapses and functional impairments, characteristic of aging and neurodegeneration [46]. 

In addition, the inflammatory responses induce other transcription factors, such as signal transducer and activator of transcription (STAT-1) and peroxisome proliferator-activated receptor-gamma (PPARγ), the latter playing an important role in mitochondrial biogenesis [47]. 

As such, the secretory phenotype associated with senescence (SASP), particularly in astrocytes, can trigger several age-related neurodegenerative diseases [48].     

3. Mitochondria in the Brain

The high cerebral metabolic activity relies mainly on oxidative phosphorylation for ATP production. However, mitochondria exert other important functions in the brain as well. 

The constant signaling leads to continuous variations in the cytosolic calcium concentrations and mitochondria in collaboration with the endoplasmic reticulum have a crucial role in regulating neurotransmission by buffering calcium in presynaptic terminals and regulating the somato-dendritic calcium levels [49,50]. Moreover, mitochondria regulate cell cycle and control cell death [51]. 

In order to accomplish these diverse functions, maintenance of mitochondrial fitness is crucial, requiring efficient quality control mechanisms [52], achieved through mitochondrial biogenesis and fission (separation of a single organelle into two or more daughter organelles, essential for populating dividing or growing cells with an adequate number of mitochondria), fusion (a process through which mitochondria share essential components), and mitophagy (elimination of damaged mitochondria before they lead to apoptosis of the whole cell) [53,54]. 

In addition, mitochondria must be trafficked along axons to provide energy all across the axon [2].       

3.1. Mitochondrial Respiratory Chain and ROS Production

A mitochondrion has a spongy outer mitochondrial membrane (OMM), which allows free movement of small ions and uncharged molecules, and an impermeable inner mitochondrial membrane (IMM), which envelops the mitochondrial matrix. 

Between the IMM and OMM lies the intermembrane space [55]. The mitochondrial electron transport chain (ETC) consists of several of protein complexes situated in the IMM which use the electrons removed by reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) from the Krebs cycle to pump protons from the matrix into the intermembrane space, thereby generating a potential gradient across the IMM, which will be used in the final step of OXPHOS to synthesize ATP [56]. 

In order to function properly, these complexes must be assembled by the folded IMM (mitochondrial cristae) into specifically configured structures [57]. Even under normal conditions, 1–2% of the total oxygen consumed leaks and generates ROS [58]. 

At least eight mitochondrial sites are able to generate ROS, with complexes I, II, and III being the main contributors [55,59]. Through lipid peroxidation, protein oxidation, and DNA damage, the generated ROS can cause alter mitochondrial function and increase the rate of ROS production, culminating in degeneration of neurons [60,61].

3.2. Mitochondria and Cellular Calcium Homeostasis

Calcium is involved in many neuronal functions, such as differentiation, vesicle release and synaptic transmission, or cell death and survival [62,63]. Transient fluctuations in the cytosolic Ca2+ act as second messengers. 

Free cytosolic Ca2+ levels are in nanomolar ranges, while extracellular levels are millimolar. Calcium influx occurs through ligandoperated or voltage-gated calcium channels (VGCCs), but Ca2+ can also be released from intracellular Ca2+ stores, among which the endoplasmic reticulum (ER) has a pivotal role [64]. Agonist binding to inositol 1,4,5-triphosphate (IP3) receptors or to ryanodine receptors (RyRs) causes a release of Ca2+ from the ER [65]. 

However, in order for the increases of cytosolic Ca2+ to be brief, calcium must be rapidly cleared through calcium efflux, binding to Ca2+-buffering proteins [66], or uptake into the ER or mitochondria [62]. 

Ca2+ efflux is achieved by the plasma membrane Ca2+-ATPase, which pumps Ca2+ against the concentration gradient while hydrolyzing ATP, and the Na+/Ca2+ exchanger (NCX), which relies the sodium gradient to extrude Ca2+ [67]. 

ER Ca2+ uptake is performed by the ATP-dependent sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) [68], while mitochondrial Ca2+ uptake is mediated by voltage-dependent anion-selective channel proteins (VDCAs), which mediate Ca2+ transfer into the intermembrane space, from where the IMM-located mitochondrial Ca2+ uniporter (MCU) further transfers the calcium into the mitochondrial matrix. 

Increases in mitochondrial calcium activate the ETC dehydrogenases and ATP production [69]. However, calcium overload can alter the mitochondrial membrane potential, open the mitochondrial permeability transition pore (MPTP), and lead to cytochrome c release [70]. 

As such, mitochondrial Ca2+ concentrations must be finely tuned. 

A mitochondrial Na+/Ca2+ exchanger located in the IMM, and termed NCLX because it can also able to exchange Li+ for Ca2+, extrudes Ca2+ from the mitochondrial matrix by using the electrochemical gradient of Na+ [71], while from the intermembrane space Ca2+ is extruded by the Na+/Ca2+ exchanger 3 and VDACs [72]. Figure 1 illustrates the mechanisms involved in cellular calcium homeostasis.

Figure 1. Intracellular calcium homeostasis. Cellular Ca2+ influx is mediated by voltage-gated calcium channels (VGCC), ligand-gated calcium channels (LGCC), and, in exceptional circumstances, by reverse functioning of the sodium/calcium exchanger (NCX). 

In addition, Ca2+ can be released from the ER following inositol-1,4,5-triphosphate (IP3) binding to specific receptors (IP3R) or the ryanodine receptors (RyR). 

IP3 is generated by binding of ligands to plasmalemmal Gprotein-coupled receptors, which activates phospholipase C to cleave phosphatidylinositol 4,5-biphosphate, resulting in the second messenger IP3. 

Excess cytosolic calcium is removed through efflux through the NCX and plasma membrane Ca2+ ATPase (PMCA) and uptake into ER by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA). 

Mitochondria buffers cytosolic calcium through the mitochondrial calcium uniporter (MCU) and extrudes excess Ca2+ through the Na+/Ca2+ exchanger (NCLX). In addition, cytosolic Ca2+ binding proteins (CBP) act as signal transducers.

In controlling the intracellular Ca2+ concentrations, mitochondria interact with the ER through mitochondria-associated ER membranes (MAMs) [73], microdomains where the OMM is just 10–100 nanometers apart from the ER [74,75]. 

These areas are enriched in inositol 1,4,5-triphosphate receptors (IP3Rs) [76] which form functional complexes with VDACs through a chaperone, Grp75 (glucose-regulated protein 75), belonging to the heat shock protein 70 family [77]. 

IP3R-Grp75-VDAC complexes regulate Ca2+ transfer from the ER to mitochondria [74]. The apposition of ER to mitochondria is controlled by phosphofurin acidic cluster sorting protein 2 (PACS2) [78]. Decreased contact sites between ER and mitochondria caused by PACS2 deletion result in mitochondrial fragmentation and apoptosis [79]. 

PACS2 is functionally linked to phosphatidylserine synthase-1 (PSS1), an enzyme located in MAMs which mediates the transfer of lipids between ER and mitochondria [80]. PACS2 and PSS1 were found upregulated in AD transgenic mice as well as human patients with late-onset AD [74,81]. Several other components of MAMs involved in calcium homeostasis and signaling cascades have been described, such as:

- Bap31 (B cell receptor-associated protein 31), which interacts with the OMM protein Fis1 [82];

- VAPB (Vesicle-associated membrane protein-associated protein B), which interacts with the OMM protein tyrosine phosphatase-interacting protein 51 [83];

- Sig-1R (Sigma non-opioid intracellular 1-receptor 1), a chaperone which binds Grp78; under ER stress conditions, Grp78 dissociates from the ER lipid rafts and activates the unfolded protein response (UPR) [84,85];

- Protein kinase-like endoplasmic reticulum kinase (PERK), which, when activated reduces protein synthesis until the accumulated unfolded protein is cleared [86].

3.3. Mitochondrial Dynamics

Mitochondria are dynamic organelles, being able to modulate their number, shape, size, and position in the cytoplasm through a careful balancing of two opposite processes: mitochondrial fusion and fission [87]. 

Fission is regulated by two proteins: Drp1 (dynamin-related/-like protein 1) and Dnm2 (dynamin 2) [88]. The initial step is wrapping of the endoplasmic reticulum around the mitochondria and reducing the diameter of the latter from 300–500 nm to about 150 nm [89]. The spatial association of replicating mtDNA with the ER-mitochondria contact sites explains the mtDNA distribution in the replicating organelles [90]. 

Following this step, the cytosolic protein Drp1 is recruited to the already marked constriction site on the OMM and bound to the phospholipid membrane by adaptor proteins such as MFF (mitochondrial fission factor) and mitochondrial dynamics proteins 49 and 51 (MiD49 and MiD51) [91]. 

Following Drp1 recruitment, a ring-like structure is formed around the mitochondria [92], after which GTP hydrolysis potentiates the constriction of the mitochondrial membrane [87]. 

The final step is recruitment of Dnm2, a GTPase which assembles at the marked site and completes the fission process [93]. The constriction and division of the IMM is calcium-dependent and occurs at ER–mitochondria contact sites, possibly even before Drp1 recruitment [94]. 

Mitochondrial fusion is the opposite process, by which the membranes of two mitochondria fuse, giving rise to one single mitochondrion and allowing for sharing of essential components between the two organelles. 

The process is regulated by two other proteins with GTPase activity, Mfn1 and Mfn2 (mitofusins 1 and 2) for fusion of the OMM, while fusion of the IMM is under control of another GTPase, OPA1 (optic atrophy 1) [95]. 

After tethering of two mitochondria (mediated through the GTP domains), the two adjacent OMMs increase their contact surface area followed by their fusion due to GTP hydrolysis, subsequent conformational changes, and oligomerization of Mfns [96]. 

Following OMM fusion, IMM fusion is mediated by IMM-inserted OPA1, which can be cleaved by two membrane-bound metalloproteases, OMA1 and YME1L, resulting in two high molecular weight fragments (L-OPA1) and three shorter fragments (S-OPA1) [97]. 

The interaction of L-OPA1 with cardiolipin, inserted in the IMM, is crucial for driving membrane fusion [98]. The balance between OMA1 or YME1L cleavage of OPA1 regulates mitochondrial fission [99]. 

Stimulation of OXPHOS induces YME1L cleavage of OPA1 and mitochondrial fusion, while OPA1 cleavage by OMA1 is a stress response, and may induce mitochondrial fragmentation as well [87].

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