**2.2. Immune response in Alzheimer's disease**

decreased activity of the respiratory chain are observed with a simultaneous increase in ROS

mutases, superoxide dismutase (SOD). Regardless the contradictory data on the contribution of COX deficiency to ROS production [44, 45], oxidative damage is an utterly feature of AD, from human samples to cellular and animal models [36, 46–48]. Evidence show that mitochondrial dysfunction and ROS production are accentuated by Aβ, a 4 kDa protein, derived from a larger protein, amyloid β-protein precursor (βAPP), that is overproduced during AD progression. Aβ interacts with mitochondrial proteins, namely ABAD, causing increased ROS production, mitochondrial dysfunction and neuronal death [49, 50]. These changes in mitochondrial metabolism seem to be related with morphological alteration of mitochondria of AD tissues and models. Electron microscopy images from AD brain tissue show mitochondria with reduced dimensions and disrupted *cristae* [51]. Similarly, mitochondria from AD subjects transferred to mtDNA depleted cell into cybrids at an ultrastructural level are small and have a swollen-like structure [52], with a fragmented mitochondrial network that correlates with increased mitochondrial content of dynamin-related protein 1 (DRP1) [53] a key protein for mitochondrial division [54]. Concerning mitochondrial content/mass in AD neurons, the matter is not as straight forward [55]. Vulnerable neurons have a decrease in functional mitochondria, but mtDNA is increased [51]. In accordance it was observed, in AD cybrids, an increase in mtDNA content [33]. This increment was first explained as a compensatory response to counteract the loss of mtDNA transcription efficiency [51], but data gathered on the subject point to decreased mitochondria degradation through autophagy (mitophagy), with imprisoned mitochondria within autophagic vacuoles that are accessible for mtDNA detection [53]. A number of studies have shown autophagy dysfunction as a driving force of AD progression, with important impact on Aβ deposition and plaque formation [56–60]. In human brain samples, it could be observed a massive accumulation of autophagic vacuoles and lysosome-related vesicles, which led to the conclusion of simultaneous induction and impairment of autophagy [56, 61]. Purified autophagic vesicles contain βAPP and the proteases responsible for its cleavage [56]. Aβ peptides are produced by sequential proteolytic processing of βAPP by β-secretase (BACE) and γ-secretase complex (presenilin and nicastrin) [62, 63]. These accumulated vacuoles cause swellings along dystrophic neurites and potentiate Aβ production and aggregation [64], which gradually form the extracellular amyloid plaques, one of the most prominent brain pathological hallmarks of AD. It is reasonable to argue that stimulating autophagy would clear the cell waste materials. Although some contradictory data were published, in opposition of ameliorating Aβ pathology, stimulating autophagy, either chemically or starvation-induced, fails to degrade accumulating Aβ and worsens cell function in *in vivo* models [65]. The driver of such failure is the microtubule network, along which autophagic vesicles are transported towards lysosomes, for degradation of cell waste. Mitochondrial metabolism failure compromises microtubule proper dynamics. Destabilized microtubule cytoskeleton negatively impacts autophagic vesicles retrograde transport towards lysosomes and promotes microtubule-associated protein Tau to detach and undergo phosphorylation [5]. Tau is the main component of paired helical filaments (PHF) that form neurofibrillary tangles found in AD brains [66] and is a microtubule-associated protein (MAP) that promotes microtubule assembly and stabilization [67–69]. Ultrastructural analysis performed in AD neurons found that the number and total length of microtubules are decreased in AD subjects [70]. In AD cybrids, microtubule network is disrupted with increased free tubulin

–., mainly

by mitochondrial dis-

O2

production [42]. The primary ROS in mitochondria is the superoxide radical anion O2

produced at complexes I and III [43], which is rapidly converted to H<sup>2</sup>

140 Mitochondrial Diseases

The role of neuroinflammation in AD dates back to 1907, to the original report of Alois Alzheimer, with microglia surrounding Aβ plaques, thus showing a close relation between the pathway and the disease [73]. Twenty-five years after the postulation of Selkoe and Hardy, the amyloid cascade hypothesis is still the main hypothesis for AD pathogenesis. It is a fact that all AD patients undergo progressive Aβ deposition, and moreover, the sequence of major pathogenic events leading to AD proposed by the amyloid cascade hypothesis is perfectly aligned with the dominantly inherited forms of AD. However, different mechanisms have to be considered to explain the development of AD in sporadic cases, which constitute the vast majority of the cases [74]. Even though Aβ peptide and tau protein oligomers are considered the major contributors to disease progression and the deposition of Aβ occurs decades before any other alterations, there are some missing links between the accumulation and oligomerization of Aβ and tau pathology, synaptic dysfunction and cognitive decline [15, 75]. In this follow-up, neuroinflammation is consistently reported to be deregulated in AD and to facilitate disease progression [76, 77]. Indeed, various forms of Aβ oligomers and aggregates are detected by numerous receptors (TLRs), receptor for advanced glycated end-products (RAGE), CD14, CD36, CD47, α6β1 integrin, class A scavenger receptor and NOD-like receptor family pyrin domains (NLRP) that activate innate immunity response (mainly via MAPK/Erk and NF-κB-mediated signaling) [22, 78–80]. In neurodegenerative diseases, such as AD, the inflammatory response starts by innate immune system activating monocytes (in periphery) and microglial cells, astrocytes and perivascular cells (in the CNS) [81].

Microglia, the resident macrophages of the CNS, play an active role surveying the brain for pathogens and maintaining neuronal plasticity and synaptic connectivity [82]. In AD, stimulation of microglia involves the microglial polarization to a M1 phenotype that triggers the production of proinflammatory cytokines (TNF-α, IL-1, IL-6, IL-12 and IL-18) [83, 84] and chemokines (CCL2, CCR3, CCR5) [85, 86] and is accompanied by impaired phagocytic capacity [87]. Interestingly, deregulation of Aβ clearance from the CNS is a key pathogenic mechanism in pathology progression, whereas microglial phagocytosis activation plays a crucial role (in combination with the endolysosomal pathway, being Aβ enzymatically digested by neprilysin, insulin-degrading enzyme and matrix metalloprotease proteases) and is controlled by two microglial cell surface receptors: TREM2 (positive regulator) and CD33 (negative regulator) [88, 89]. Moreover, caspases are known mediators of apoptosis, but they also regulate inflammation. Upon binding of Aβ to NLRP, there is an inflammasome-dependent activation of caspase-1 that mediates the production of mature IL-1β by cleavage of an inactive pro-IL-1β peptide [90, 91]. Therefore, elevated concentrations of active caspase 1 detected in the brains of patients with AD [92] are in accordance with the increased NLRP3 activation observed in monocytes from AD patients [93]. In addition, mitochondrial DAMPs were shown to increase AD-associated biomarkers, such as App mRNA, APP protein and Aβ1–42 levels, in SH-SY5Y and mice brains [94, 95]. Together, these studies suggest that mitochondria and mitochondrial DAMPs have the potential to promote inflammation in the brain, with important consequences relevant for neurodegenerative disorders such as AD.

Pathological responses of astrocytes include reactive astrogliosis directed at recovery of injured neural tissue and neuroprotection [96]. In AD, astrocytes, like microglia, are a major source of cytokines (TNF-α and IL-1β are readily released upon astrocytic Aβ detection) [97, 98] and chemokines. Indeed CCL4 has been detected in reactive astrocytes near Aβ plaques [99] and has a high capacity to degrade Aβ1–42 in a more efficient way than their microglial counterparts [100]. In addition, post-mortem brains from AD patients are characterized by hypertrophic reactive astrocytes, elevated GFAP and S100B expression surrounding senile plaques [101]. Interestingly, studies have shown that reactive astrogliosis occurs early in the course of pathogenesis and correlates with the severity of cognitive impairment in AD patients [102]. Furthermore, resident microglia and astrocytes in AD have been shown to stimulate inducible nitric oxide synthase (iNOS) and NADPH oxidase [103]. These upregulations lead to the production of high concentrations of ROS (such as nitric oxide, superoxide, hydrogen peroxide, peroxynitrite), which not only further promote microglia activation but also lead to posttranslational modifications (nitration, S-nitrosylation, and dityrosine formation), including Aβ nitration leading to a higher propensity to aggregate and seriously suppress hippocampal LTP [103–105]. Likewise, the complement system is another major constituent of the innate immune system that shows enhanced levels in disease settings. In the brain, activated microglia and astrocytes are responsible for the production of proteins of the complement system, which in turn are associated with Aβ deposits [106]. Additionally, complement receptor 1 (CR1) modulates the impact of the APOE ε4 allele on brain fibrillar amyloid burden [107]. Furthermore, there are other players with neuroinflammatory actions in AD, such as perivascular macrophages promoting Aβ clearance [108], endothelial cells contributing to the transport of Aβ species between the brain and the periphery [109, 110], oligodendrocytes [111] and neurons [112] that contribute to neuroinflammation by expressing complement components.

of the innate immune system in AD on their update on the status of the amyloid hypothesis [74]. Accordingly, three risk genes have been highlighted: TREM2, CD33 and CR1, and all are involved in some way in microglial response, being upregulated during Aβ plaque development [117–119]. Another important aspect is the timeline involvement of the immune system response in AD's development. Analyses from both patients with early AD and mild cognitive impairment (MCI), which precedes AD stage, have identified a correlation between clinical symptoms and the presence of inflammatory markers in the cerebrospinal fluid (CSF), suggesting a much early involvement of the immune system in the disease [120, 121]. Noteworthy, a study in wildtype mice found that chronic inflammatory conditions triggered the development of AD-like neuropathology during aging, demonstrating a case where immune response not only precedes fibrillary Aβ plaque deposition and neurofibrillary tangle formations but also is responsible for their induction [122]. Thus, the possibility to manipulate inflammatory pathways, thereby changing the course of the disease, is yet another indication of the role of inflammation as a driving force of AD pathology. The questions we should make previously of that said manipulation are: Which cells and immune molecules should be modulated? And when should modulation occur? As the activation of microglia and the neuroinflammatory environment are constantly changing depending on the stage of the disease, the time window for modulation and for therapeutically potential is very important [81]. Inefficiency in clinical trials with nonsteroidal antiinflammatory drugs (NSAIDs) in AD could be largely due to wrong timing of intervention [123], since epidemiological and preclinical studies show a reduction up to 80% in the risk of AD onset

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and decrease in microglial activation and amyloid burden with NSAID use [124, 125].

or MCI subjects, increased microglia activation has been detected by PET scans [133].

The induction of neuroinflammatory effects is not restricted to factors of the CNS and can result from systemic influences [125]. On the one hand, traumatic brain injury is an example of a CNSintrinsic neuroinflammatory condition that facilitates the development of AD pathology [134]; on

As aforementioned, there is an uncontrolled production of cytokines and chemokines that may be used as effective tools for inflammatory biomarkers in AD. Early assessment of neuroinflammation in the AD patients may be an important preventive strategy to act before the detrimental aspects of neuroinflammation, thus averting or delaying any cognitive decline [126]. Several studies have investigated the levels of proinflammatory and anti-inflammatory markers in the CSF, plasma and serum of AD patients. IL-1β, TNF-α and IL-6 have been observed to be altered in the three types of samples in AD, although the results vary according to the time point of sampling [126]. Once again, the stage of the disease is a crucial factor for any therapeutic intervention. Moreover, an increase in TGF-β [127] and S100B [128] levels in the CSF from AD compared to controls has also been reported. Regarding blood-based biomarkers of inflammation, α-1-antichymotrypsin (ACT) [129] and C-reactive protein (CRP) [130] have been shown to be increased in AD. Noteworthy, α-2-macroglobulin (α-2 M) [131] and clusterin (or apolipoprotein J) [132] have been implicated in the pathology of AD, with significant increases in patients, showing promising results as potential plasma biomarkers of AD. Interestingly, many of these inflammatory mediators are also altered in MCI subjects. The levels of IL-8, monocyte chemoattractant protein-1 and interferon-γ-inducible protein 10 are found to be increased in CSF, while IL-1β and TNF-α are increased and apolipoprotein A-1 and complement C1 inhibitor are decreased in blood [126]. Besides the detection of neuroinflammatory markers, inflammation may be also monitored through imaging methods. In patients with AD

In the end, the recruited microglia and astrocytes fail to resolve the Aβ insult effectively, resulting in an excessive proinflammatory cytokine and chemokine production, as well as enhancing DAMPs secretion, ultimately leading to deleterious microglial and astrocytic reactivity [113]. This chronic neuroinflammatory environment thus starts a vicious cycle altering APP processing towards a further increase in Aβ production, culminating in neuronal loss and perpetuating inflammation, which with the advance of the disease compromises blood-brain barrier (BBB) permeability, allowing the invasion of peripheral inflammatory cells that exacerbate the deleterious neuroinflammation and facilitate neurodegeneration [114]. Therefore, neuroinflammation in AD was firstly attributed exclusively to these innate immune sensors of Aβ, contributing to the exacerbation of the disease and viewed only as a response, but in reality the pathway is much more complex.

A decade ago, a significant change in this thought was brought by Wyss-Coray who reviewed the hypothesis that inflammation may serve as a cause and driving force for AD [115]. As seen by the significant immune response later on in the disease and as a response to the Aβ accumulation, it is accurate to state that inflammatory pathways are a driving force in AD. However, for a causative role, inflammation should have an early impact or precede the pathogenesis of the disease [81]. In support of inflammation as a primary contributor for the disease, recent genome-wide association studies (GWASs) of sporadic AD cases (or LOAD—late-onset AD) have found associations between AD and genes that are involved in cholesterol metabolism and in innate immunity [116]. Surprisingly, even Selkoe and Hardy drew attention to the importance of the innate immune system in AD on their update on the status of the amyloid hypothesis [74]. Accordingly, three risk genes have been highlighted: TREM2, CD33 and CR1, and all are involved in some way in microglial response, being upregulated during Aβ plaque development [117–119]. Another important aspect is the timeline involvement of the immune system response in AD's development. Analyses from both patients with early AD and mild cognitive impairment (MCI), which precedes AD stage, have identified a correlation between clinical symptoms and the presence of inflammatory markers in the cerebrospinal fluid (CSF), suggesting a much early involvement of the immune system in the disease [120, 121]. Noteworthy, a study in wildtype mice found that chronic inflammatory conditions triggered the development of AD-like neuropathology during aging, demonstrating a case where immune response not only precedes fibrillary Aβ plaque deposition and neurofibrillary tangle formations but also is responsible for their induction [122]. Thus, the possibility to manipulate inflammatory pathways, thereby changing the course of the disease, is yet another indication of the role of inflammation as a driving force of AD pathology. The questions we should make previously of that said manipulation are: Which cells and immune molecules should be modulated? And when should modulation occur? As the activation of microglia and the neuroinflammatory environment are constantly changing depending on the stage of the disease, the time window for modulation and for therapeutically potential is very important [81]. Inefficiency in clinical trials with nonsteroidal antiinflammatory drugs (NSAIDs) in AD could be largely due to wrong timing of intervention [123], since epidemiological and preclinical studies show a reduction up to 80% in the risk of AD onset and decrease in microglial activation and amyloid burden with NSAID use [124, 125].

Pathological responses of astrocytes include reactive astrogliosis directed at recovery of injured neural tissue and neuroprotection [96]. In AD, astrocytes, like microglia, are a major source of cytokines (TNF-α and IL-1β are readily released upon astrocytic Aβ detection) [97, 98] and chemokines. Indeed CCL4 has been detected in reactive astrocytes near Aβ plaques [99] and has a high capacity to degrade Aβ1–42 in a more efficient way than their microglial counterparts [100]. In addition, post-mortem brains from AD patients are characterized by hypertrophic reactive astrocytes, elevated GFAP and S100B expression surrounding senile plaques [101]. Interestingly, studies have shown that reactive astrogliosis occurs early in the course of pathogenesis and correlates with the severity of cognitive impairment in AD patients [102]. Furthermore, resident microglia and astrocytes in AD have been shown to stimulate inducible nitric oxide synthase (iNOS) and NADPH oxidase [103]. These upregulations lead to the production of high concentrations of ROS (such as nitric oxide, superoxide, hydrogen peroxide, peroxynitrite), which not only further promote microglia activation but also lead to posttranslational modifications (nitration, S-nitrosylation, and dityrosine formation), including Aβ nitration leading to a higher propensity to aggregate and seriously suppress hippocampal LTP [103–105]. Likewise, the complement system is another major constituent of the innate immune system that shows enhanced levels in disease settings. In the brain, activated microglia and astrocytes are responsible for the production of proteins of the complement system, which in turn are associated with Aβ deposits [106]. Additionally, complement receptor 1 (CR1) modulates the impact of the APOE ε4 allele on brain fibrillar amyloid burden [107]. Furthermore, there are other players with neuroinflammatory actions in AD, such as perivascular macrophages promoting Aβ clearance [108], endothelial cells contributing to the transport of Aβ species between the brain and the periphery [109, 110], oligodendrocytes [111] and neurons [112]

that contribute to neuroinflammation by expressing complement components.

reality the pathway is much more complex.

142 Mitochondrial Diseases

In the end, the recruited microglia and astrocytes fail to resolve the Aβ insult effectively, resulting in an excessive proinflammatory cytokine and chemokine production, as well as enhancing DAMPs secretion, ultimately leading to deleterious microglial and astrocytic reactivity [113]. This chronic neuroinflammatory environment thus starts a vicious cycle altering APP processing towards a further increase in Aβ production, culminating in neuronal loss and perpetuating inflammation, which with the advance of the disease compromises blood-brain barrier (BBB) permeability, allowing the invasion of peripheral inflammatory cells that exacerbate the deleterious neuroinflammation and facilitate neurodegeneration [114]. Therefore, neuroinflammation in AD was firstly attributed exclusively to these innate immune sensors of Aβ, contributing to the exacerbation of the disease and viewed only as a response, but in

A decade ago, a significant change in this thought was brought by Wyss-Coray who reviewed the hypothesis that inflammation may serve as a cause and driving force for AD [115]. As seen by the significant immune response later on in the disease and as a response to the Aβ accumulation, it is accurate to state that inflammatory pathways are a driving force in AD. However, for a causative role, inflammation should have an early impact or precede the pathogenesis of the disease [81]. In support of inflammation as a primary contributor for the disease, recent genome-wide association studies (GWASs) of sporadic AD cases (or LOAD—late-onset AD) have found associations between AD and genes that are involved in cholesterol metabolism and in innate immunity [116]. Surprisingly, even Selkoe and Hardy drew attention to the importance As aforementioned, there is an uncontrolled production of cytokines and chemokines that may be used as effective tools for inflammatory biomarkers in AD. Early assessment of neuroinflammation in the AD patients may be an important preventive strategy to act before the detrimental aspects of neuroinflammation, thus averting or delaying any cognitive decline [126]. Several studies have investigated the levels of proinflammatory and anti-inflammatory markers in the CSF, plasma and serum of AD patients. IL-1β, TNF-α and IL-6 have been observed to be altered in the three types of samples in AD, although the results vary according to the time point of sampling [126]. Once again, the stage of the disease is a crucial factor for any therapeutic intervention. Moreover, an increase in TGF-β [127] and S100B [128] levels in the CSF from AD compared to controls has also been reported. Regarding blood-based biomarkers of inflammation, α-1-antichymotrypsin (ACT) [129] and C-reactive protein (CRP) [130] have been shown to be increased in AD. Noteworthy, α-2-macroglobulin (α-2 M) [131] and clusterin (or apolipoprotein J) [132] have been implicated in the pathology of AD, with significant increases in patients, showing promising results as potential plasma biomarkers of AD. Interestingly, many of these inflammatory mediators are also altered in MCI subjects. The levels of IL-8, monocyte chemoattractant protein-1 and interferon-γ-inducible protein 10 are found to be increased in CSF, while IL-1β and TNF-α are increased and apolipoprotein A-1 and complement C1 inhibitor are decreased in blood [126]. Besides the detection of neuroinflammatory markers, inflammation may be also monitored through imaging methods. In patients with AD or MCI subjects, increased microglia activation has been detected by PET scans [133].

The induction of neuroinflammatory effects is not restricted to factors of the CNS and can result from systemic influences [125]. On the one hand, traumatic brain injury is an example of a CNSintrinsic neuroinflammatory condition that facilitates the development of AD pathology [134]; on the other hand,systemic inflammation may be induced from several chronic diseases [135], such as obesity and T2D, all characterized by CNS inflammation and microglia activation [136, 137]. Therefore, in AD, neuroinflammation can cause and drive pathogenesis [22].

MPTP is able to cross the blood-brain barrier, is metabolized to 1-methyl-4-phenylpyridinium

decreased in PD individuals in the occipital cortex compared to control individuals [157].

Oxidative damage driven by mitochondria malfunctioning is a prominent aspect in PD [158]. Mitochondrial complex I is one of the most important sites of ROS production in the cell, pri-

proposed as the cause for dopaminergic neurons death in the SNpc [160, 161]. The same authors found in post-mortem samples from PD subjects increased lipid peroxidation whereas glutathione pathway, an antioxidant defense, is impaired [160]. Mitochondria are the main producers and are also the primary targets of ROS. PD brain biopsies revealed complex I itself is oxidatively damaged, which prevents its proper assembly and function [149]. Although it is incontestable that oxidative stress contributes to PD pathology, it is now generally accepted that ROS are a by-product of mitochondrial dysfunction that contributes to worsen cell demise [162].

Familial forms of PD bearing mutations in mitochondrial proteins reinforced the involvement of mitochondrial dysfunction in PD etiology and shed light into the mechanisms leading to neuronal death, unifying both familial and sporadic cases. Rare mutations causing juvenile PD are related to mitochondrial degradation by mitophagy created an opportunity for clarification of the disease mechanisms. The first identified mutation in *PARK2* (Parkin), an E3 ubiquitin ligase, cause early onset PD [163]. The second mutation was identified in *PARK6,* PTEN-induced kinase 1 (PINK1) and a mitochondrial kinase [164]. PINK1 and Parkin act together in a tightly regulated process to target dysfunctional mitochondria for degradation, named mitophagy. This process is crucial for the maintenance of a healthy pool of mitochondria, potentially protecting cells in early stages of mitochondrial dysfunction [165]. In healthy mitochondria, PINK1 levels are maintained low as this protein is degraded within mitochondrial matrix after its import from cytosol [166]. When mitochondria lose their membrane potential, PINK1 is stabilized at their surface recruiting Parkin that, in turn, ubiquitinates and targets mitochondria to undergo mitophagy [167–169]. PD caused by PINK1 and Parkin mutations is not clinically differentiated from idiopathic PD [170]. Morphologically, PINK1 mutations have drastic repercussions in mitochondria from *Drosophila melanogaster* to mouse models, with larger, swollen and disrupted cristae [171, 172]. In cybrids from sporadic PD subjects, mitochondria also present abnormal structure with enlarged and scarce cristae [52, 173]. Mitochondrial network images show that in PD models it presents a fragmented structure. From PD cybrids [174] to dopaminergic neurons treated with MPTP [175], a number of models show early mitochondrial fragmentation that precedes cell death. Although DRP1 has been implicated in the fragmentation of mitochondria in PD [174], studies point to SNCA directly interacting with mitochondria inducing fragmentation, in a process that does not require DRP1 [176]. Recently, it was found a common mechanism for mitophagy failure, besides Pink1-Parkin axis, that is shared by familial and sporadic PD, with potential

[159]. The consequences of oxidative damage are such in PD that oxidative stress was

) and is uptaken by dopaminergic neurons, inhibiting mitochondrial respiration at complex I [148]. Complex I activity was shown to be decreased in PD brain samples [149], in peripheral tissues namely platelets and lymphocytes [150] and in PD cybrids [151]. The inhibition of complex I, with MPTP and rotenone, is widely used as *in vitro* and *in vivo* models of PD since these recapitulate the main features of the disease [152–154]. Mitochondrial dysfunction in PD tissues and models is also characterized by a decrease in ∆Ψmit [52, 155, 156]. Accordingly, at a functional level, brain bioenergetics is compromised in PD where PET scans showed glucose utilization are

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(MPP+

marily O2

–
