Normal Aging Induces Changes in the Brain and Neurodegeneration Progress: Review of the Structural, Biochemical, Metabolic, Cellular, and Molecular Changes

1Due to improved economic status and public health care systems, global populations have expanded, and life expectancy has increased. In Korea, the aging population is increasing very fast. It has been reported that approximately 5% of the global elderly population is affected by dementia. The number of patients with dementia has doubled or tripled within the last few decades (2020). Including dementia and neurodegenerative diseases are associated with brain aging. Brain aging is associated with anatomical and functional deterioration, which can result in neurodegenerative diseases.

2Neurons in the brain are post-mitotic cells that require regulation systems that keep the levels of neuronal electrical activity consistently to maintain their neuronal network. However, dysfunction of energy metabolism could jeopardize this system and lead to aging of the brain.

3According to previous studies, brain aging shows several hallmarks in other tissues as follows: (1) mitochondrial dysfunction; (2) intracellular accumulation of oxidatively damaged proteins, nucleic acids, and lipids; (3) dysregulated energy metabolism; (4) impaired cellular waste disposal mechanisms; (5) impaired adaptive stress response signaling; (6) compromised DNA repair; (7) aberrant neuronal network activity; (8) dysregulated neuronal Ca2+handling; (9) stem cell exhaustion; and (10) inflammation. Chronic positive energy balance accelerates brain aging and leads to neurodegenerative diseases through mitochondrial dysfunction, neurotoxic protein accumulation, and neuro-inflammation.

4This review summarizes the structural, biochemical, metabolic, cellular, and molecular changes that occur with normal brain aging (Table 1). In terms of structural changes, both gross and microscopic changes occur with aging. Biochemical and metabolic changes can be categorized into different neurotransmitter groups such as acetylcholine, monoamine, and hormones. Cellular and molecular changes in the brain affect the nucleus and mitochondria, oxidatively damage molecules, lysosome and proteasome function, electrophysiological regulation and neuronal calcium homeostasis.

5The brain undergoes various morphological changes with aging, such as cerebral atrophy, gray and white matter changes (GM and WM, respectively), volume loss, ventricular enlargement, and sulci widening. It is widely known that the volume of the brain and its weight decreases with age at a rate of around 5% per decade after 40 years of age. Furthermore, the rate of decline may acutely increases after 70 years old. For example, a large cross-sectional study of 2200 participants aged 34–97 years suggests lobar volume loss during aging. The frontal lobe volume decrease about 12% across the cohort. The temporal lobe volume declined about 9%. The occipital and parietal lobes shows no significant age-related volume change. It is known that the volume loss is accompanied by expansion of ventricular volume and other cerebrospinal fluid spaces.

6In GM, cerebral atrophy occurs through morphological alterations related to a decrease in the complexity of dendrite arborization. Dendritic shortening, loss of dendritic shortening, and decreased dendritic spines trigger a progressive reduction in synaptic density and synaptic transmission, with major consequences on cognitive decline. In contrast, in WM, the common features of tissue changes are partial loss of myelin, axons, oligodendroglial cells, and mild reactive astrocytic gliosis linked to WM lesions. Furthermore, WM changes are related to arteriolosclerosis of small vessels, resulting in incomplete ischemia and cell death and the emergence of perivascular spaces that interfere with the glymphatic drainage of the brain’s waste products. During normal aging, the GM volume fraction has been reported to drop from 52.35% in those in their 40s to 50.49% in those in their 80s. The WM has been reported to decrease from 47.63% in those in their 40s to 40.29% in those in their 80s, whereas the ventricular volume fraction increases from 3.22 to 5.66%. In addition, ventricular enlargement is caused by an increase in the space between folds and loss of gyrification.

7With respect to the sulci and gyrus of the cerebral cortex, the grooves and folds or ridges, respectively, become wider and shallower with aging. Sulci modifications result from shrinkage in the gyri, both nearby and distally. They may affect the global shape of the brain with many regional outcomes. These integral forces are provoked by the combined changes in the cortical GM and WM, along with other subcortical structures. Sulci morphology differences indicate that the sulci are wider (17.3%) in older people. In addition, the sulci depth has been reported to be shallower in older participants in the intraparietal sulcus. Moreover, sulci width is generally associated with local brain volumetric differences.

8A sufficient supply of blood and structural and functional blood vessels is important for normal brain function. In the brain, cerebrovascular reactivity is crucially important in maintaining cerebral autoregulation and cerebral blood flow over a large range of arterial pressures. However, the aging process changes complex interactions between the brain parenchyma and cerebrovascular system, and these changes have effects on health and functioning that negatively impact cognition. Along with arterial inflammation, arteriosclerosis is one of the earliest measurable changes in vascular function. Arterial stiffening occurs before many negative changes to the rest of the cerebrovascular system, brain, and cognition related to dementia, including Alzheimer’s disease (AD) in the elderly. Specifically, elastic fibers, collagen, and smooth muscle cells comprise the arterial wall, and these components deteriorate over one’s life due to aging-associated mechanisms. Carotid artery dispensability and compliance start to decrease around 30 years old. However, elastin fibers are not normally generated when they are impaired, whereas collagen, which increases the stiffness, is produced. CBF pulsatility is directly related to carotid pulse pressure and predictive WM hyperintensities. Systolic blood pressure has been associated with GM volume loss in a cross-sectional study, and can result in cerebrovascular injuries, accelerated atrophy, WM abnormalities, and asymptomatic infarcts. In addition, cerebral vasculature is associated with cognitive function, as metabolic demand decreases with increasing age and functional adult neurogenesis. Specifically, large-vessel factors, such as atherosclerosis, increase the risk of AD and may play a role in vertebral vessel amyloid deposition.

9Microscopically, lipofuscins, neurofibrillary tangles, and senile plaques form and increase with age. Lipofuscin accumulates in some neurons. Lipofuscin contains peroxidased proteins and lipids and may express the frequent failure of cells to clear these products of peroxidation-induced cell damage. With aging, cellular proteostasis declines and disturbs the degradation of misfolded proteins that are prone to aggregating into aggrosomes. Normally, lipofuscin is located in the lysosome, as macroautophagy triggers lysosomes to uptake these aggregates. However, when macroautophagy pathways are blocked for reasons such as aging, lipofuscin can accumulate in the cytosol. In normal aged mammalian brains, lipofuscins correlate with a specific senescence pattern that alters the neuronal cytoskeleton and cellular trafficking. The normal brain aging process involves intraneuronal deposits of lipofuscin and neuromelanin pigment. In contrast, in neurodegenerative disorders, lipofuscin accumulation increases with age and pathological processes such as neuronal loss or cellular alterations related to oxidative stress, proteasome, lysosomal, and mitochondrial dysfunction, which will be described below.

10In the process of aging, toxic proteins, such as amyloid-beta (Aβ) protein in AD and tau in frontotemporal dementia, are potential contributory factors in cognitive decline. Normally, amyloid plaques and neurofibrillary tangles are considered hallmarks of AD. However, it is observed during normal aging. Aβ is a protein deposited in the brain in some individuals with aging. In addition, Aβ is higher in some normal individuals with mild cognitive impairment (MCI) than in normal older adults. When neuropsychologically normal, healthy older adults have significant neuropathology at autopsy, and amyloid is common because of the form of amyloid deposition. The number of tangles located in the cell body of affected neurons is low and restricted to the hippocampus, amygdala, and entorhinal cortex in normal aging. However, when minimal neurofibrillary tangles are present, they are located in the transentorhinal region of individuals without dementia. When neurofibrillary changes increase in the brain, the entorhinal cortex is also affected, and cognitive impairment, related to the hippocampus, is triggered with further progress. Paired helical filaments (PHF) and occasional straight filaments comprise neurofibrillary tangles, and it has been shown that patients with AD have neurons severely affected by PHF, as the normal cytoskeleton of microtubules and neurofilaments totally disappear. Therefore, it is believed that a shortage of functional cytoskeleton probably triggers neuronal loss. The contrast between normal and pathological aging is the presence and distribution of neurofibrillary tangles. The number of tangles in each affected tissue is lower in normal aging than in pathological cases and is limited to the olfactory nucleus, para-hippocampal gyrus, amygdala, and entorhinal cortex. In contrast, in pathological aging, the neurodegenerative way, in other words, neurofibrillary tangles are often found with amyloid plaque in patients with AD.

11Other microscopic changes include morphological alterations in neurons. It has been revealed that neuronal loss during normal aging occurs slightly (no more than 10%). However, morphological changes in neurons, especially dendrites and axons, are involved in cognitive decline and behavioral changes. With increasing age, the dendritic tree underwent regression. Dendritic shafts decrease in number, become shorter and less branched, and have fewer spines. Moreover, not every spine is affected to the same extent, and in the specific area of the cortex, thin spines are lost. These spines have high motility and plasticity and are believed to be related to learning. Axons may have glycogen inclusion, degenerated mitochondria, and accumulations of filaments. These modifications may lead to degeneration. Regeneration of axons follows degeneration, and these axons enable regeneration in old age. However, it is delayed and proceeds at a slower rate in the elderly than in younger people. The materials move along the axon by slow transport, and the speed of this transport decreases with aging. The decreased rate of transport is believed to explain the process of degeneration of axons. Furthermore, the myelin sheaths of nerve fibers and synapses are also affected by aging. The myelin sheath plays a crucial role in the rapid propagation of action potentials by providing insulation to axons. However, they are altered by demyelination, remyelination, and myelin decomposition. Through these changes, myelin sheaths lose conduction velocity, and the degrees are slowed with aging. In addition, the number of synapses is reduced, with the rate ranging from 15 to 50% depending on the species and the region of the nervous system. This alteration is associated with the regression of the postsynaptic structure and loss of presynaptic structures. Although the number of synapses may decrease, the structure of synapses does not change with old age.

12Aging is accompanied by several biological changes in the brain. The manner in which neurons transmit information through nerve impulses is called the action potential. When an action potential is transmitted to the presynaptic terminal of the synapse, it may trigger the release of neurotransmitters. These neurotransmitters are released by synaptic cleavage and bind to synaptic receptors. This process affects other neurons in an exciting or inhibitory manner, causing other neurons to induce action potentials. It revealed that various neurotransmitters and receptors change in several regions of the brain during the aging process. Specifically, major neurotransmitter systems can be classified as cholinergic systems, monoamine systems (catecholamine: norepinephrine and dopamine, and indole amine: serotonin), and others such as amino acids, nitro oxide, and hormones.

13Acetylcholine activates skeletal muscles in the somatic nervous system and may affect internal organs in the autonomic systems. In particular, cholinergic pathways control cognitive processes and behaviors such as wakefulness, mood, learning motor function, motivation, short-term memory, and a minor part of reward responses. According to the cholinergic hypothesis, dysfunction of the cholinergic system plays a role in the memory decline often observed in aging and dementia. With normal aging, the nicotinic binding ability is lost. In particular, cholinergic neurons and certain nicotinic acetylcholine receptor (nAChRs) subtypes are specifically reduced or eliminated during normal aging. It has been revealed that high-affinity nicotine-binding sites in the entorhinal cortex and presubiculum are largely lost in those over 40 years. Moreover, considering that nAchRs trigger the reactivation of neurogenesis by endogenous neural stem/progenitor cells, it is easy to believe that reduction in nAchRs may affect neurogenesis. In fact, it has been identified that a decreased cholinergic input disturbs proliferation, short-term survival, and differentiation into mature neurons, whereas it promotes apoptosis in the hippocampus in mice. Cognitive deficits associated with aging and AD are also associated with cholinergic deficits. Therefore, the maintenance of nicotinic receptors may be important for neuronal survival.

14Norepinephrine and dopamine are the most important neurotransmitters that are associated with aging. They play important roles in the regulation of synaptic plasticity and neurogenesis in the adult brain. Serotonin and brain-derived neurotrophic factor levels decrease with aging. Furthermore, monoamine oxidase increases with age and results in the generation of excess free radicals that exceed the inherent antioxidant process.

15Norepinephrine or epinephrine functions in the central nervous system to regulate sleep patterns and alertness. These charges regulate adrenal glands and fight-or-flight responses. Norepinephrine is associated with cognitive processes and behaviors such as anxiety, arousal, circadian rhythm, cognitive control, and working memory. They also regulate feeding and energy homeostasis, medullary control of respiration, negative emotional memory, nociception and play a minor role in the reward related system.

16Dopaminergic pathways are related to cognitive processes and behaviors such as wakefulness, aversion, cognitive control, working memory (along with norepinephrine), emotion and mood, motivation, motor function, positive reinforcement, reward, sexual arousal, and the refractory period. In age-related changes in the dopaminergic system in the brain, dopamine synthesis, binding ability, and its receptors decrease. According to positron emission tomography (PET) of the normal aged brain, dopamine synthesis is significantly reduced in the striatum and parastriatal region with aging. In addition, D1, D2, and D3 dopamine receptors also decreased. Dopamine levels decrease by 10% per decade from early adulthood, and in accordance with this change, cognitive and motor performance decline. That is, with increasing age, the levels of dopamine decline, and synapses/receptors and binding ability are also reduced in the dopaminergic pathway between the frontal cortex and striatum. Moreover, dopamine levels are associated with various brain regions, such as the anterior cingulate cortex, frontal cortex, lateral temporal cortex, hippocampus, medial temporal cortex, amygdala, medial thalamus, and lateral thalamus. These deficiencies cause age-related neurological symptoms such as a decrease in arm swing, increase in rigidity, and cognitive flexibility changes.

17The serotonergic pathway is associated with cognitive processes and behaviors such as wakefulness, body temperature regulation, emotion, and mood, including aggression, feeding and energy homeostasis, and sensory perception. It affects appetite, sleep, memory, learning, temperature, mood behavior, muscle contraction, and function of the cardiovascular and endocrine system. The number of serotonin receptors and transporters decreases with increasing age. According to PET studies, the number of S1 receptors in the caudate nucleus, putamen, and frontal cerebral cortex decreases. In the same context, the binding capacity for serotonin transporters in the thalamus and midbrain is also diminished.

18The brain is a steroidogenic organ that retains steroidogenic enzymes and produces neurosteroids. Neurosteroids are steroids produced from various regions of the brain, mainly in the hippocampus, and they are the endogenous regulator of neuronal excitability. The precursors of neurosteroids are the circulating steroid hormones. Neurosteroids belonging to the sex hormones may have neuroprotective effects against brain aging features, especially Aβ- or tau-related toxicity and oxidative stress. For example, with the neurosteroid estrogen receptors widely distributed in the brain, estrogen plays a role as a neuroprotective antioxidant against several toxins, which in turn encourage the production of free radical. The brain consumes a high rate of oxygen and consists of the neuronal membrane with a high concentration of polyunsaturated fatty acids, factors that leads to the risks of lipid peroxidation. Therefore, the antioxidant activity of the brain is required for its homeostasis. Estrogen may play important role in the regulation of mitochondrial function both directly and indirectly, yet this process has not been identified clearly. Their effects are important to the central nervous system (CNS) which demands high energy. Along with the metabolism regulation, estrogen also affects mitochondria in neuronal tissues during biogenesis, apoptosis, and morphology. Further, estrogen protects mitochondria from oxidative damage, which may cause mitochondrial DNA mutations.

19In the senescence process, the decline of neurosteroids, especially that of testosterone in men and estrogen in women after menopause, may impair neuronal function and cause significant age-associated neurodegenerative diseases. Testosterone regulates brain functions including dendritic spine morphology, neurogenesis, and learning and memory. When testosterone level declines in the serum and brain, the blood–brain barrier (BBB) integrity and the expression of tight junction proteins are disrupted, which may in turn induce an inflammation activity. Androgen, a metabolite of testosterone, also carries neuroprotective function. Its depletion, which comes with that of testosterone, may stretch into the decline of CA1 spine synapse density. Again when age, the local synthesis of estrogen is declined due to the reduced expression of aromatase, the enzyme that synthesizes estrogen utilizing androgen. Additionally, the expression of estrogen receptors in the brain drops during aging, successively causing detrimental impacts on memory and learning by retarding the signaling pathways in the brain areas where estrogen receptors are mediated, that is hippocampus and prefrontal cortex. The reduction of synapse number and spine density in the CA1 area of the hippocampus is observed in multiple animal models, especially those with rats, when their ovarian hormones decreased either naturally or artificially. Estrogen’s effects on the hippocampus are also shown to be less responsive for older models than younger ones, for there is a significant gap in the number of estrogen receptors between the two groups.

20Sex hormones, or neurosteroids, interact with the insulin receptor, which regulates glucose and carbohydrate metabolism. Glucose being the main energy source of the brain, approximately 20% of the total metabolized glucose in the body at a rest state is consumed by it. Therefore, disturbed glucose metabolism can lead to various brain malfunction. Considering that peptides and steroid hormones control the influx of glucose in cells, the decrease in neurosteroids during aging may pervert glucose homeostasis of the brain.

21Associated with aging, a number of microarray studies have been implemented to identify genome-wide changes in gene expression, particularly in the brain. Specific biological pathways change due to the aging process, as opposed to the genome-wide dysregulation of transcription. In addition, the expressed genes triggered by the induction of stress are related to aging. In a study that conducted transcriptional profiling of the aging human frontal cortex in 30 individuals from 26 to 106 years of age, approximately 4% of the genes expressed in the brain were found to be age-regulated. Specifically, age-associated changes in gene expression become obvious in middle age and are most clear after 70 years of age. Synaptic function-related genes, which mediate memory and learning, were significantly downregulated. These genes include glutamate receptor subunits, synaptic and vesicle proteins, and members of major signal transduction systems that mediate long-term potentiation (LTP). For example, synaptic calcium signaling systems are probably affected by decreased expression of calmodulins 1 and 3, several Ca2+/calmodulin-dependent protein kinases (CAM kinases), and multiple protein kinase C isoforms. Other categories of age-reduced genes are vesicle-mediated protein transport and mitochondrial function. In addition, genes related to stress responses, such as antioxidant defense, DNA repair, and immune function, represent the largest category and have been found in several different cortical areas of the aging human brain. Gene expression alterations affect the susceptibility of the aging brain to neurodegenerative disorders. For instance, a microarray study of AD indicated that a substantial number of expressions are related to pathological markers and cognitive test scores. Specifically, it is upregulated in signaling and tumor suppressor genes and downregulated in protein folding, metabolism, and energy-related genes. Therefore, neurodegeneration and cognitive decline may be involved in specific alterations in gene expression.

22One of the primary hallmarks is telomere shortening. According to the telomere theory of aging and cellular senescence, cells have a definite number of divisions and define when replication is suitable. Telomeres, which have a role in the biological clock, have thousands of tandem DNA repeats, TTAGGG at the end of each linear chromosome. Telomeres are important because they are engaged in genome maintenance and promote stability through replication procedures, preventing chromosomal fusion and unnecessary recombination. Telomerase is a ribonucleoprotein enzyme consisting of two essential subunits: telomerase reverse transcriptase protein (TERT) and telomerase RNA (TER). It reverses telomere shortening like stem cells. Telomere shortening and its role in the healthy aging process of the brain and neuronal senescence at the cellular level have not been explained.

23Age-related alterations in neurons are not fully understood. As neurons are post-mitotic, cell division, the main factor for telomere shortening, has been regarded as absent in neurons when they reach terminal replication. However, this view has been opposed by the investigation of DNA content variants, describing cell cycle activity in approximately 10–20% of post-mitotic neurons in the cortex of healthy aging brains and AD. Telomerase activity restores various features of aging in somatic cells, such as senescence. However, in some somatic tissues, including the central nervous system, the telomerase enzyme has downregulated transcript levels and activity, which are related to protein levels. In particular, telomerase function in post-mitotic cells, such as neurons, is not related to telomere elongation but is involved in cell survival-promoting function. Furthermore, hippocampal TERT plays a role in modulating mood behaviors by controlling the proliferation of neural progenitor cells (NPCs) and is needed for spatial memory formation. In this respect, hippocampal-dependent learning and memory functions and neurogenesis in the hippocampus are downregulated in telomerase-knock mice. In addition, when human NPCs are cultured, it is shown that NPCs have a restricted amount of cell divisions and undergo senescence. Telomere erosion occurs in the aging brain in a cell cycle-independent and dependent way. In addition, one controversial study reported that leukocyte telomere length is associated with structural changes to the brain and impaired cognitive capacity during aging.

24Mitochondria are intracellular organelles known for producing cellular energy substances (ATP, adenosine triphosphate) and managing cell functions by controlling secondary messengers in cell signaling mechanisms (Calcium ion, Ca2+) and reactive oxygen species (ROS). Mitochondria drive the derivation and storage of energy using the respiratory chain via oxidative phosphorylation. A single neuron contains hundreds or thousands of mitochondria. However, abnormal ROS and calcium ion levels induce mitochondrial damage, which triggers mitochondrial dysfunction. Specifically, mitochondrial damage disturbs mitochondrial DNA (mtDNA) maintenance and ATP production and promotes apoptosis.

25The aging process is accompanied by mitochondrial alterations, and key reported features are somatic point mutations and large deletions in mitochondrial mtDNA. These mtDNA mutations alter the transcription of electron transfer complex proteins, which are used in the mitochondrial respiratory system. This alteration has been shown to be accountable for mitochondrial dysfunction, especially in interfering with ATP production and increasing ROS generation. Moreover, mtDNA mutations affect mitochondrial morphology. mtDNA integrity is maintained by mitochondrial dynamics, fusion, and fission. The mtDNA heteroplasmy, a mixture of mutated and normal mtDNA, is formed during mitochondrial dynamics and is controlled by proteins such as mitofusins (Mfs1 and Mfs2), mitochondrial dynamin-like GTPase (Opa1: fusion), and dynamin-related protein 1 (Drp1: fission). Prevalent fission triggers respiratory impairment and an increase in ROS levels, whereas excess fusion can hinder mitochondrial autophagy induced by large and defective mitochondria.

26From the perspective of ATP production, since neuronal cells are highly energy-consuming, mitochondrial dysfunction is considered a serious factor in neuronal disease. In terms of brain energy metabolism, ATP is the main source of the cellular system. Specifically, ATP is consumed by the cell membrane pumps Na+/K+-ATPase and Ca2+ATPase, which regulate ion gradients during neuronal signaling. ATP is also used in the arrangement of neuronal cell organelles. When axons transport mitochondria, RNA, proteins, vesicles, and other cargo to the presynaptic terminal, the process is controlled by calcium, motor protein, and microtubules, ATP is required. Additionally, neuronal networks related to fast-spiking interneurons demand high metabolic energy, and this process is supported by mitochondrial oxidative phosphorylation (OXPHOS). However, at the cellular level, mitochondrial dysfunction, specifically mutated mtDNA, occurs, leading to a decrease in ATP production generated by OXPHOS, beta-oxidation, and the Tricarboxylic acid cycle(TCA) cycle. In particular, AD has features of mitochondrial dysfunction with a lower uptake of glucose and a decrease in the TCA cycle. Many PET studies have shown that MCI is related to a 10–12% deficit in glucose uptake. Furthermore, this deficit becomes more widespread with the advent of AD and worsens during its progression.

27Furthermore, mitochondrial dysfunction induces apoptosis and programmed cell death. Mitochondria play a role in the intrinsic apoptotic pathway. The intrinsic apoptotic pathway is the prevalent mechanism of neuronal death. This mechanism involves increased production of ROS, cytochrome c release, which mediates the TCA cycle in mitochondria, ATP depletion, and caspase 9 and 3 activation. Probable triggers of apoptosis include the interaction with α-synuclein. α-Synuclein is abundant in the central nervous system and is a major factor in Lewy bodies, a pathological indicator of PD. Aggregation of α-synuclein in dopaminergic neurons decreases the activity of mitochondrial complex I and increases ROS. ROS and the release of cytochrome c into the cytosol evoke mitochondria-mediated apoptosis as mitochondrial dysfunction occurs. Another factor that causes apoptosis is dopamine metabolism. Dopamine blocks mitochondrial complex I, which leads to mitochondrial dysfunction, and produces anti- and pro-apoptotic factors. Dopamine metabolism is also accompanied by the generation of ROS and dysfunction of the mitochondrial complex I. Damage to the activity of mitochondrial complex I has been suggested to increase the vulnerability of dopaminergic neurons to neuronal disorders by downgrading the threshold for activation of the intrinsic apoptotic pathway. Although it is not clear whether apoptosis induces neurodegeneration in AD, it is certain that several molecules of this pathway are triggered in the AD brain. For instance, caspase 3, an executive caspase that regulates apoptosis, is cleaved and activated in AD and is linked to tau cleavage and neurofibrillary tangle (NFTs) formation.

28Cellular ROS are generally produced by both exogenous and endogenous sources. Exogenous sources of ROS production are ultraviolet (UV) radiation, ionizing radiation, and drugs that mediate their mechanism through ROS production. Additionally, environmental toxins and chemicals are responsible for ROS, which are metabolism by-products. In the case of endogenous sources, mitochondrial and non-mitochondrial ROS-generating enzyme produces ROS. Non-mitochondrial ROS enzymes include nicotinamide adenine dinucleotide (NADH) phosphate oxidase (Nox), xanthine oxidase, cytochrome P450 from the endoplasmic reticulum, and flavin oxidases from peroxisomes.

29However, most cellular ROS (90%) are generated through mitochondrial ATP production by OXPHOS. While the mitochondrial electron transfer chain system (ETC) operates during OXPHOS and produces ATP, complexes I and III of the ETC mainly generate ROS. If this damage is not compensated by the antioxidant process, elevated generation of ROS triggers neuronal damage. Antioxidant systems protect against ROS and consist of antioxidant enzymes such as glutathione peroxidase (GPX), non-enzymatic antioxidant factors, superoxide dismutase (SOD), and catalase. During aging, it has been reported that antioxidant capacity decreases.

30Molecules damaged by oxidative stress are present in lipids, proteins, DNA, and RNA. Lipid peroxidation (LPO) is triggered by lipids that are attacked by ROS through a free-radical chain mechanism to generate LPO products. In particular, 4-hydroxy-2,3-non-enal (HNE), a common cytotoxic product of LPO, damages neurons and functions of membrane proteins, such as the neuronal glucose transporter GLUT.

31In terms of damaged proteins, those vulnerable to oxidative stress can be classified into several groups: metabolic pathways responsible for glycolysis and metabolism, energy metabolism, mitochondrial proteins, cytoskeleton, chaperones, and members of the ubiquitin-proteasome system (UPS). Protein oxidative damage may result in abnormalities in the nervous system, such as abnormal glycolysis and energy metabolism, abnormal protein folding and oxidative stress responses, abnormalities in the cytoskeleton, and damaged protein degradation. For example, a number of proteins are sensitive to oxidative stress, such as the chaperone and ubiquitin-proteasome systems, and this damage results in abnormal neuronal function in AD. Therefore, it is appropriate to think that some of the metabolic disturbances monitored at the advent of degenerative processes are engaged with oxidative damage of selected proteins other than neuron loss.

32In the case of mtDNA damaged by oxidative stress, as mentioned above, most ROS are generated from mitochondria, and impaired mtDNA is the common oxidative damaged molecule. Whether mitochondrial ROS affects mtDNA mutations has not been confirmed, but mtDNA changes are more likely to increase ROS beyond normal concentrations, causing neuronal damage. According to the mitochondrial theory of aging proposed by Harman in the 1970s, somatic mtDNA mutations injure OXPHOS complexes, resulting in ROS production (complex I and III). Subsequently, these ROS impair proteins, lipids, and DNA, including mtDNA, and again, these impaired molecules produce new ROS and damage normal molecules. Through this process, a vicious cycle is formed.

33From the perspective of cellular DNA, oxidative stress can induce DNA double-strand breaks, DNA/protein or DNA/DNA cross-linking, and base modification. DNA bases are sensitive to oxidative damage such as hydroxylation, nitration, and protein carbonylation. Similarly, RNA is commonly single-stranded and is a target of oxidative damage/modification, similar to DNA. Generally, oxidative damage to DNA/RNA is increased in AD. Specifically, 8-hydroxy-2-deoxyguanosine and 8-hydroxyguanosine levels, which indicate DNA and RNA oxidation, are elevated, and these markers are localized in Aβ plaques and NFTs. In addition, the levels of oxidized rRNA or mRNA are also increased in AD.

34The features of neuronal cells are retained in cellular machinery for protein synthesis and degradation. In contrast to other cells, neuronal cells have a unique morphology, which is a specific area of presynaptic neurotransmitter release, postsynaptic receptor activation, and plasticity of synapses related to alterations in the synaptic proteome. Since molecular machinery proteins play a role in mediating signal transduction, protein synthesis and degradation are important for maintaining the plasticity and memory of neuronal cells. In eukaryotic cells, there are two major degradation systems that provide cell recycling of cellular components, ranging from soluble proteins to intracellular organelles: autophagy, which plays a role in the degradation of long-lived, insoluble or accumulated proteins and cellular organelles, and UPS, which usually degrades the most soluble, short-lived protein. Although they have different mechanisms of action, both play important roles in regulating cell homeostasis.

35Cells can digest cytosolic components via autophagy lysosomal degradation. In addition to clearing the cytosol for macromolecules and impaired organelles, the autophagy process provides cells with amino acids and energy by recycling, which is energy efficient. Since autophagy plays an important role in the production of long-lived proteins and the elimination of damaged organelles and cellular debris, it is considered a part of the antiaging process. However, lipofuscins, which are aggregates of insoluble particles formed during the aging process in post-mitotic cells, accumulate in autophagosomes and hinder the autophagic system, which is a critical protective mechanism in the cell. Impairment of the autophagic system, which is induced by aging, causes excessive ROS production from the mitochondrial respiratory system, interrupts recycling, and increases oxidative stress. For example, mitophagy reduction (mitochondrial autophagy) is related to aging-induced diseases such as Parkinson’s disease (PD). Mitophagy reduction disturbs the removal of Lewy bodies and the aggregation of filamentous intracytoplasmic inclusions, which could trigger brain disorders, such as AD or PD. Another neurodegenerative disease related to autophagy reduction involves genetic alterations and protein expression modification. For instance, changes in PTEN-induced novel kinase 1(PINK1) and parkin, which are related to mitophagy, correspond to 5% of PD. Normally, when mitochondria are impaired, PINK1 aggregates in the outer membrane of mitochondria, parkin is recruited, and mitochondria start to degrade. However, if the PINK1-parkin pathway is dysfunctional due to aging, the homeostatic process conducted by the mitochondria is disturbed.

36The other perspective of protein degradation is the proteasome. Proteins that are fated for degradation are tagged with ubiquitin by UPS. The UPS targets intracellular, soluble, and transmembrane proteins that are extracted from the membrane into the cytosol. The UPS plays a significant role in the regulation of memory and neurotransmitter release. The balance between protein synthesis and degradation is responsible for long-term plasticity and memory. In addition, synaptic transmission at the pre- and postsynaptic terminals is controlled by UPS. For example, in hippocampal neurons at presynapse, the frequency of miniature excitatory postsynaptic currents increases when the proteasome is inhibited. In animal models, transgenic animals lacking SCRAPPER showed a large increase in excitatory postsynaptic currents. Considering that SCRAPPER is an E3 enzyme located in the presynaptic membrane and regulates vesicle release, the UPS at the presynaptic membrane plays an important role in controlling the size of the vesicle pool and vesicle release. Furthermore, the UPS is responsible for the abundance of proteins that regulate postsynaptic responses, including ionotropic glutamate receptors and proteins related to postsynaptic density (PSD). Chronic inhibition of action potentials, such as tetrodotoxin, or of inhibitory neurotransmission, bicuculline, changes PSD proteins. Interestingly, these changes are blocked by proteasome inhibitors, which indicates the significance of proteolysis in restructuring the synapse corresponding to changes in neural activity. In the process of aging, cellular proteostasis declines, misfolds, and damaged proteins aggregate. These failures in proteostasis are involved in the stabilization of correctly folded proteins and protein clearance systems.

37Electrolytes, located in extracellular and intracellular fluid, form electrical currents in the body. These ions exercise a crucial role in maintaining homeostasis. Along with Ca2+, the movement of ions through ionic channels engages in the action potential (AP), the process which is essential for neuronal signals. In neurons, ions move rapidly through a whole course of depolarization, repolarization, and signal propagation, producing AP. Such ionic channel actions are categorized by which ions participate and how the ion channel gating permeation works. The voltage-gated ion (Na+, K+, Cl–, and Ca2+) channels and ligand-gated ion channels are two examples. When depolarization is activated, ions cross the membrane through the channels that match their electrochemical gradients. Ion channels in membranes trigger nerve impulses and synaptic transmission.

38The intrinsic electrophysiological properties change with age. Normally, AP has three main stages: depolarization, repolarization, and hyperpolarization. Depolarization occurs when the influx of Na+ exceeds the AP threshold and the voltage-gated Na+ channels are open. Repolarization happens with the closing of Na+ channels and the opening of K+ channels. Hyperpolarization is when excessive K+ is accumulated and moves outside the cell through the opened K+ channels. Nonetheless, recent studies show that the AP threshold of the hippocampal CA1 pyramidal cell is higher for aged rats. Similarly, even though it has not been fully proven, this age-based variation in the AP threshold may also be applied to humans, affecting the voltage-gated Na+ channel activation properties and channel subtype expression patterns. This variation in the AP threshold may disturb the excitability of neurons and repress neuronal activities by lowering the transmissive function of neurons. Such impairment in the brain might have a correlation with cognitive decline during senescence.

39Action potential amplitude takes up a critical role in evoking Ca2+ currents and regulating the neurotransmitter release through axon terminals. Some studies show AP amplitude decreases in primates while they age. This phenomenon can be explained by either a reduction of Na+ channels or an increase in K+ channels. Age-based changes in AP amplitude could be a result of the altered expression of voltage-gated Na+ channel subunits, reduced expression of Na+, or altered expression of K+ channel which involves the K+ currents. Additionally, several studies reveal that AP axonal conduction velocity decreases alongside the aging process. The cause of such a decrease might be demyelination, the process that induces an ion leakage and reduces the efficiency of transduction.

40Calcium (Ca2+) is a secondary messenger in the cell signaling mechanism. The optimal concentration of Ca2+ remained constant in the cell and extracellular space at the expense of energy. Through this process, cells promote Ca2+-induced signaling pathways and inhibit Ca2+ driven excitotoxicity. Ca2+ is mainly regulated by Ca2+influx through ligand-gated glutamate receptors, such as N-methyl-D-aspartate receptors (NMDARs) and various voltage-dependent Ca2+channels (VDCCs). Moreover, intracellular organelles, such as mitochondria or Endoplasmic Reticulum (ER), and Ca2+-binding proteins (CBP) also manage homeostatic mechanisms. Intracellular organelles store Ca2+, and CBP functions as a buffering agent that reduces the peak intracellular calcium ion concentration.

41N-methyl-D-aspartate receptors (NMDARs), which are ionotropic non-selective cationic glutamate receptors, play a major role in the rapid control of synaptic plasticity. NMDARs are composed of ubiquitously expressed essential subunits (NR1) and modulatory subunits (NR2A-NR2D). NMDAR activation requires glutamate (binding of ligand), membrane depolarization (to eliminate the Mg2+ that blocks the channel), and binding of glycine, which is a co-agonist. Although NMDARs are non-selective, they are the most permeable to Ca2+ ion. NMDAR functions within the brain region decline with aging, including learning and memory. There are several reasons why a decrease in NMDAR function due to aging may occur. One of the mechanisms is a decrease in the level of NMDAR protein expression, especially its subunits, in the hippocampus with aging. The decrease primarily occurs in the CA1 region. Specifically, the expression of NR1 and NR2 (NR2A, NR2B) subunit proteins and their mRNA levels have been found to decrease in the aged hippocampus. The alteration in the expression of specific NR2 subunits may severely affect NMDAR function in relation to the regulation of the average channel open time and conductance of NMDARs. Modified NR2 subunits induce changes in the time course and magnitude of Ca2+ signaling, resulting in reduced Ca2+ influx. Therefore, a shift in NR2A and NR2B expression triggers developmental changes in cognition and synaptic function. Moreover, the NMDAR binding affinity of glutamate decreases during aging and is related to memory decline. There are several changes in the binding affinity of NMDAR, including glycine or other antagonist. Additionally, post-translational modifications of receptors are related to NMDAR functions. For example, the phosphorylation state of NMDAR, which is triggered by several kinases such as tyrosine kinase, protein kinase C, and protein kinase A, increases NMDAR-mediated current. In contrast, protein phosphatases such as calcineurin act vice versa. Aging is associated with a disturbance of kinase/phosphatase activity, which is inclined to phosphatase activity, which decreases NMDAR function. Therefore, NMDAR function is disturbed by aging due to altered phosphorylation. In terms of VDCC, VDCCs are ion channels in the plasma membrane and open during membrane depolarization. VDCC enables cellular Ca2+influx from the extracellular space. VDCC is classified into L-, P/Q-, and N type channels. Interestingly, during aging, L-channels increase in the hippocampus, which inhibits the blockade of L-type VDCCs. Increased L-channels lead to an increase in after-hyperpolarization (AFP) in accordance with an increase in Ca2+influx. Considering that long-term potentiation (LTP) occurs from depolarization and LTP requires blocking specific Ca2+ sources, an aging-related increase in the L-channel impairs the LTP threshold and LTP is inhibited. Ca2+ channels suggest a point of crosstalk between age-related Ca2+ regulatory disorders and signaling in neurodegenerative diseases.

42Intracellular Ca2+ reserves include cellular organelles and CBP. Cellular organelles, mitochondria, ER, and lysosomes have Ca2+ buffering systems, which release and sequester Ca2+. In the case of mitochondria, mitochondrial Ca2+ induce the TCA cycle to generate energy by neutralizing the polarized negative membrane potential, which is produced from ATP production in the mitochondrial matrix. However, aged mitochondria have structural changes in the mitochondrial DNA and mitochondrial membrane, which results in a net reduction in the Ca2+ buffering capacity. Decreased Ca2+ uptake capacity directly contributes to decreased electrochemical gradients across the mitochondrial membrane. This mitochondrial depolarization may increase the threshold level of Ca2+ required to initiate mitochondrial uptake. Consequently, this triggers an age-dependent delay in Ca2+ sequestration or the recovery of intracellular Ca2+. However, from the perspective of ER, ER regulates Ca2+ in two pathways: the inositol (1,4,5)-triphosphate (IP3) pathway activated by G protein-coupled receptors (GPCRs) and Ca2+induced Ca2+release (CICR). In short, the GPCR and CICR pathways regulate Ca2+ through IP3R and ryanodine receptors (RyRs), respectively. However, with aging, the effect of Ca2+ dysregulation due to activation of these receptors is cell-specific and depends on other Ca2+cellular regulating mechanism. For example, CICR is reduced during aging in peripheral synapses because of decreased expression of RyRs, and the reduction in Ca2+ amplification in basal forebrain neurons is compensated by mitochondrial buffering. Furthermore, recent studies have proposed the possibility of lysosomes acting as Ca2+ storage organelles. Although altered lysosomal control of Ca2+ is due to aging remains unclear, it has been revealed that the aging brain shows increased lysosomal markers and decreased lysosomal function. CBP, such as parvalbumin, calbindin-D28K, calretinin, calmodulin, and hippocalcin, which handle the intracellular Ca2+ level by rapid Ca2+ buffering in cytosol, is regarded to be neuroprotective. Generally, with aging, changes include a decrease in the expression of CBP, which is related to the loss of function, and these changes are cell and region specific. A decrease in Ca2+ buffering or delayed elimination could cause larger or prolonged Ca2+responses, which are common in aged neurons. Slight alterations in the concentration of Ca2+ are fatal for neurons. However, Ca2+ homeostasis in neurons is disturbed by changes in physiological and molecular levels in accordance with increasing age, finally provoking neurodegeneration.

43Aging is a risk factor for a number of neurodegenerative diseases. There are mechanisms in the normal brain aging process that can converge to neuronal disorders. Under conditions of normal brain aging, the changes were reviewed in terms of structural, biochemical, metabolic, cellular, and molecular mechanisms.

44Both gross and microscopic structural changes have been reported. Gross changes include multiple morphological changes, including cerebral atrophy, sulci widening, and cerebrovascular changes. In contrast, microscopically, lipofuscins are accumulated, occupying a large space of cytoplasm where glycoproteins, lipoproteins, and neurotransmitters are synthesized. However, from another perspective, lipofuscin has a positive role in energy production when synergized with myoglobin and respiratory enzymes. Other microscopic changes are associated with amyloid-beta and neurofibrillary tangles, which are regarded as neurotoxic proteins. These changes starve the neurons and have the potential to cause cognition loss. In addition, morphological changes in neuronal cells can be considered aging-related degeneration. Overall, structural changes initiate the destruction of the surrounding neuropil, leading to cognitive damage in dementia. Histopathologic changes appear, especially in the limbic system and most of the CNS.

45Regarding the biochemical and metabolic changes involved in aging, several types of neurotransmission molecules are involved, such as acetylcholine, monoamine, and neurosteroid. They affect the intracellular condition by diminishing related enzymes, receptors, and protein functions and triggering an imbalance of neurotransmitters. This disrupted balance influences various regions of the brain, such as the hypothalamus, pituitary gland, and pineal gland, and cascades of dysfunction occur.

46Lastly, cellular and molecular changes with aging largely occur in the nucleus, mitochondria, oxidatively managed molecules, lysosomes, proteasomes, electrophysiological regulation and Ca2+ homeostasis. During normal brain aging, DNA expression changes, and telomere length are shortened in the nucleus (Figure 1). Conversely, mitochondrial dysfunction is largely caused by mtDNA, ROS, and calcium alterations. These changes have been attributed to abnormal mtDNA integrity, ATP production, and apoptosis. Oxidatively damaged molecules have been reported to be promoted mainly by the respiration of mitochondria. This alteration affects the mitochondrial respiratory system itself, especially in complexes I and III. In addition, these damaged molecules, such as lipids, proteins, and DNA/RNA, along with successive damage, form a vicious cycle that triggers neurodegenerative disorders. As neurons have exotic morphology and function compared with other cells, efficient utilization of energy systems is essential. Lysosomal autophagy and ubiquitin-proteasome functions are important for the reutilization of useful molecules and the removal of cellular waste. However, during the aging process, the functions of the clearance system decrease, and abnormal proteins accumulate. This disturbance contributes to age-related neurodegenerative disorders such as AD and PD. Electrophysiological regulation is controlled by the homeostasis of electrolytes. During senescence, however, the regulation is disturbed by the alteration of channel expression and the modification of the conductive rate of currents. In respect of Ca2+ homeostasis, Ca2+ alteration is fatal to neurons, and its regulation is tightly regulated through NMDAR, VDCC, mitochondria, ER, and Ca2+related proteins. However, due to aging, homeostasis is attributed to alterations in ion exchange systems, a decline in metabolic events, and Ca2+related proteins such as receptors. They are also associated with abnormal neuronal functions.

47Therefore, normal brain aging entails neurodegenerative changes in the brain. It is worth concentrating on how and why normal aging processes are correlated with neurodegenerative disorders. Through this analysis, a profound understanding of aging in the normal brain and the related factors that generate age-induced neuronal disorders is expected. As everyone ages, it is important to comprehend and limit risk factors to promote healthy brains and quality of life. The mechanism of normal aging-induced neuronal disorders should be considered in the future.

48H-JK: study concept and design and critical revision of the manuscript. H-JK and JL: acquisition, analysis, and interpretation of data and drafting of the manuscript. Both authors contributed to the article and approved the submitted version.

49The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

50All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.