Chapter 1: Alzheimer's is a Multifactorial Disease

The central nervous system (CNS) consists of the spinal cord and the brain is the sophisticated body system that controls most of the body's functions. The complex structure, function, and operating efficiency of the mysterious brain have attracted philosophers, scientists, and engineers to gain deeper insights and emulate them for biomedical and technological advancements. Advanced neurological and behavioral research has revealed some of the structural and functional mysteries of the brain and associated disease mechanisms. The brain cells (neurons) are highly specialized in establishing complex connections and networks to efficiently relay the sensory signals and information to-and-fro from the brain to coordinate the physiological or psychological body functions. The loss of neuronal functions and developing atrophy due to neurodegeneration is the leading cause of several neurodegenerative disorders and the condition is collectively termed dementia. Dementia is characterized by the symptoms of impairment in memory, communication, behavior, and thinking that severely affect the ability to perform day-to-day activities. More than a century ago, Alois Alzheimer, a German physician, described a unique progressive neurodegeneration, which was subsequently named Alzheimer's disease (AD) by his colleague Emil Kraepelin.^1,2 

AD is a chronic neurodegeneration-related disorder and rapidly growing health calamity worldwide with 70–80% contribution to all dementia cases.³  Aging is one of the leading risk factors of AD, while genetic factors and family heredity are known to have a definite role. The absence of accurate diagnosis and treatment is contributing to its fast rise as a global health and economic burden. There are currently ∼50 million people suffering from AD, and this number is expected to cross over 130 million by 2050.⁴  Besides, the statistics reveal that the number of deaths by AD increased by ∼146% between 2000 and 2018, while all other major diseases such as heart diseases, HIV, and cancer showed an appreciable decrease owing to the availability of improved detection and treatment options.²  The evidence accumulated through multidisciplinary academic and clinical studies have indicated the multifactorial nature of AD (Figure 1.1). The multifactorial nature characterized by multiple toxicities hampers the development of accurate diagnosis and disease-modifying therapies.⁵  There are two types of AD, namely (i) early-onset, a rare form of AD contributing to <5% of total AD, and (ii) late-onset, the most prevalent form of AD. Both AD types show a multifactorial nature and are associated with multifaceted toxicity.^3,4  The abnormalities linked to various vital physiological processes are responsible for the multifaceted toxicity, which includes cholinergic toxicity, amyloid burden, tau toxicity, metal ion toxicity, oxidative stress, biomolecular damage, immune outrage, calcium ion (CaII) dyshomeostasis, neurovascular toxicity, lymphatic dysfunction, mitochondrial dysfunctions, α-synuclein mediated toxicity, synaptic dysfunctions, membrane toxicity, apoptosis dysfunctions, impairment in telomerase activity, aberrant post-translational modifications, microbial imbalance and infection, glucose hypometabolism, endoplasmic reticulum stress, the role of cholesterol, autophagy dysfunctions, genetic risk, and insulin resistance and diabetes among others. This chapter provides a concise overview of AD onset and progression concerning various individual pathophysiological processes and their interplay (Figure 1.1). We briefly refer to the current status of biomarkers, diagnosis, targets, and therapy as a prelude to the detailed discussion of each topic in the subsequent chapters.

The amyloid burden plays a central role in AD pathogenesis.⁶  Decades of academic and clinical research have established that the accumulation of amyloidogenic amyloid beta (Aβ) and its deposition in the brain is a major event in the etiology of AD.^6,7  The pathogenic mutation in the amyloid precursor protein gene (chromosome 21) encoded for essential transmembrane neuronal glycoprotein, amyloid precursor protein (APP) was identified. The proteolysis of APP by the specific combination of proteases (secretases) leads to generation, misfolding, self-aggregation, and deposition of insoluble Aβ plaques in the AD brain.⁷  Normally, the expression of unmutated APP is associated with neurite development, cell growth, ions transport, and healthy synapse.⁸  The proteolysis of APP through α-secretase and γ-secretase generates soluble and nontoxic peptide fragments that are cleared from the healthy brain. Under the pathologically prone conditions, mutated APP undergoes sequential proteolysis by β-secretase and γ-secretase to produce Aβ peptides of variable length (37–43 amino acids).^6–8  Aβ peptides (mainly Aβ42) are highly aggregation-prone and undergo self-assembly to form toxic polymorphic aggregation species (alloforms) viz., oligomers, protofibrils, and insoluble fibrils. As a result, the concentration levels of Aβ monomers rise and decline in the brain and cerebrospinal fluid (CSF), respectively, with the disease progression.³  Accumulating evidence has demonstrated that oligomers are the most toxic form of Aβ compared to protofibrils or fully grown fibrils (Figure 1.2).⁴  In other words, the surface of fully grown fibrillar aggregates acts as an active catalytic surface to produce highly toxic Aβ oligomers through the secondary nucleation process.⁹  The toxic Aβ aggregation species interact and damage the neuronal plasma membrane, which prompts the internalization of misfolded Aβ peptides into the healthy neurons.³  The insoluble Aβ aggregation species are highly neurotoxic to mature neurons as they cause axonal and dendritic atrophy.⁷  Aβ oligomers disrupt neuronal signaling cascade leading to synaptic dysfunction, weakening in neuronal circuits, and neuronal death. The N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid (AMPA) receptors are blocked by Aβ aggregation species causing memory impairment and cognitive dysfunctions. In addition, regulation of endocytosis and surface expression of NMDA-type glutamate receptors by Aβ reduce the synaptic plasticity and hippocampal long-term potentiation (LTP) formation.¹⁰  As a result, Aβ is designated as one of the definite biomarkers by the National Institute on Aging and Alzheimer's Association (NIA-AA) Research Framework 2018, and the levels of Aβ species are directly correlated with the disease progression and decline of cognitive functions in AD.¹¹  Further, the amyloid burden is closely associated with various other pathological events such as metal dyshomeostasis, excess ROS production, oxidative stress, inflammation, mitochondrial damage, tau phosphorylation, and aggregation among other neurotoxic processes (Figure 1.2).³  Overall, the involvement of Aβ burden is widely studied and accepted for understanding the multifactorial nature of AD and became a promising therapeutic target to modify AD pathology (see Chapter 16).^12–16 

Microtubule-associated proteins (MAPs) are engaged in the maintenance and stabilization of the microtubule assembly in matured neurons. Tau, a MAP, interacts with tubulin and stimulates its assembly into microtubules (Figure 1.2).¹⁷  The activity of tau is regulated through the extent of phosphorylation and one mole of tau phosphorylated with 2–3 moles of phosphate under physiological conditions. However, hyperphosphorylation of tau (pTau) reduces its normal physiological function leading to several neuronal disorders like AD and tauopathies (see Chapters 3 and 17).¹⁸  The self-aggregation of pTau forms intracellular neurofibrillary tangles (NFTs) and paired helical filaments (PHFs) that impair the axonal functions and degenerate neuronal cells (see Chapters 3–6).¹⁸  The pTau significantly reduces the necessary O-glycosylation modifications (of Ser and Thr) and impedes its activity. Initially, the tau hypothesis in AD was considered an independent pathway, while recent studies have established overlapping links with the amyloid hypothesis.⁴  Aβ-mediated glycogen synthase kinase 3β (GSK3β) activation triggers the tau hyperphosphorylation under AD conditions.¹⁹  It has been shown that misfolded tau transport from neuron to neuron and spread in the brain, which is significantly influenced by the amyloid load.²⁰  Further, apolipoprotein E4 (APOE4) increases the accumulation of pTau by decreasing its clearance from the AD brain.²¹  Considering the prominent role of tau in AD pathology, the NIA-AA Research Framework 2018 has declared tau (pTau and NFT) as one of the core biomarkers (Figure 1.2).¹¹ 

Metal ions play a crucial role in functioning biological systems and their dyshomeostasis is linked to several disease conditions. In the CNS, bioactive metal ions (Cu^II, Zn^II, and Fe^III) serve as cofactors for enzymatic activity, mitochondrial, and neuronal functions.²²  However, the accumulation of high concentrations of these metal ions in their free form and as inclusion complexes with insoluble amyloid aggregates aggravate AD pathology. In 1994, Bush et al. showed the possible link between Aβ and Zn^II.²³  Cu^II and Zn^II are revealed to coordinate Aβ and accelerate the amyloid aggregation to form metal-dependent plaques. On the other hand, the concentration of Fe^III reaches ∼1 mM in Aβ deposits, causing ferroptosis, which is an iron-induced cell death process.²⁴  In a fortuitous discovery, aluminum salts (Al^III) exhibited NFT degeneration in rabbit brain that showed a close link of Al^III with AD pathogenesis and a few other reports showed elevated levels of Al in AD brains.^25,26  The redox-active metal ions (Cu^II and Fe^III) stabilize the toxic forms (oligomers) of Aβ aggregates. The soluble oligomeric species interact with various synaptic receptors (NMDA and AMPA) and induce synaptic and cognitive dysfunctions through metal-dependent amyloid toxicity.¹⁰  Aβ-Cu and Aβ-Fe complexes undergo redox cycling in a reducing environment to produce excess reactive intermediate species (RIS), which are the key elements to trigger oxidative stress and neuroinflammation.³  In addition, biometal ions (Cu^II, Zn^II, Al^III, and Fe^III) play a crucial role in the synthesis and processing of APP resulting in excessive production of pathological Aβ (Figure 1.3A and B).²⁷  Thus, development of metal chelators that chelate or sequestrate and maintain the Aβ-bound metal ions in a redox-dormant state is a promising therapeutic approach (see Chapters 7 and 16).⁴  The design of multifunctional therapeutic agents that target Aβ-bound toxic metal ions along with other pathological toxicity (amyloid, tau, oxidative stress, mitochondrial dysfunction, inflammation, and others) is gaining the attention of researchers.^{4,12,14,15,28} 

The RIS include reactive oxygen species (ROS) and reactive nitrogen species (RNS), which play a crucial role in cell signaling cascade and combatting infections.²⁷  However, excessive production of RIS (superoxide radical, O₂˙⁻; hydrogen peroxide, H₂O₂; hydroxyl radical, HO˙; nitric oxide, NO; peroxynitrite, ONOO˙ and hypochlorous acid, HOCl) instigates severe oxidative stress that damages biomolecules (DNA, lipids, and proteins) and causes neuronal death (Figure 1.3C).³  Oxidative stress is a physiological condition caused by an imbalance in the pro-oxidants and antioxidants associated with disruption of redox circuitry and macromolecular damage.²⁹  Elevated levels of RIS are observed in the brain under the pathophysiological conditions of AD.³  The redox-active metal ions (Cu^II and Fe^III) chelate with Aβ peptide, stabilize oligomeric species and act as a depot to produce excess RIS (Figure 1.3C and D).²⁹  Aβ-bound Cu^II and Fe^III undergo a Fenton-type reaction in the presence of Aβ peptide or other reducing agents to generate H₂O₂ and other RIS from molecular oxygen.^30,31  Besides, RIS can be produced from cellular sources, including endoplasmic reticulum, mitochondria, monoamine oxidases, peroxisomes, and NADPH oxidases. The mitochondria are the primary RIS source, and mitochondrial dysfunction and high levels of cytochrome oxidase are observed under AD conditions.³²  The expression and activity of antioxidant enzymes like dehydrogenase complexes for α-ketoglutarate (α-KG)are downregulated in AD. Therefore, oxidative stress is the crux of AD progression and a potential target in a disease modifying approach employing natural or synthetic antioxidant molecules.^13,15,28,33 

The optimum physiological concentration of RIS (ROS and RNS) and their homeostasis is crucial for neurons functioning. However, elevated RIS levels cause oxidative stress and extensive damage to several biomolecules like DNA, lipids, and proteins.³⁴  The life span of RIS is short, while their reactivity is exceptionally high, allowing the rampant oxidative damage of biomolecules (Figure 1.3). The neuronal plasma membrane consists of high levels of polyunsaturated lipids that are vulnerable to attack by RIS. Under AD conditions, the unsaturated lipids undergo peroxidation and produce neurotoxic elements like 4-hydroxynonenal (HNE), among others.³⁵  The enzymatic activity in cells is regulated by oxidative stress, e.g., mitogen-activated protein kinases (MAPKs) showed Aβ-dependent oxidative stress-induced activation. The protein kinase p38, a family of MAPK, is associated with tau phosphorylation in the AD brain.³⁶  Amyloid-mediated oxidative stress damages the nucleic acids, adversely altering the DNA/RNA structure and function. In particular, mitochondrial DNA is highly susceptible to oxidative damage as mitochondria have no histone proteins. Oxidative damage of the guanosine base during DNA or RNA oxidation produces 8-hydroxyguanosine (8-oxoG), found in high concentrations in neurons of the AD brain.³⁷  A lipid rafts analysis of AD brain tissue showed the formation of oxidative stress-induced cholesterol microdomains that block the antioxidant vitamin E inside the lipid membrane. RIS also oxidize proteins that lead to the incorporation of OH or carbonyl groups. The generation of carbonyl groups is a potential marker for protein oxidation, which occurs by oxidizing side-chain hydroxyls into the aldehyde or ketone derivatives.³⁸  Overall, the destructive oxidative damage in neurons affects the physiological functions and causes neurodegeneration (Figure 1.4).³⁹ 

The plasma membrane comprises a semipermeable lipid bilayer that separates the interior cellular components from the extracellular environment.⁴⁰  The physiological functions of the membrane are associated with cell (neuronal) survival and integrity. Under AD conditions, toxic Aβ aggregation species interact and punch holes (pore formation) in the neuronal membrane, leading to an influx of metal ions and an imbalance in their homeostasis (Figure 1.4).⁴⁰  The proposed mechanism of membrane damage by Aβ aggregation species is comparable to the action of antimicrobial peptides (AMPs).³  Experimental evidence shows that the membrane pores facilitate the internalization of Ca^II, elevating its cytoplasmic concentration triggering neuronal degeneration.⁴¹  Besides, membrane Aβ peptides also interact and block the membrane-bound receptors such as α7-nicotinic, which is closely associated with LTP formation that internalizes Aβ into neuronal cells (Figure 1.4).⁴²  Under AD conditions, the membranes of essential organelles (like mitochondria) are also affected by RIS that triggers the organelle (mitochondrial) dysfunctions and neuronal damage.^14,15,28,38 

Khachaturian suggested the involvement of Ca^II in AD pathogenesis, and Mattson et al. experimentally showed the calcium dyshomeostasis in AD.^43,44  Ca^II is essential for the functioning of several key enzymes such as kinases, phosphatases, and proteases in the brain. Ca^II homeostasis is strongly associated with learning, short-term memory (STM), and LTP formation.⁴¹  Aβ elevates intracellular Ca^II concentration in neurons, which leads to synaptic dysfunction and neuronal damage (Figure 1.7).⁴⁵  The postmortem analysis of the human AD brain has shown selective alteration to calcineurin. It is a calcium- and calmodulin-dependent serine/threonine protein phosphatase that controls T-cells signaling activation and causes Aβ-mediated cognitive decline. Recent studies have revealed the accumulation of excess Ca^II inside the mitochondrial matrix through receptor-mediated internalization (Figure 1.4).⁴⁶  Overall, the dyshomeostasis of Ca^II is one of the prominent contributing factors in developing multifactorial AD.

Cholesterol is a sterol and type of lipid essential for maintaining the structural integrity of the cell membrane. It is a necessary precursor for the biosynthesis of vitamin D, bile acids, and steroid hormones.⁴⁷  The biosynthesis of cholesterol is a long and complicated process starting from mevalonate in the HMG-CoA reductase pathway. Cholesterol is essential to normal body functioning, while its high levels are speculated to raise the risk of AD progression (Figure 1.4).⁴⁷  Cellular studies have revealed the overexpression of human APP and decline in APPsα production with exogenous cholesterol treatment. Interestingly, methyl-β-cyclodextrin, a cholesterol removing agent, treated cells showed an elevated activity of α-secretase.⁴⁸  Cholesterol also influences the activity of β- and γ-secretase and triggers the amyloidogenic pathway to upregulate Aβ production.⁴⁹  Under AD conditions, the aggregation of Aβ peptides is positively affected by cholesterol levels. The plasma membrane with low cholesterol content allows greater internalization and degradation of Aβ peptides. In contrast, high cholesterol levels promote extracellular amyloid aggregation by blocking the Aβ internalization.⁵⁰  The amino acid residues (20–35) of Aβ act as a cholesterol-binding domain, in which K28, A21, V24, and I32 play a significant role in the binding that triggers the membrane pore formation. There is concrete evidence to show the involvement of cholesterol in multifactorial AD.

Mitochondrion (plural, mitochondria) is an important organelle present in the cytoplasm and powerhouse of eukaryotic cells.⁵¹  Mitochondria drive aerobic respiration to produce adenosine triphosphate (ATP), a source of chemical energy, from nutrients and molecular oxygen. Mitochondria are membrane-bound, rod-shaped organelles that contain circular DNA known as mitochondrial DNA (mtDNA). The mitochondrial membrane consists of two layers: (i) a permeable outer membrane, which allows the passing of ions and small molecules, and (ii) a mostly impermeable inner membrane that separates the mitochondrial matrix from the cytosol.⁵¹  The mitochondrial inner membrane forms cristae, a specific fold to generate wrinkled shapes to increase its surface area and maximize aerobic respiration. The required high metabolic energy in the brain is extremely dependent on mitochondria as neurons have minimal glycolytic capacity. In 2004, Swerdlow and Khan hypothesized the involvement of mitochondrial dysfunction in AD pathology. They proposed that the inherent genetic factors that control mitochondrial baseline activity can be manipulated with other external factors to damage the mitochondria and cause neurodegeneration.⁵²  It has been established that the essential mitochondrial functions, including ATP synthesis, glucose metabolism, and ROS production, are altered before the accumulation of Aβ plaques and tau tangles in the AD brain (Figure 1.4). Therefore, early mitochondrial dysfunction potentially aids AD progression and plays a central role in multifactorial AD pathology.⁵³  Mitochondria contain an array of redox-enzymes capable of generating ROS by transferring electrons to molecular oxygen during the respiratory chain reaction. The detoxification of oxidants is taken care of by the mitochondrial antioxidant defense system, which is compromised in damaged mitochondria and impairs the cellular redox balance (oxidative stress). Cellular studies have shown that the Aβ-mediated interruption of the electron transport chain, membrane potential, and ATP synthesis causes mitochondrial dysfunction and neuronal toxicity.⁵⁴  The internalization of Aβ has been proposed to occur through the translocase of the outer membrane (TOM) and inner membrane (TIM) complexes.⁵⁵  Aβ complexed with alcohol dehydrogenase induces excess ROS production by impairing the complex IV, ATP synthesis and suppressing the expression of cytochrome c.⁵⁴  The mtDNA is highly exposed to ROS due to the lack of histone protein that hinders the mitochondrial protein synthesis.⁵⁵  On the other hand, neuronal oxidative stress triggers the secretase (β)expression by activating p38 mitogen-activated protein kinase (MAPK) and c-Jun aminoterminal kinase pathways that lead to elevated Aβproduction. Under oxidative stress conditions, tau phosphorylation is promoted by activation of GSK3β. Mitochondrial biogenesis is an essential physiological process that maintains the population of healthy mitochondria in eukaryotic cells. The PGC-1α-NRFTFAM pathway regulates mitochondrial biogenesis in response to the damaged mitochondria or energy demands. An in vitro (APPswe M17 cell) and animal (AD hippocampus tissue) study demonstrated the downregulation of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α), nuclear respiratory factor (NRF)-1 and -2 and mitochondrial transcription factor A (TFAM), which leads to the impaired mitochondrial biogenesis followed by neurodegeneration.⁵⁶  Mitochondria are highly dynamic organelles that translocate throughout the cell body and undergo frequent fusion and fission depending on the physiological stimuli. A very short contact can promote mitochondrial fusion, which extensively exchanges the essential proteins and biomolecules in each compartment of the mitochondrion. Recent studies have suggested that the impairment in mitochondrial dynamics, such as increase and decrease of fission and fusion, respectively, are early events found in neurodegenerative diseases, including AD. The fusion dynamin-related protein 1 (Drp1), a protein that controls mammalian mitochondrial dynamic, interacts with Aβ and tau (phosphorylated), resulting in excessive mitochondrial fragmentation, impaired translocation of mitochondria in the neurons under AD conditions.⁵⁴  The overexpression of APP influences the mitochondrial dynamic causing elevated fragmentation in neurons. The alteration of mitochondrial dynamics under AD condition is supported by the reduced and enhanced expression of proteins involved in fusion (OPA1, Mfn1, and Mfn2) and fission (Fis1) processes, respectively.⁵⁷  The programmed cell (neuronal) death (apoptosis, autophagy, or necrosis) is facilitated by increased mitochondrial membrane permeability, which results in the reduction of mitochondrial membrane potential (ΔΨm), increased mitochondrial swelling, and triggers the mitochondrial outer membrane rupture. Cyclophilin D (CypD), a mitochondrial peptidylprolyl cis-trans isomerase associated with the mitochondrial permeability transition pore regulation, and plays an important role in programmed cell death. The cortical mitochondria of transgenic mice and AD patients displayed Aβ-CypD complexation resulting in ΔΨm reduction, compromised respiration, increased oxidative stress, alleviated cytochrome c expression, and impaired axonal transport.⁵⁸  It was also observed that Aβ interacts with the mitochondrial membrane and triggers mitochondrial fragmentation and mitophagy, a process that digests mitochondria (Figure 1.5).^15,59  Overall, the growing evidence on mitochondrial dysfunction indicates its involvement in AD pathology and has become a promising therapeutic target.

Endoplasmic reticulum (ER) is associated with the synthesis, folding, quality control, and subcellular trafficking of proteins. Modification like glycosylation occurs in the ER and decides the fate of protein folding, trafficking, and function.^60,61  One of the crucial functions of ER in neuronal cells is to regulate the Ca^II homeostasis that is required for healthy synaptic communication. It is the major reservoir of Ca^II and contains a large number of Ca^II channels to regulate intracellular Ca^II levels and dynamics in neuronal cells.⁶²  The reduced levels of Ca^II in pre- and post-synaptic neurons alter the neurotransmitter release and intracellular signaling, respectively.⁶³  Dysregulation of calcium homeostasis has a role in many neurodegenerative diseases. The protein quality control is maintained at various stages of protein processing, and ER plays a major role in sorting functional and damaged proteins to the target site and degradation pathways, respectively (Figure 1.6). Many neurodegenerative diseases involve misfolding and aggregation of functional proteins into toxic species. Numerous studies have suggested that the pathological events viz., accumulation of Aβ, tau, and Ca^II dyshomeostasis, play a prominent role in the ER stress leading to cell death under progressive AD conditions.⁶⁴ 

Anatomically, ER extends from soma towards axon and dendrites and plays a major role in maintaining cytosolic Ca^II levels in neurons. In ER, Ca homeostasis is maintained by the Ca-ATPase actively mediated intake, and the inositol trisphosphate receptor (IP3Rs) and ryanodine receptors (RyRs) facilitate release to cytosol.⁶²  Several studies have demonstrated the dysregulation of Ca^II influx in AD mice models. mRNA analysis in post-mortem brain samples showed an increased expression of RyRs in MCI and AD cases.⁶⁵  In vivo studies of deleting the RyRs gene in young mice enhanced AD phenotypes; on the contrary, old mice showed reduced AD phenotypes.^66,67  Studies have shown Aβ mediated cell surface mGluR5 receptor activation, which results in an elevated Ca^II level.⁶⁸  ER contains integral membrane protein sensors such as inositol requiring enzyme 1 (Ire1), protein kinase RNA (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6), which identify the misfolded proteins and trigger signaling to activate the unfolded protein response (UPR).^69,70  Immunostaining analysis revealed elevated levels of activated UPR kinases, pPERK, peIF2α, and pIRE1α proteins in degenerated (AD) hippocampal neurons.⁷¹  Several in vitro and in vivo studies have demonstrated that Aβ oligomers induced ER stress-mediated cell death (Figure 1.6).

An in vivo study in an APP/PSEN1 mice model concluded that Aβ mediated ER stress along with mitochondrial cholesterol trafficking significantly contribute to AD disease progression.⁷²  The events of APP processing and sorting to the target compartment are mediated by ER. ER stress causes abnormal APP processing and sorting, which contribute to AD pathology. Besides, pPERK activated neurons contain high levels of GSK-3β, a kinase associated with hyperphosphorylation of tau, that indicates the role of ER stress in enhancing the formation of NFTs.⁶⁴ 

ER stress triggers an inflammatory response in the cells via toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors’ (NLRs) activation. Further, ER is involved in the cellular synthesis of cholesterol, which is essential for physiological functions. Dysfunction in cholesterol synthesis and its intracellular concentration is directly associated with AD pathology (vide supra).⁷¹  The degeneration of ER under stress conditions alters the neuronal functions leading to neurodegeneration. A wide variety of factors contributing to ER stress and AD pathology suggests ER stress as a probable target to combat AD.⁷³  Various strategies can be adapted to target the ER stress and halt AD progression. Synthetic chaperons have been studied in a few reports, which reduce ER stress and UPR. Calcium homeostasis is one of the tangible targets for therapeutics to reduce ER stress-induced neuronal cell death. Molecules targeting calcium sensors and regulating proteins are potential strategies. While the evidence on targeting ER stress to treat AD is rare, the significant contribution of ER stress and related pathways to AD pathology designate them as potential therapeutic targets.

Chromosomes contain noncoding telomere (TTAGGG repeats) sequences at their ends as protective caps, shortened with each cell division.⁷⁴  The continuous reduction of a telomere's length with each cell division plays a critical role in cell senescence through inflammation and oxidative stress.⁷⁵  Telomerase, a ribonucleoprotein consisting of telomerase reverse transcriptase (TERT) and an RNA template (TER) adds a TTAGGG repeats unit to the shortened telomere end of the chromosomes to protect it from erosion during cell division.⁷⁴  Emerging evidence indicates that the shortening of telomere length plays a vital role in AD progression (Figure 1.4).⁷⁵  The elongation of telomeres is hampered by soluble Aβ oligomers as they bind and block the DNA-telomerase complex formation.⁷⁶  Thus, the amyloid toxicity inhibits telomerase activity and stimulates neuronal senescence resulting in neurodegeneration.

Cholinergic toxicity is associated with the impairment of cholinergic neurons (Figure 1.5). Cholinergic neurons are involved in brain functions such as learning, memory, attention, response, sleep, and administering sensory information.⁷⁷  In 1976, Peter Davies and A. J. F. Maloney reported the dysfunction of cholinergic neurons in AD. They studied the critical enzymes involved in the synthesis of neurotransmitters (γ-aminobutyric acid, acetylcholine, dopamine, 5-hydroxytryptamine, and noradrenaline)in different regions of AD brains and healthy brains.⁷⁸  Under AD conditions, most of the enzymes, including glutamic acid decarboxylase, aromatic amino acid decarboxylase, tyrosine hydroxylase, and dopamine-β-hydroxylase are found to be unaffected. However, this study identified abnormality in choline acetyltransferase (ChAT) activity, which is significantly reduced in the AD brains.⁷⁸  ChAT is associated with acetylcholine (ACh) synthesis from acetyl-CoA, choline, and ATP.⁷⁷  Therefore, abnormality in ChAT activity causes the AD brain to suffer from ACh deficiency that hampered the synapses and synaptic transmission. On the other hand, ACh is hydrolyzed into choline and acetic acid in the presence of acetylcholinesterase (AChE) enzyme at the synaptic cleft. ACh production and breakdown are tightly balanced by a feedback loop mechanism involving ChAT and AChE enzymes.⁷⁹  The compromised activity of ChAT or overactivity of AChE under AD conditions lead to a significant decline of ACh level at the synaptic cleft in the cortex, hippocampus, and amygdala. The impairment in the cholinergic neuronal circuit critically affects the cognitive functions during AD progression (Figure 1.7).⁸⁰  Thus, targeting cholinergic neuronal dysfunction is considered a potential target to decrease the risk of cognitive dysfunctions in AD patients.⁷⁹  Tacrine is the first FDA (Food and Drug Administration) approved (1995) AChE inhibitor for the treatment of AD. Currently, all the FDA approved AD drugs are AChE inhibitors.⁸¹ 

Neurons are the basic cells in the CNS that coordinate and transmit information within and among other cell types through a specialized connection known as the synapse.⁸²  The transmission of information through synapses is the core of memory formation, and cognitive functions are severely impaired in the AD brain (Figure 1.7).⁸³  The synaptic dysfunction correlates well with cognitive decline rather than pathogenic markers like Aβ plaques. It has been reported that the synaptic loss starts long before the Aβ plaques deposits, while evidence in the literature suggests a definite role of Aβ in synaptic dysfunction.⁸⁴  In 1997, Lambert et al. showed the involvement of soluble Aβ oligomers in synaptic loss. Subsequent studies have shown Aβ oligomers-mediated blocking of essential synaptic receptors such as NMDA and AMPA receptors in the synaptic cleft.¹⁰  The learning (hippocamps-mediated) and LTP formation are closely associated with SUMOylation that is impaired by the soluble Aβ oligomers resulting in cognitive decline.^83,85  Aβ oligomers reduce the release of neurotransmitters in the presynaptic neurons by blocking the Stx1a and SnpI protein, which controls the synaptic vesicles.⁸⁶  The progressive cognitive decline established the critical role of synaptic dysfunction in AD progression and its multifactorial nature (Figure 1.7).

Research in recent literature demonstrates that activation of microglia causes reactive gliosis and neuroinflammation, which plays a crucial role in AD progression.⁸⁷  Inflammation is a physiological defense mechanism or response of the immune system to counter the health hazard from harmful debris, pathogens, damaged proteins, and damaged cells, and pledges their degradation and clearance (Figure 1.8). Microglial cells are the brain-specific macrophages in the CNS. The accumulation of microglia near insoluble Aβ plaques is typically 2–5 times higher than the healthy brain, which indicates the role of microglia in AD.⁸⁸  Aβ interacts with microglia through the CD36-TLR4-TLR6 receptor complex that helps in clearing Aβ aggregates from the brain. Amyloid plaques, NFT, and damaged or dead cells activate microglia in the AD brain to initiate inflammation.⁸⁷  The primed microglia (activated) release cytokines, acute-phase reactants, and other inflammatory mediators such as TNF-α, IL-1β, TGF-β, IL-12, and IL-18 in the CNS and cause acute neuroinflammation (Figure 1.8).⁸⁸  Therefore, anti-inflammatory drugs that reduce neuroinflammation are good candidates to target AD pathogenesis. It has been shown that treatment with anti-inflammatory drugs significantly improves AD conditions.⁸⁷  CD22 is a B-cell receptor, and its upregulation reduces the phagocytosis. Thus, the antagonist of the CD22 receptor can enhance the phagocytosis to clear debris and Aβ fibrils in AD conditions.⁸⁸  Recent findings have revealed the essential involvement of inflammasome, which is accountable for cytokine maturation and cell pyroptosis, in the development of AD. The Aβ aggregation species activates inflammasomes leading to the initiation of innate immune responses. A recent study has revealed that the silencing of TREM2 function intensifies tau pathology in the presence of Aβ, from which we can infer the probable protective role of TREM2 against AD pathogenesis (Chapter 8).⁸⁹ 

The human brain is about 2% of total body weight, while it consumes ∼20% of the entire body O₂ and glucose through a corresponding volume of blood supply by the neurovascular system. An efficient energy supply through glucose metabolism is critical for the brain's normal functioning, and any deficiency in the energy supply contributes to a range of neurological disorders. In 1994, it was demonstrated that neurovascular dysfunction leads to cerebrovascular pathologies and AD.⁹⁰  The imaging of the brain showed neurovascular dysfunction before the onset of AD and Aβ induced constriction of cerebral arteries during AD progression.⁹¹  The neurovascular system consists of vascular, glial, and neuronal cells. It maintains the microenvironment of the brain by regulating blood–brain barrier (BBB) permeability and cerebral blood flow. APP overexpressed mice showed impairment in the cortical microcirculation, leading to alterations in cerebral blood flow and neuronal damage. The BBB is directly associated with the clearance of senile Aβ plaques and maintains the balance between the production and removal of Aβ in the CNS.⁹²  The dysfunction in the neurovascular system fails to clear excess Aβ from the AD brain that accelerates the AD progression. On the other hand, the accumulation of Aβ plaques damages the BBB's integrity, which allows undesirable chemicals into the brain and impedes neuronal functions. Studies in AD mice models have revealed that the circulating Aβ binds to the endothelial receptor (receptor for advanced glycation end products; RAGE), triggers the RAGE-mediated Aβ transport into the brain, thereby enhancing the amyloid load in the AD brain. It was observed that the neurovascular system is ineffective in the clearance of Aβ under AD conditions, as it is poorly bound to low-density lipoprotein receptor-related protein 1 (LRP1), which results in Aβ accumulation and aggregation.⁹²  The rise in Aβ levels in the AD brain causes Aβ plaques deposition in the cerebrovascular system and instigates cerebral Aβ angiopathy.^91,93  This range of evidence supports the role of neurovascular dysfunction in the development of multifactorial AD.

The lymphatic system (LS) is part of the immune and circulatory system and consists of an extensive network of lymphatic organs, vessels, and tissues. It helps remove toxins, waste, and unwanted debris through the extracellular fluid (lymph) and maintains the balance of circulation of immune cells and fluid in the tissue.⁹⁴  The CNS is covered with three layers (dura, arachnoid, and pia mater) containing meninges, which maintains the network of the LS. The meningeal lymphatic system flowing from the interstitial fluid to CSF through the meninges and ventricles plays a crucial role in removing the brain's waste materials, and its dysfunction is linked to various neurodegenerative disorders, including AD. The brain astrocytes express aquaporin-4, a membrane protein that transports water molecules (water channel), and is essential for exchanging materials between interstitial fluid and CSF. The aquaporin-4 knockout animals displayed a significant decline in large solute (Aβ) removal from the brain. However, the efflux of Aβ depends on LRP1 and p-glycoprotein expressed in the brain's endothelial cells. P-Glycoprotein helps in the formation of Aβ-ApoE complexes that transport through LRP1. The reduction of LRP1 protein expression in the pericytes and endothelial cells effectively suppresses the ApoE dependent clearance of Aβ.⁹⁵  The genetic variants of phosphatidylinositol binding clathrin assembly protein (PICALM) are linked as the risk factor of sporadic AD. PICALM is proficient in controlling the Aβ efflux, and the reduction of its expression in endothelial cells is associated with Aβ pathology and cognitive impairment in AD mice and AD patients. Recent evidence has demonstrated the role of lymphatic dysfunction in BBB breakdown under progressive AD conditions.⁹⁴  These findings have established the role of LS in the multifactorial AD and a promising therapeutic target.

Besides pathogenic Aβ and tau, the literature reports suggest the involvement of α-synuclein (αSyn)in AD pathology.⁹⁶  αSyn is a small presynaptic protein (140 amino acid residues) mostly associated with Parkinson's disease (PD) and Lewy bodies (LB) dementia. The primary structure of αSyn can be divided into three parts: (i) amphipathic N-terminal (1–60 amino acid residues), (ii) non-amyloid component (NAC) in the middle (61–95 amino acid residues), and (iii) an unstructured C-terminal segment (96–140 amino acid residues). Under normal physiological conditions, the N-terminal and NAC segments adopt α-helical secondary structures. However, the NAC part adopts a β-sheet secondary structure under pathological conditions and is mainly responsible for pathogenic αSyn aggregation and LB formation.⁹⁷  The point mutations in the NAC region showed a drastic reduction in the amyloidogenic nature of αSyn. The αSyn knockout AD mice (APP/αSyn-KO) showed a significant reduction of amyloid load and improved memory and cognitive function that demonstrated the involvement of αSyn in AD pathology. The unstructured C-terminal end interacts with the microtubule-binding domain of tau, which triggers the toxic aggregate formation. Further, the existence of LB in more than 50% of AD patients’ brains confirms the contribution of αSyn to multifaceted AD toxicity.⁹⁷ 

Apoptosis is a genetically programmed cell death mediated by an intercellular proteolytic cascade and is essential for tissue homeostasis, development, and removing damaged or infected cells.³  It is well accepted that massive neuronal and glial cell death caused by apoptosis is a common feature in brains of neurodegenerative disease (AD) patients. Aβ-mediated activation of the IκBα/NF-κB signaling pathway resulted in the reduction of cytochrome c oxidase subunit expression that instigates mitochondrial dysfunction and apoptosis. An in vivo study in Tg2576 mice showed elevated levels of apoptosis signal-regulating kinase 1-interacting (ASK1-interacting) protein-1 (AIP1). However, the activation of ASK1 is prompted by Aβ42 that facilitated neuronal apoptosis in the AD brain.⁹⁸  Aβ42-induced phosphorylation (Tyr219, Thr211, and Ser207) of mitogen-activated protein kinase 6 (MKK6) is associated with P66shc-MKK6 interaction, which facilitates p66shc phosphorylation (Ser36) and ROS production, triggering neuronal apoptosis.⁹⁹  Studies have shown that AICD-mediated neuronal cell-specific apoptosis probably occurs through the p53 and GSK3β pathway.³  It was observed that mutated (M146 L) presenilin 1 containing neurons are more susceptible to degeneration than other neurons (expressing wild-type presenilin 1) after inducing apoptosis. Apoptotic cells exhibit various physiological changes like DNA fragmentation, caspase activation, and expression of phosphatidylserine on the plasma membrane, which is observed in the AD brain. Therefore, alterations in the fundamental cellular process like apoptosis results in neuronal death and neurodegeneration.⁴ 

Most of the proteins undergo post-translational modifications (PTMs) following their translation from mRNA on the ribosome and trafficking to functional destinations in the cell.¹⁰⁰  The polypeptides synthesized on the ribosome are subjected to PTMs at the N- or C-terminal and on the side chains of amino acids to transform them into matured and functional proteins. In the process, various organic and inorganic moieties are covalently attached to the translated proteins through different enzymatic transformations, including phosphorylation, glycosylation, methylation, acetylation, ubiquitination, SUMOylation, nitrosylation, and hydroxylation, among others.^100,101  These chemical modifications are essential in protein localization, stability, structure, and functions. However, abnormal cleavage and maturation or aberrant PTMs of proteins lead to pathological conditions, including neurodegenerative disorders (see Chapter 9). The phosphorylation, incorporation of phosphate groups in proteins through ester, amide, and anhydride bond formation on Ser/Thr, is the most abundant PTM. Tau regulates the microtubular assembly and its hyperphosphorylation resulting in abnormal folding and aggregation to form pathogenic NFTs. The phosphorylation of beta-site APP cleaving enzyme 1 (BACE1) enzyme (β-secretase)regulates the amyloidogenic processing of APP and Aβproduction.¹⁰¹  High levels of phosphorylated BACE1 (Thr252) elevates its proteolytic activity in the AD brain. The phosphorylation of APP (Thr668) triggered by Aβ in a reverse feedback mechanism promotes the production of excess Aβ peptides.¹⁰⁰  Aβ peptide is phosphorylated at Ser8 and Ser26 and propagates aggregation to form toxic oligomeric species. Glycosylation, enzymatic incorporation of sugar moieties through O- and N-glycosidic linkages to the proteins, is another important PTM. The glycosylation regulates protein folding, trafficking, stability, secretion, and function. The altered glycosylation pattern on the protein plays a major role in the disease pathology of AD. The glycosylation of tau stabilizes tau aggregates, while de-glycosylation reduces aggregation propensity. Interestingly, the O-glycosylation of APP predominantly at serine residues (396, 400, and 404) regulates the pathogenic phosphorylation of selective sites of tau. The pathological phosphorylation at selective sites and specific glycosylation patterns of tau inversely regulates each other. Ubiquitin is a small regulatory protein consisting of 76 amino acid residues, and its addition to the target proteins is known as ubiquitination. This modification influences the proteins in various ways such as (i) labeling the protein for degradation, (ii) modifying the intracellular location and activity, and (iii) modulating (prevent or promote) protein–protein interactions. The dysregulation in ubiquitination alters the degradation of misfolded proteins and promotes their accumulation. Acetylation is the incorporation of the acetyl group and a crucial PTM of a protein to control its functional activity. The selective mono-acetylated (Lys280) tau impairs the microtubule assembly and promotes the formation of toxic aggregates. The histone acetylation is elevated in the AD brain and is associated with the accumulation of toxic aggregation species of Aβ and tau.¹⁰⁰  Nitrosylation of protein is elevated in the AD brain and regulates proteins related to mitochondrial dysfunctions. Other aberrant PTMs (palmitoylation, prenylation, methylation, and SUMOylation) of proteins have been shown to play key roles in AD progression and multifaceted toxicity.¹⁰¹  The signature of PTMs and modulating AD-related aberrant modifications are considered as the potential diagnostic and therapeutic targets, respectively.¹⁰⁰  For example, the phosphorylation states of tau at Thr181, Thr217, and Thr231 residues have emerged as potential AD biomarkers, as the levels of these ptau variants increase in CSF.¹⁰² 

The human gut microbiota consists of microbes (archaea, bacteria, and fungi) in the digestive tract that regulate immune and metabolic homeostasis and protect against pathogens. The biochemical bi-directional signaling between the gastrointestinal tract (gut microbiota) and the CNS is termed as the gut–brain axis or microbiome–gut–brain axis.¹⁰³  The healthy gut or R13 (prebiotic supplement) treated mice gut showed less accumulation of Aβ in the gut, reducing AD development risk. The disruptions in the microbiota-gut–brain axis led to brain and gastrointestinal disorders and cognitive decline. The increasing evidence suggests that there is a definite link between gut microbiota with AD development. The pathological changes, including brain atrophy, amyloid accumulation, neuroinflammation, immune anomalies, and cognitive and memory impairment observed in AD, are associated with microbial and viral infections. Recently, the presence of herpes simplex and picornavirus was reported in AD brains. This shines a light on the pathogenic etiology and transmissible nature of AD. The role of these viruses in triggering AD pathology needs to be further studied to understand the pathophysiological mechanism. A few clinical trials have been initiated to evaluate antiviral drugs for the treatment of AD.^104,105  There is evidence for the pathogenic bacteria Porphyromonas gingivalis in AD brains, which causes inflammation-mediated pathology (see Chapter 12). Further, this aspect needs to be studied to develop novel therapeutics.¹⁰⁶  The short-chain fatty acids and metabolites of the microorganisms can regulate the pathological processes in the CNS.¹⁰³  Experimental studies have revealed that acetate can alter neurotransmitter levels such as glutamate and gamma-aminobutyric acid (GABA). The pro-inflammatory bacteria (bad bacteria) are known to secrete excess lipopolysaccharides (LPS) and Aβ under AD conditions contributing to amyloid-mediated AD pathology.¹⁰⁷  Besides, the disruption of the tight junction in the gastrointestinal tract (leaky gut) under progressive AD conditions triggers neuroinflammation and promotes the BBB permeability. 5-Hydroxytryptamine (serotonin), a crucial neurotransmitter to regulate cognition functions, is mostly (∼ 95%)synthesized in the gut through gut microbiota. The treatment with serotonin improves the health of mouse gut microbiota that reduces Aβproduction, thereby decreasing the risk of AD. A detailed understanding of the microbiome–gut–brain axis is anticipated to aid the effective modulation of AD.¹⁰⁷ 

The human body organ sources the energy required to perform normal physiological functions through glucose, protein, or fat metabolism, while the brain is mostly dependent on glucose metabolism. Glucose is stored in the form of polysaccharide glycogen in the liver, and glycogen breaks down to glucose through gluconeogenesis. The human brain is an energy drain and utilizes more than 20% of total body energy delivered in the form of glucose and O₂ through ∼20% of blood supply. Thus, brain functions are strongly affected by disruptions in glucose metabolism. Glucose hypometabolism, a physiological condition that exhibits an unusually low metabolic rate, is one of the early onsets during the prodromal AD conditions associated with cognitive decline.¹⁰⁸  Positron emission tomography (PET) imaging using 2-deoxy-2-fluorine-18-fluoro-d-glucose (¹⁸FDG) has emerged as a promising tool to diagnose neurodegenerative disorders, including AD.¹⁰⁹  Several research findings have demonstrated that glucose hypometabolism plays a significant role in the cognitive dysfunction-mediated development of AD pathology.¹⁰⁸ 

Diabetes (type 1 and 2) is a metabolic disorder that affects blood glucose levels. Insulin, a peptide hormone produced by the pancreatic β-cells, maintains the blood sugar level by glucose internalization into the cells or storage form. The inability to make or use insulin (insulin resistant) effectively causes type 1 and type 2 diabetes.¹¹⁰  The insulin produced in the pancreas is transported to the CNS by crossing the BBB (receptor-mediated process). Recent findings have shown insulin production in the hypothalamus controlled by the Wnt/β-catenin/NeuroD1 pathway.¹¹¹  The meta-analysis, a statistical assessment that merges multiple scientific results, has revealed an increased risk of AD by ∼56% for individuals with type 2 diabetes. The hyperinsulinemia (type 2 diabetes) decreases insulin levels and insulin-receptors’ sensitivity in the brain that promotes the pathogenic events of AD.¹¹²  The soluble Aβ species blocks insulin signaling by binding to the receptor resulting in LTP disruption and memory impairment.¹¹⁰  The pre-treatment of insulin can prevent this phenomenon of blocking insulin signaling under AD conditions. Type 2 diabetes triggers the neuronal degeneration independent of Aβ and tau by manipulating apoptosis, oxidative stress, caspases activity, and mitochondrial function.¹¹²  The term type 3 diabetes used under AD conditions adequately justifies the correlation between diabetes and AD because AD patients develop diabetes-related neuronal damage and degeneration.¹¹³ 

Autophagy (self-eating in Greek), a natural intracellular process to degrade and recycle the toxic protein or debris and damaged organelles, was described by Christian de Duve in 1963.¹¹⁴  Yoshinori Ohsumi was awarded the Nobel Prize (2016) in Physiology or Medicine for identifying the genes and mechanisms of autophagy.⁴  Healthy cells exhibit a basal level of autophagy, which plays a crucial role in maintaining the cellular homeostatic turnover of organelles and proteins. The autophagy process produces mature-autolysosome, a fusion product of autophagosomes (the vesicles containing degradable cellular material) and lysosomes that help suppress the trapped contents (organelles and proteins) and its inner membrane. Research findings over the years have demonstrated that dysfunction of the autophagy process promotes multifactorial AD progression.^115,116  In fact, autophagy is an essential regulator of intracellular Aβ production and clearance. However, the maturation of autolysosomes is hindered inside the neurons under AD conditions. Malfunctioning of the autophagy process leading to the accumulation of excess autophagic vesicles, toxic proteins, and aggregation species stimulates neurodegeneration by blocking the endosomal sorting complexes required for transport (ESCRT)-III complex.¹¹⁵  The accretion of autophagic vesicles within the neurons has been observed in AD phenotype mouse brains. The inhibition of autophagy stimulates microglia activity, including the secretion of cytokines and toxic ROS production. The activation of microglia or neuroinflammation plays a critical role in the multifaceted toxicity of AD. Human studies indicate the mammalian target of the rapamycin (mTOR) signaling pathway in AD.¹¹⁶  The reduction of mTOR signaling results in the amelioration of Aβ burden and memory impairment. Interestingly, rapamycin treatment (blocks mTOR signaling) improved cognitive function and reduced the Aβ burden by triggering autophagy within the brain cells. The literature reports show that autophagy is an essential physiological process in the degradation of pathogenic proteins (Aβ and tau) and stimulates the mTOR pathway and neuroinflammation.¹⁰¹  In general, autophagy dysfunction is a significant contributor to AD progression and a promising therapeutic target (see Chapter 10).¹¹⁷ 

The mutations in several genes are responsible for numerous disease conditions. Carrying the variants of some genes increases the risk of developing disease conditions and are called genetic risk factors. Although AD is considered as a chronic disorder, the genetic risk factors cannot be overlooked in the disease development. There is a greater risk of developing AD by inheriting certain genes (FAD: Familial AD). However, the etiology of FAD is mostly dependent on environmental factors and epigenetic variations along with genetics.¹¹⁸  FAD is very rarely found in individuals, contributing less than 5% of the total AD cases.¹¹⁹  Advanced experimental analysis showed that the inheritance of essential gene variants like APP, PSEN1, PSEN2, and APOEε4 alleles increases the genetic risk factor for AD (see Chapter 2). The most prevalent form of AD is late-onset, which is associated with multiple genes such as APOE, ABCA7, CD2AP, BIN1, CD33, CR1, CLU, EPHA1, MS4A4E/MS4A4A/MS4A6E, SORL1, and PICALM.¹¹⁸  Several mutations in APP have been identified, which are mostly in the transmembrane domain (exons 16 and 17). These mutations imply the modification of the secretase cleavage sites of APP that control the length of Aβ peptides. For example, the specific mutated APP at the γ-secretase domain (V717I and L723P) is associated with pathogenic Aβ production and accumulation in FAD. The PSEN1 mutations influence the ratio of Aβ42/Aβ40 by modulating the γ-secretase activity. In particular, mutated PSEN1 (L166P) carrying individuals exhibit exceptionally high production of Aβ42 and impair the production of the notch intracellular domain. It was observed that individuals who inherit a copy of the APOE ε4 allele are at increased risk of developing AD, and those who inherit more than one copy of this gene are at greater risk.¹¹⁸  However, not all AD patients have the APOE ε4 allele, reiterating the multifactorial nature of AD.¹²⁰  A detailed genetic study and identification of novel gene variants would lead to understanding of the multiple factors involved in AD. The genetic data have made a route for identifying the role of microglia in AD, and many such newer insights are anticipated to be unraveled in the future.¹²¹  Genetic testing of AD-associated genes from blood can estimate the risk of developing AD before the onset. This approach can be undertaken to estimate the possible early onset of AD, which facilitates the personalized clinical diagnosis and planning of appropriate treatment options.

An effective therapeutic strategy or treatment demands an early and accurate diagnosis of the disease. The early diagnosis of AD with precision has been a big puzzle, and the scientific and clinical community is directing enormous effort in finding a solution. As discussed (vide supra), the decline in memory processing and cognitive functions is primarily employed as a tool for the clinical assessment of AD. The progressive deterioration of cognitive functions is assessed through the Alzheimer's Disease Assessment Scale–Cognitive Subscale (ADAS-Cog) process, which was established in the 1980s.¹²²  There are eight different phases to diagnose AD: prodromal AD, AD dementia, typical AD, atypical AD, mixed AD, preclinical states of AD, Alzheimer's pathology, and mild cognitive impairment (MCI). However, the NIA-AA Research Framework 2018 has proposed the use of definite biomarkers such as Aβ (A) and tau (T) aggregation (core biomarkers) and related neurodegeneration (N) to ensure the effective diagnosis of AD (see Chapter 13).^4,5  The different alloforms of Aβ (monomers, oligomers, protofibrils, and fibrils) appear in the extracellular matrix under pathological conditions. Similarly, hyperphosphorylated tau protein aggregates coexist in different alloforms like oligomers, truncated tau, phosphorylated tau, PHFs, and NFTs. Therefore, fluorescent-based imaging techniques were employed to monitor different alloforms of Aβ and tau under progressive AD conditions. After an enormous effort, fluorescent probes were developed to target these pathogenic (Aβ and tau) aggregation species with excellent sensitivity and selectivity.^5,93,123  The in vivo fluorescent imaging of pathological events with near-infrared (NIR) probes allow the possibility of near-infrared fluorescence (NIRF) imaging with clinical applications in AD diagnosis.⁹³  Further, the molecular tools for utilizing PET and magnetic resonance imaging (MRI) techniques targeting core biomarkers have shown substantial success in the clinical diagnosis of AD (Figure 1.9). In the last decade a couple of PET probes targeting Aβ and tau have been approved by the FDA for the clinical diagnosis of AD. Besides the core biomarkers, extensive research has demonstrated that many indirect biomarkers correlate with the different AD brain pathological events.⁵  The multifactorial nature and ambiguity in the diagnosis of AD underline the importance of multiplexing and multimodal detection of core and indirect biomarkers for AD diagnosis through characteristic fingerprint analysis. In fact, the NIA-AA Research Framework has kept the list of biomarkers open-ended to include new biomarkers as and when they are identified and validated.¹¹  To overcome the major drawbacks of detecting the core and indirect biomarkers in the brain involving invasive procedures and complex imaging techniques (like PET and MRI), assessment of circulating biomarkers in peripheral fluids like CSF and blood are gaining importance in AD diagnosis (see Chapter 14). Recently, many novel biomarkers of core AD pathology (Aβ and tau) and other associated biomarkers (novel protein, RNA, metabolites) have been demonstrated to have potential to aid the diagnosis of AD. Biofluids such as saliva, urine, and tears are being assessed for the identification of novel biomarkers, which are anticipated to revolutionize the diagnosis of AD owing to the simplicity of non-invasive sample collection, rapid testing capability, and cost-effectiveness.¹²⁴  The development of various molecular tools, techniques, and strategies to fulfill the unmet need for reliable AD diagnosis is covered extensively in Chapters 13–15. We have also proposed the accurate diagnosis of AD by generating a characteristic fingerprint of core and indirect biomarkers through multiplexed and multimodal detection to distinguish between AD patients and healthy individuals with precision (Figure 1.9).^4,5,31 

AD is a progressive neuronal disorder, and there are no promising therapies to prevent or halt the disease progression. Considering the disease severity, the FDA has approved four drugs for AD that target acetylcholinesterase and NMDA receptor activity. These drugs treat symptomatic dysfunctions with temporary effects rather than targeting the actual disease casing routes of AD. The FDA has approved donepezil (Aricept^®), rivastigmine (Exelon^®), galantamine (Razadyne^®), and memantine (Namenda^®) for the preliminary treatment of AD. These drugs are mainly cholinesterase inhibitors and are prescribed to treat individuals with cognitive dysfunction symptoms such as impairment in language, thinking, judgment, and memory. Unfortunately, these drugs have several side effects like vomiting, nausea, headache, confusion, loss of appetite, muscle cramps, constipation, and dizziness. The current status of AD drugs and AD-associated death uphold the requirements of effective disease-modifying therapies. Multiple pathological events are involved in AD progression (vide supra) that significantly prevent therapeutic developments.^3,4  Notwithstanding several constraints, researchers (academia and pharma) from across the globe have directed enormous efforts to discover drug candidates targeting individual disease routes of AD. Natural products such as curcumin, berberine, epigallocatechin gallate (EGCG), physostigmine, rutaecarpine, and geissospermine, among others, are found to exhibit disease-modifying effects. However, most of the drug candidates exhibit low to moderate effect, and some of them have failed in different stages of clinical trials, which has forced researchers to rethink the drug design and targeting strategies. A promising solution is to target multiple pathological events using multifunctional modulators/inhibitors (Figure 1.10).^13,15,28,38  Various aspects of targeting the multifaceted toxicity of AD using multifunctional molecules are discussed in Chapters 12 and 16–20. The therapies targeting tau pathology have attracted considerable attraction in recent years, and some candidates are undergoing clinical trials. A few small molecules and antibody-based drugs are in the advanced stage of clinical trials with mixed results.¹²⁵  Apart from core AD pathology like Aβ and tau, neuroinflammation and oxidative stress are some of the other main culprits, causing severe damage to neurons. Researchers in AD drug discovery are looking for novel drug candidates which significantly reduce neuroinflammation to ameliorate brain damage.¹²⁶  Recent findings have highlighted the role of the gut microbiome (gut–brain axis) in brain health and AD pathology. China has approved a seaweed-based drug, which is shown to alter the gut microbial population to ameliorate AD pathology and restore memory deficits.¹²⁷  In a landmark development, an anti-amyloid immunotherapy (Aducanumab, Biogen) has been given conditional approval by the FDA for the treatment of AD. There is an unmet need to identify novel and tangible targets and smart strategies to target multifaceted toxicity employing multifunctional/multipronged therapeutic candidates to effectively treat and manage AD.

AD was reported more than a century ago, and yet there are no reliable tools for its diagnosis and therapy. Nevertheless, valiant multidisciplinary efforts by academic, pharma, and clinical researchers have provided greater insights into the multifactorial nature of AD. The multiple pathophysiological processes are the major hurdle in the development of reliable diagnosis and therapeutics, while several intricate disease mechanisms and their interplay are yet to be understood. This chapter has briefly summarized various pathophysiological events and their relationship to provide the current status of AD in terms of pathophysiological understanding, diagnostics, and therapeutic modalities. The individual topics on pathophysiological events are elaborated thoroughly in the subsequent chapters. The current status of AD biomarkers and diagnostic strategies are discussed in detail with future directions. Various therapeutic approaches targeting individual and multiple toxicity targets of AD are described with an optimistic outlook. Overall, this chapter and the book is compiled to provide critical insights into the multifactorial nature of AD with respect to the current understanding of disease mechanisms, diagnostic, and therapeutic advancements with perspectives for future developments to address the unmet need for early and reliable diagnosis and effective therapy.