Alzheimer's disease: Innate immunity gone awry?

    Alzheimer’s disease: innate immunity gone awry? Theodore B. VanItallie PII: DOI: Reference:

S0026-0495(17)30018-5 doi: 10.1016/j.metabol.2017.01.014 YMETA 53531

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Metabolism

Please cite this article as: VanItallie Theodore B., Alzheimer’s disease: innate immunity gone awry?, Metabolism (2017), doi: 10.1016/j.metabol.2017.01.014

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ACCEPTED MANUSCRIPT Alzheimer’s disease: innate immunity gone awry?

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Theodore B. VanItallie, MD1

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Professor Emeritus of Medicine Columbia University College of Physicians & Surgeons New York, NY

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Home address: 16 Coult Lane Old Lyme, CT 06371 USA

5984 words.

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Email address: [email protected]

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Telephone number (Landline): +1-860-434-5662

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ACCEPTED MANUSCRIPT Abstract Inflammation is an immune activity designed to protect the host from pathogens and noxious

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agents. In its low-intensity form, presence of an inflammatory process must be inferred from

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appropriate biomarkers. Occult neuroinflammation is not just secondary to Alzheimer’s disease (AD) but may contribute to its pathogenesis and promote its progression. A leaky blood-brain

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barrier (BBB) has been observed in early AD and may play a role in its initiation and

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development. Studies of the temporal evolution of AD’s biomarkers have shown that, in AD, the brain’s amyloid burden correlates poorly with cognitive decline. In contrast, cognitive deficits in

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AD correlate well with synapse loss. Oligomeric forms of amyloid-beta (oAβs) can be synaptotoxic and evidence of their deposition inside synaptic terminals of cognition-associated

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neurons explains early memory loss in AD better than formation of extracellular Aβ plaques. Among innate immune cells that reside in the brain, microglia sense danger signals represented

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by proteins like oAβ and become activated by neuronal damage such as that caused by bacterial endotoxins. The resulting reactive microgliosis has been implicated in generating the chronic form of microglial activation believed to promote AD’s development. Genome-wide association studies (GWAS) have yielded data from patients with sporadic AD indicating that its causes include genetic variation in the innate immune system. Recent preclinical studies have reported that β-hydroxybutyrate (βOHB) may protect the brain from the adverse effects of both the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome and the deacetylation of histone. Consequently, there is an urgent need for clinical investigations designed to test whether an orally administered βOHB preparation, such as a ketone ester, can have a similar beneficial effect in human subjects. Keywords: neuroinflammation; blood-brain barrier; β-hydroxybutyrate; microglia; 2

ACCEPTED MANUSCRIPT NLRP3 inflammasome; apoliprotein E4 allele; amyloid β oligomers; astrocytes; histone

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deacetylase inhibitor; synaptic pruning; complement activation;ketone esters

1. Introduction

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In Book III of his classic De Medicina, published in the 1st century, the Roman encyclopedist,

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A. Cornelius Celsus, listed the four cardinal signs of inflammation in one concise sentence.

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“Now the signs of an inflammation are four: redness (rubor) and swelling (tumor), with heat (calor) and pain (dolor)” [1]. A fifth sign, “disturbance of function” (funcio laesa), is thought to

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have been added by Galen a century and a half later [2].

As inflammation research became more comprehensive, the traditional concept of

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inflammation was found to be seriously deficient [3,4]. It is now accepted by authorities in the field that, at the lower end of a spectrum of intensities, there are grades of inflammation which

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are not necessarily visible, painful, or localized. In such instances, the existence of a chronic, low-grade inflammatory process is inferred from the concurrent presence of inflammationrelated mediators or biomarkers in the circulation and/or in the affected part [5]. Inflammation is a protective immune activity, regulated by the host in response to threats and incursions by potentially harmful pathogenic organisms, agents and substances. Such threats can be endogenous (e. g., from a gout attack [6]) as well as exogenous. Heppner et al. [7] have summarized recent evidence that neuroinflammation is not simply an epiphenomenon of Alzheimer’s disease (AD) and several other brain-affecting disorders. To the contrary, new data indicate that, instead of just being responses to the pathophysiological events associated with AD, immune system-mediated actions may actually contribute to and drive AD’s pathogenesis [7]. As people age, occult inflammation can be remarkably destructive,

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ACCEPTED MANUSCRIPT contributing to the development of such metabolic and neurodegenerative disorders as type 2 diabetes mellitus (T2D), impaired cognition, AD, and Parkinson’s disease [PD] [8-10].

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De Felice and Ferreira [11] have called attention to the fact that inflammation, insulin resistance, and mitochondrial dysfunction are “common molecular denominators that connect

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type 2 diabetes (T2D) to AD.” In T2D and obesity (both significant AD risk factors), low-grade

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chronic inflammation—for example, overproduction of pro-inflammatory mediators (such as

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tumor necrosis factor-α [TNF-α])—gives rise to peripheral inflammation and systemic insulin resistance. In the brain, pro-inflammatory signaling, triggered by toxic amyloid-β oligomers

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[oAβs] that localize within synapses in the AD brain [12], causes hippocampal insulin resistance, diminished glucose utilization, and contributes to the synapse deterioration, and memory

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impairment observed in T2D, obesity and AD [13]. Given the growing evidence that low-grade inflammation can be progressively injurious to

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key areas of the brain, it is essential that neuroscientists strive to understand the mechanisms that drive neuroinflammation and seek remedies that will enable the treating physician to abort or greatly reduce the activity of the damaging inflammatory process. 2. The principal risk factor for AD: advancing age Advancing age is AD’s principal risk factor [14]. The odds of developing sporadic AD increase rapidly after age 65. In the United States, 2016, 11% of people age 65 and older have AD, while 32% of people age 85 and older have AD. Among women age 71 and older, about 16% have AD and other dementias compared to 11% among men in the same age range. Among people with AD, 4% are <65 years, 15% are 65-74 years, 44% are 75-84 years, and 37% are 85+ years—a decline that seems to reflect the increased mortality among elderly people with

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ACCEPTED MANUSCRIPT AD. Among individuals aged 70 years, 61% of those with AD are expected to die before the age of 80 compared with 30% of those without AD.

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Examples of aging-related changes that increase an individual’s vulnerability to AD include

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the following:

2.1 Impairment of the integrity of the blood-brain barrier (BBB)

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In experimental studies of the BBB, Zlokovic observed that, as the BBB began to deteriorate

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with aging, the leakiest part was usually the section shielding the hippocampus. This was also the place where AD plaques formed [15]. Experiments conducted by D.K. Kumar, et al. in mouse

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and worm models of AD have suggested that, when the BBB is unable to prevent entry into the CNS of invading pathogens such as viruses, fungi and bacteria, the brain’s defense forces

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counter with production of Aβ, which then envelops interlopers in a dense sticky, predominantly amyloid mass. The result is accumulation of plaques typical of AD [16]. Using dynamic,

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contrast-enhanced magnetic resonance (MR) imaging with dual time resolution, van de Haar et al. [17] found that, in contrast to age-matched control subjects, global BBB leakage was occurring in patients with early AD associated with cognitive decline. They concluded that “…a compromised BBB may be part of a cascade of pathologic events that eventually leads to cognitive decline and dementia”.

According to Zlokovic [15,18], approximately 25% of all individuals are carriers of one or two copies of the apolipoprotein E4 (apoE4) allele, AD’s most influential genetic risk factor. In apoE4 heterozygotes the risk of developing AD is increased by about 400%; in homozygotes the increased risk is about 1,500%. BBB breakdown is more pronounced in AD patients carrying the apoE4 allele. ApoE4 expression (compared with apoE2 and 3) is associated with a significant

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ACCEPTED MANUSCRIPT increase in amyloid plaques in brain at earlier ages. ApoE4 impairs Aβ clearance from brain and across the BBB in animal models and patients at risk of developing AD [15,18].

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2.2 Reductions in the resting-state cerebral metabolic rate of glucose (CMRgluc) have been

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measured in individuals at high risk of developing AD decades before the onset of AD symptoms

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The decreases in cerebral glucose metabolism commonly found during AD’s premonitory and

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preclinical phases have been attributed to such upstream events as aging-linked mitochondrial dysfunction, infiltration of soluble oAβs into mitochondria, and increased production of reactive

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oxygen species (ROS) [19,20]. The brain regions that manifest the earliest signs of deterioration in advanced AD are also the ones in which an ongoing preclinical reduction in glucose utilization

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can be demonstrated by means of fluorodeoxyglucose-positron emission tomography (FDGPET); namely, medial temporal lobe (MTL) structures, the entorhinal and perirhinal cortex, and

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the CA-1 and subiculum regions of the hippocampus. It is worthy of note that, although glucose uptake may be impaired in the MTL and other memory-critical brain regions in aged individuals and in early dementia, these same regions do not exhibit any metabolic impairment when the blood level of ketone bodies (KBs) is substantially increased by adherence to a strict ketogenic diet or ingestion of certain ketone esters [21]. The reduced glucose metabolism measured in cognitively normal elderly individuals and in patients with early AD cannot readily be attributed to neuronal atrophy. A more likely explanation might be the presence of a metabolic lesion such as insulin resistance, which (for example) can impair the function of the rate-limiting mitochondrial enzyme complex, pyruvate dehydrogenase (PDH)[22]. Nevertheless, when the brain’s memory-critical structures (like synapses which have a high energy requirement) are subjected to reduced supply of energy, even

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ACCEPTED MANUSCRIPT a modest degree of fuel deprivation—over time—can be expected to have a damaging effect on memory and cognition.

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Causation of synaptic dysfunction and memory loss by oAβs may depend—at least in part—

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on their ability to overstimulate N-methyl-ᴅ-aspartate-type glutamate receptors (NMDARs) in the hippocampal and cortical regions associated with cognition. One effect of such

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overstimulation is an abnormal rise in extrasynaptic glutamate levels. Adverse downstream

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occurrences include aberrant elevation of cytoplasmic Ca2+, with serious disruption of cellular homeostasis. Unregulated calcium entry into mitochondria uncouples ATP energy transfer and

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increases generation of free radicals. The homeostatic imbalance produced by these events can then lead to mitochondrial dysfunction, resulting in synaptic and neuronal loss, and cognitive

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decline [23].

2.3 Aging-related decline in mitochondrial efficiency

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According to Gaziev et al. [24], oxidative damage does not substantially contribute to the frequency of mitochondrial DNA (mtDNA) mutations; to the contrary, mtDNA replication errors of DNA polymerase gamma (POLG) are the principal source of mtDNA mutations. When mitochondria become dysfunctional owing (at least in part) to aging-related mtDNA mutations, they increase their production of ROS thereby increasing oxidative stress in adjacent brain regions. However, in their 2013 analysis of the role of mitochondria in aging, Bratic and Larsson [25] concluded that oxidative damage does not play an important role in age-related neurodegenerative diseases such as AD. In their view, mtDNA mutations in somatic stem cells may give rise to progeroid phenotypes without increasing oxidative stress. They suggest that mtDNA mutations could bring about a bioenergetic deficiency capable of driving the aging process.

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ACCEPTED MANUSCRIPT Hara et al. [26] have found that “delayed response (DR) accuracy”—an indicator of dorsolateral prefrontal cortex (dlPFC)-dependent working memory—correlates inversely with

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the prevalence of abnormal, donut-shaped mitochondria in dlPFC presynaptic terminals (synaptic

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boutons). Synaptic terminals are specialized presynaptic areas which contain synaptic vesicles that embody neurotransmitters, mitochondria, and endoplasmic reticulum. In older male rhesus

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monkeys and in menopausal rhesus monkeys, the increased prevalence of such donut

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mitochondria correlates directly with working memory decline. Aged ovariectomized monkeys were found to exhibit a significant reduction in working memory and a 44% increase in

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presynaptic donut-shaped mitochondria, both of which were reversed with estradiol treatment [26]. Accumulation of donut mitochondria was also associated with fewer synaptic vesicles.

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C.1. Beneficial effect of regular physical activity on mitochondrial function and cognition In their commentary on the Hara, et al. 2014 PNAS report, Picard and McEwen [27] conclude

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that, in primates, mitochondrial function affects brain behavior and cognition by influencing synaptic transmission. It seems likely that dysmorphic mitochondria also accumulate in synapses of the aging or diseased human brain and could therefore be targets of various treatments, including regular physical activity known to be capable of improving mitochondrial function and cognitive performance [28]. Sleiman, et al [29] recently published evidence showing that brain-derived neurotrophic factor (BDNF) increases in the brains of previously sedentary mice given free access to a running-wheel for 30 consecutive days. Apparently, this effect only occurs when histone deacetylase enzymes stop inhibiting BDNF production. It was also noted that the livers of the exercising mice were producing extra β-hydroxybutyrate (βOHB), a known inhibitor of class I histone decacetylases (HDAC2 and 3). The resulting HDAC inhibition would be expected to act

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ACCEPTED MANUSCRIPT positively on BDNF promoter activity. Sleiman et al. found that direct ventricular application of βOHB was followed by enhancement of hippocampal BDNF expression. Taken together, these

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observations suggest that beneficial responses to exercise that occur in the brain, such as cognitive improvement and reduced depression and anxiety, may arise because of the increase in

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brain BDNF made possible by exercise-induced increases in circulating levels of βOHB.

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2.4 Complement- and microglia-mediated aberrant loss of hippocampal synapses correlates

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with memory impairment in early AD

During normal development, complement constituents C1q and C3 mediate timely synapse

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elimination by phagocytic microglia. The same pruning pathway—normally down-regulated in the mature brain—is activated early in the J20 AD mouse model. The premature synapse loss

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that occurs helps explain the early memory loss in clinical AD. Hong et al. [30] found a significant loss of synapses in the hippocampus of the J20 transgenic mouse at 3-4 months, prior

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to Aβ plaque deposition. C1q immunoreactivity in J20 brains was elevated by 1 month, well before synapse loss. The C1q rise in pre-plaque brains was stimulated by oAβ. Experimental reduction of oAβ levels resulted in a corresponding reduction of C1q deposition. C1q is a principal mediator of oAβ-induced synaptic damage and loss and microglia are a major source of C1q in these preplaque brains. Thus, Hong and associates have provided further evidence that the classical complement cascade mediates premature synapse loss in AD mouse models.

3. Increased Aβ production and deposition: a triggering event? Liu et al [5] have suggested that the earliest event in the AD process is abnormal deposition of Aβ in cognition-involved brain regions. It is their view that Aβ deposition activates microglia, astrocytes, and complement—events followed by release of pro-inflammatory cytokines from the

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ACCEPTED MANUSCRIPT activated glial cells. Normally, the cytokines so produced are supposed to help repel invading pathogens, but when microglial and astrocytic activation becomes chronic, the cytokines’

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protective role is largely replaced by the damaging effects of rampant neuroinflammation on neurons and other key brain structures that support memory and cognition. Experimental

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evidence of Aβ’s initiating role in AD has been found in the AD (PS1V97L-Tg) mouse model

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[31]. In these animals, brain Aβ shows a significant increase at 6 months—an occurrence

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associated with memory dysfunction and aberrant hyperphosphorylation of tau. According to the authors, these changes (which did not occur in wild-type littermate control mice) are promoted

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by Aβ-induced oxidative stress and inflammatory reactions. Apart from activating glial cells and causing release of excess cytokines, Aβ42 and its

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oligomers appear to have a directly destructive action on neurons. Like the pore-forming toxins that attack neuronal membranes, Aβ also seems capable of perforating such membranes and

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causing synaptic failure and neuronal death. These Aβ actions seem to be potentiated by neuroinflammation [32].

4. Immune system activation occurs in tandem with AD development In their review of inflammation’s role in AD, Heppner et al. call attention to earlier reports that the levels of various mediators of inflammation are increased in the body fluids and tissues of persons with AD [7]. Correlative analyses of these observations and the clinical symptoms that presage mild cognitive impairment and the presence of asymptomatic inflammatory markers in the cerebrospinal fluid (CSF) disclosed a much earlier involvement of the immune system in AD’s development than was previously suspected. As one example, a recent study in wild-type mice found that systemic immune challenge can trigger and drive the development of AD

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ACCEPTED MANUSCRIPT neuropathology consisting of Aβ plaques and aggregations of tau, together with activation of microglia and reactive gliosis. Also, the upregulation of inflammatory genes found in tissues of

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patients with early AD further supports the concept of a cause-and-effect relationship between

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inflammation and neurodegeneration, with early involvement of immune actions. Thus, inflammation could play a role in initiating AD and in promoting and accelerating its

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progression. A study in wild-type mice has found that immune processes are capable of causing

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bioenergetic deficit and driving AD-like neuropathology in the absence of Aβ deposition [33]. Neuroinflammatory effects can be induced by factors limited to the central nervous system

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(CNS) and/or by systemic influences that arise outside the CNS. For instance, systemic inflammation may result from psoriasis (a chronic immune system disease)[34], from the effects

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of obesity [35], and from obesity-generated T2D [36]. Acquired obesity in humans is associated with several metabolic perturbations, low-grade inflammation (e.g., elevation of the acute phase

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reactant, serum high-sensitivity C-reactive protein (hs-CRP), and insulin resistance[37]. Within the CNS, traumatic brain injury (TBI) is a familiar example of a condition attended by brain inflammation [38].

5. Innate immune-driven versus adaptive immune-driven neuroinflammation All metazoans possess a genetically-encoded surveillance system designed to protect them from potentially dangerous invaders. This mechanism, known as the innate immune system (IIS) originated 600 million or more years ago, being conserved in both plants and animals throughout their subsequent evolution. The IIS is the body’s first line of defense against invading pathogens. Some authors have suggested that two parts of the IIS—the defensins and the toll-like

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ACCEPTED MANUSCRIPT receptors (TLRs)—are very ancient, predating the split between animals and plants more than a billion years ago [39]. In contrast to the adaptive immune system (AIS), which originated100

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million years or more later than the innate system, the responses of the IIS do not take a week or

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longer to become effective against microbial intruders. IIS responses depend on the ability of the organism to recognize conserved features of pathogens not present in the intact host. To fulfill

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their mission, innate immune responses require the ready availability of proteins and phagocytic

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cells preprogrammed to recognize the generic patterns of encroaching pathogens and quickly attack and destroy them. In this way, the IIS can rapidly protect the body from a microbial

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incursion that might otherwise overwhelm the body’s defenses and become lethal. The IIS uses toll-like receptors (TLRs) responsive to membrane characteristics to identify

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extracellular microbes and promptly initiate effective defensive measures. Pathogen-specific molecules are recognized by TLR proteins found in plants and invertebrate and vertebrate

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animals. Intracellular microbial sensors, known as NOD-like receptors (NLRs) detect nonmicrobial as well as microbial danger signals. When that happens, NLRs form large cytoplasmic complexes known as inflammasomes. The NOD-like receptor protein 3 (NLRP3) inflammasome (for example) activates caspase-1 in concert with activation of the inflammatory cytokines, pro-interleukin (pro-IL)-1β and pro-IL-18. In vertebrates, microbial surface molecules activate complement—a family of circulating proteins that produce an inflammatory response and conjointly disrupt the membranes of bacteria and target them for destruction by neutrophils and macrophages. In IIS-propelled neurodegenerative diseases, the inflammatory process is principally driven by monocytes (arising outside the CNS) and by microglial cells, astrocytes, and perivascular macrophages (arising inside the CNS)[7].

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ACCEPTED MANUSCRIPT 6. Adaptive immune system Heppner et al.[7] have proposed that it is the nature of the associated inflammation that

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distinguishes traditionally defined neurodegenerative from neuroinflammatory diseases. Neuro-

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degenerative disease—of which AD is an example—is innate immune system (IIS)-driven while neuroinflammatory disease—exemplified by multiple sclerosis (MS)—is adaptive immune

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system (AIS)-driven. In contrast to the IIS, the AIS adaptive responses are exquisitely attuned to

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the particular pathogen that elicits them. The mission of the AIS is to destroy the invaders and any toxic molecules that accompany them. This all-out immunological warfare makes it essential

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that the AIS distinguish unequivocally between molecules peculiar to the host and those that identify the invading pathogen. The immune cells from outside the CNS that drive the AIS

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include B lymphocytes and T lymphocytes. However, blood-derived monocytes make up the largest proportion of the outside cell population that, when needed, enter the CNS. Once inside

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the CNS these monocytes change into inflammatory phagocytes (macrophages or dendritic cells) and are believed responsible for much of the tissue damage that occurs in the encephalidities and MS.

Dendritic cells (DCs) are known as “professional” (formerly “accessory”) antigen processing cells. Two categories of DC have been described: (i) the conventional dendritic cell (cDC), which resembles the monocyte and secretes interleukin 12 (IL-12), and (ii) the plasmacytoid dendritic cell (pDC), which looks like a plasma cell and produces substantial amounts of interferon-α, which recruits increasing numbers of activated macrophages primed for phagocytosis. Resident brain dendritic cells (bDC) show an age-related rise in number in the aging mouse brain, together with a concomitant increase in the expression of markers of immune

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ACCEPTED MANUSCRIPT activation. A 2- to 5-fold rise in immunolabeled bDC was measured in the cortex, corpus callosum and cerebellum of the aged brain [40].

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Gao, et al. [41] have reported that mature DC-derived exosomes increase endothelial

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inflammation and atherosclerosis via a membrane TNF-α-mediated NF-кᴃ pathway. As

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described by Steinman (discoverer of “dendritic cells” [42]), DCs patrol the body seeking out foreign invaders such as bacteria, viruses, or toxins. After capturing and degrading these

ᴃ cells and killer ᴛ cells to identify, attack and destroy the

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or spleen where they stimulate

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intruders, DCs display the antigenic fragments on their surfaces and carry them to lymph nodes

invading agents.

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Heppner et al. point out differences between AD and MS at the pathogenic level [7]. To begin

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with, as mentioned earlier, the genetics of MS provides evidence that T cells play an etiologic

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role in the disease. In contrast, genome-wide association studies (GWASs) of sporadic AD have disclosed associations between AD and other genes that are linked to innate immunity. Examples include AD-linked TREM 2 mutations in the microglial or myeloid genes which encode the triggering receptor expressed on myeloid cells as well as mutations in the complement receptor genes. Moreover, in MS, the disease process begins with T cell autoimmunity; in contrast, the process in AD may start with abnormal protein processing and, early on, involve aberrant innate immunity. Finally, the quantitative trait loci implicated in AD are expressed in monocytes, while those involved in MS are principally found in T lymphocytes.

7. Astrocytes Astrocytes—the most abundant cells in the brain—“are responsible for regulation of blood flow, maintenance of the BBB, synaptic function and plasticity and maintenance of the 14

ACCEPTED MANUSCRIPT extracellular environment of ions, fluids, and neurotransmitters” [43]. Astrocytes play a special role in removing glutamate, the brain’s most important excitatory transmitter, from the

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extracellular space (ECS)—the general setting from which its neurotransmitter function is

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exercised. Because glutamate receptor proteins are expressed on the surfaces of astrocytes and neurons, they can only be activated from such surfaces. Hence, to a considerable degree, control

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of glutamate’s activation of its receptor proteins (notably N-methyl-ᴅ-aspartate [NMDA]) is a

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function of its concentration in the ECS, determined in considerable part by the dynamic balance of glutamate’s release into, and removal from, the ECS [44]. Glutamate removed by astroglial

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cells can be metabolized in the tricarboxylic acid (TCA) cycle, and can be converted to glutamine, a form unable to activate glutamate receptors.

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ECS glutamate levels that are too low or too high are potentially harmful; therefore, it is essential that they be kept within safe limits at all times. Glutamate is constantly released from

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neuronal terminals into the ECS. For the most part, this process involves exocytosis of glutamate-containing synaptic vesicles from nerve terminals. But, there are other, non-exocytotic mechanisms of glutamate release; for example, via anion channels or by reversed function of glutamate-transporting proteins at the cells’ plasma membrane. Inhibition of glutamate’s clearance rate can lead to its rapid buildup in the ECS, leading to “excitotoxicity” of nerve cells—a process which may “overexcite” and thereby damage or kill glutamate-exposed nerve cells [44]. The intact BBB protects the brain from serum glutamate levels in the peripheral circulation (50-200 μM) that may be orders of magnitude higher than the concentrations which are toxic to neurons [44,45]. Given the mounting evidence that AD dementia arises from the degeneration of glutamatergic corticocortical neurons that normally enable memory and cognition, it has become increasingly

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ACCEPTED MANUSCRIPT urgent to understand more fully the neuroplastic changes in glutamatergic circuits that occur in aging and dementia, and to develop approaches that can prevent and treat cognitive decline,

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whether it is age- or AD-related. In the pursuit of this goal, Pereira et al. [46] reported studies

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indicating that glutamatergic regulation, accomplished by administering riluzole— a glutamate modulator—to aged rats, can prevent hippocampal-dependent age-related memory loss. The

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riluzole appears to exert its therapeutic effects by “promoting synaptic NMDA receptor

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activation while preventing extrasynaptic NMDA activity, thereby protecting against age-related cognitive decline through induction of neuroplastic changes in the hippocampus and PFC.” The

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authors demonstrated that the riluzole induced clustering of dendritic spines, which “significantly empowered neural circuits with nonlinear summation of synaptic inputs,” in effect, increasing

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synaptic strength.

Insults to the CNS from injury or disease can result in molecular, cellular and functional

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changes in astrocytes, leading to what is known as ‘reactive astrogliosis’, epitomized by an alteration in gene expression, hypertrophy, and proliferation of astrocytes. Molecular triggers of reactive astrogliosis include peptide growth factors, cytokines such as FGF2, IL-6, TNFα and IL1, glutamate, noradrenaline, and disease-associated products such as Aβ42. These factors can be released by all cell types of the CNS, including neurons, microglia, oligodendrocytes, endothelia, and astrocytes. Negative effects of reactive gliosis include neurotoxicity, inflammation and inhibition of axon regeneration.

8. Microglia Microglia are innate immune cells that reside in the brain making up ~12% of the brain’s mass. Microglia do not arise from the neuroectoderm. In humans, they start their existence as

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ACCEPTED MANUSCRIPT fetal macrophages—the earliest microglial precursor cells in the embryonic brain. Thus, they are distinct from neurons, oligodendrocytes and astrocytes. They sense danger signals like those

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represented in AD by protein aggregates and become activated by various kinds of neuronal

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damage, like that caused by exposure to the bacterial endotoxin, lipopolysaccharide (LPS). LPSs are large molecules present in the outer membrane of gram-negative bacteria. Administered to an

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animal, LPS elicits a strong immune response. Reactive microgliosis—the microglial response

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to neuronal injury and exposure to toxic substances, such as ROS and LPS—has been implicated in the initiation of the chronic form of microglial activation that appears to play a significant role

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in promoting development of AD. In effect, microglia foster the neuroinflammation that accompanies AD. When microglia are activated they become chronic sources of TNFα, NO, and

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ROS (including superoxide [O2¯ ])—molecules designed to destroy pathogenic invaders [47]. However, these cytotoxic substances can also contribute to the inflammation, cellular damage,

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atrophy, and loss of function found in AD dementia [48]. Post-mortem studies of brains from AD patients have found microglia-derived macrophages intimately associated with the damaged brain tissues, suggesting a role for these cells in clearing accumulated Aβ or other harmful debris [7].

9. Roles of astrocytes and microglia in neuroinflammation Under normal conditions, activated microglia can protect the brain from traumatic injury via modulation of neuronal synapses and by promoting neurogenesis, clearing debris, and suppressing inflammation. SH Cho, et al [49] have reported a mechanistic link between chronic inflammation and aging microglia, identifying a causal role of aging microglia in neurodegenerative cognitive deficits. They showed that the NAD-dependent protein, sirtuin1

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ACCEPTED MANUSCRIPT (SIRT1), which is known to have protective effects against neurodegenerative diseases, is reduced in aging microglia, and that microglial SIRT1 deficiency plays a causative role in

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aging- or tau-mediated memory deficits via IL-1β upregulation in mice. These findings disclose a

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special epigenetic mechanism in aging microglia that may contribute to cognitive deficits in aging and neurodegenerative diseases.

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There are variants in the microglial-expressed receptor TREM2 that are associated with a 2-4-

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fold increased risk of developing AD. The most striking and consistent observation related to TREM2’s role in regulating Aβ deposition inside the hippocampus has been a robust decrease in

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microgliosis surrounding Aβ plaques in TREM2-deficient mice. These findings suggest that TREM2 plays a key role in promoting the microgliosis that occurs in response to CNS

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parenchymal damage. Microgliosis is a term that describes the protective migration of microglia

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to regions in the brain’s parenchyma that have been injured or threatened by potentially

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damaging invaders like fibrillar Aβ42 and aberrantly phosphorylated tau [50]. Microglia—which express receptors for inflammatory modulators on their cell surface—can also receive inflammatory signals from neighboring astrocytes, neurons, endothelium and leukocyte infiltrates. Astrocytes may be the major source of these inflammatory modulators. For example, astrocytes exposed to neuron-derived α-synuclein (the principal filamentous ingredient of Lewy bodies) produce pro-inflammatory cytokines and chemokines that can activate microglia [43]. Inflammatory modulators increased in astrocytes by extracellular αsynuclein regulate microglial chemotaxis, activation, and proliferation. However, some of the inflammatory modulators expressed in astrocytes can have opposite effects on microglia and may act on them to suppress inflammatory responses. “Production of these suppressive modulators … may play an important role in fine-tuning microglial activation” [43].

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ACCEPTED MANUSCRIPT 10. Possible role of neuroinflammation in the initiation and/or progression of AD A decade ago, Wyss-Coray reviewed the evidence that inflammation may serve as a cause or

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driving force of Alzheimer disease [51]. He concluded that “we need to determine whether

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inflammation in Alzheimer disease is: (i) an early event; (ii) whether it is genetically linked with the disease; and (iii) whether manipulation of the inflammatory pathways changes the course of

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the pathology.” As mentioned above, Heppner et al. [7] have made a convincing case for

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inflammation as an early event in AD’s development. Also, as reported by Jones, et al. [52], genetic evidence obtained from large, genome-wide association studies (GWAS) have identified

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the first “LOAD” (late-onset Alzheimer disease) susceptibility genes since the APOE-ɛ4 allele was reported to be a major genetic contributor to AD risk in 1993 [53]. The same GWAS

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datasets were used to disclose key LOAD pathophysiological processes [52].

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The GWAS data analyzed by Jones and co-workers [52] suggest “… that the primary

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causes of LOAD (or sporadic AD) include genetic variation in cholesterol metabolism and the innate immune system.” In their comprehensive update on the status of the amyloid hypothesis twenty-five years after its original proposal in 1991, Selkoe and Hardy [54] call attention to the brain’s innate immune system as an important factor in AD’s pathogenesis. They specifically refer to the microglial response to plaque formation and the complement cascade that occurs in and around neuritic plaques. In their words, “In the last few years, genetic variability in the IIS (as reported by Jones et al. [52]) has emerged as a compelling determinant of AD risk, implicating many components of innate immunity and the complement cascade as risk factors in the disease.” Unlike the situation in multiple sclerosis (MS) where the inflammatory response is typical of the adaptive immune system (AIS) and in which the disease process starts with T cell

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ACCEPTED MANUSCRIPT autoimmunity, inflammation in AD is characteristic of the IIS in which the quantitative trait loci are expressed in monocytes [7].

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11. Pathways of inflammation

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Recently published laboratory studies primarily in mice, have reported that βhydroxybutyrate may protect the brain from the adverse effects of both the NLRP3

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inflammasome and the deacetylation of histone [55,56]. Thus, it would seem urgently important

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to conduct clinical investigations designed to test whether an orally administered βOHB preparation, such as a ketone ester [57], can have similarly favorable actions in human subjects.

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A. The NLRP3 inflammasome: activation and deactivation It is now well understood that the NLRP3 inflammasome is an important innate immune

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sensor that becomes activated by exposure to damage-associated molecular patterns (DAMPs), including toxins, ceramides, urate and cholesterol crystals, amyloid, and glucose excess. If

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NLRP3 is ablated in various animal models of human illness, the manifestations of T2D, atherosclerosis, multiple sclerosis, AD, age-related functional decline, osteoporosis and gout become attenuated [55]. Thus, as Youm et al. [55] state, “ … identification of endogenous mechanisms that control NLRP3 inflammasome deactivation may provide insights into the control of several chronic diseases”. Using as their rationale the evidence that prolonged fasting reduces inflammation, and that plasma levels of the ketone body, ᴅ-β-hydroxybutyrate (abbreviated here as ‘βOHB’), are elevated by total energy deprivation, Y-H Youm and co-workers [55] conducted tests to determine whether βOHB affects NLRP3 inflammasome activation. They treated lipopolysaccharide (LPS)-primed mouse bone marrow-derived macrophages (BMDMs) with the NLRP3 activator ATP, along with βOHB, and measured caspase-1 activation. βOHB dose-

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ACCEPTED MANUSCRIPT dependently inhibited both the ATP-induced cleavage of caspase-1 into p20 and the processing of the biologically active p17 form of IL-1β. The inhibition occurred at βHB concentrations

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similar to those induced by 2 days of fasting. In sum, they found that βOHB, but neither acetoacetate (AcAc) nor the structurally related fatty acids, butyrate and acetate, suppresses the

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NLRP3 inflammasome in response to urate crystals, ATP and the lipotoxic fatty acids. βOHB

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inhbits the NLRP3 inflammasome by preventing K+ efflux and reducing ASC (apoptosis-

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associated speck-like protein containing a CARD) oligomerization and speck formation. βOHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1β and IL-18 in human monocytes. In

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mouse models of NLRP3-mediated diseases such as urate crystal-induced peritonitis, MuckleWells syndrome, and cold autoinflammatory syndrome, βOHB attenuates caspase-1 activation

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and IL-1β secretion [55]. The authors conclude that use of dietary or pharmacologic approaches

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to elevate βOHB—without concurrently restricting energy intake—holds promise in reducing

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chronic inflammatory diseases mediated by the NLRP3 inflammasome. B. Effect of changes in histone acetylation on neuroinflammation Histones are the principal protein constituents of chromatin—a complex of DNA and histone present in eukaryotic cells. Chromatin is usually dispersed in the interphase nucleus, but during mitosis or meiosis it is condensed into chromosomes. Chromatin subunits consisting of DNAhistone complexes are known as nucleosomes. Histones serve as “spools” around which DNA winds. Such winding enables the compaction needed to fit the large genomes of eukaryotes inside cell nuclei (thereby producing a ~40,000-fold reduction in molecular length). Important functional changes occur when the histone’s lysine moiety is acetylated or deacetylated [58]. Lysine acetylation reduces the electrostatic attraction between the negatively charged DNA spine and the histone, loosening the chromatin structure and making it more accessible to active

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ACCEPTED MANUSCRIPT transcription. Deacetylation results in a tightening of the chromatin structure making it resistant to active transcription. Agents that block the removal of acetyl radicals from histone are known

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as histone deacetylase (HDAC) inhibitors. In 2013, Zhang and Schluesener [59] reported that,

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after 10 days of treatment of an animal model of cerebral amyloidosis with an efficient benzamide histone deacetylase inhibitor (MS-275) there was significant amelioration of

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microglial activation and β-amyloid deposition in cerebral cortex and/or hippocampus. This was

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associated with improved nesting behavior and an attenuated inflammatory activation of a mouse macrophage line in vitro.

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Shimazu et al [56] reported that βOHB is an endogenous and specific inhibitor of class I (1-3) histone deacetylases (HDACs). They found that administration of exogenous βOHB increased

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global histone acetylation in mouse tissues and that βOHB-induced inhibition of HDAC was correlated with global changes in transcription of the genes encoding oxidative stress resistance

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factors FOXO3A and MT2. Substantial protection against oxidative stress was obtained when mice were pretreated with βOHB.

Closing comments

It is impossible to imagine a plausible explanation of AD’s pathogenesis that fails to include consideration of the striking increase in AD risk that occurs after the age of 65. This review has briefly described some of the anatomic and physiological changes that accompany advancing age and how they might increase vulnerability to AD development. At the same time, attention has been given to the evidence that aging is associated with increased concentrations in the circulation of inflammatory mediators and biomarkers. Systemic inflammation may result from

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ACCEPTED MANUSCRIPT conditions like psoriasis, obesity or type 2 diabetes (T2DM). Sometimes there is no evident reason other than aging for the rise in the elderly of blood levels of cytokines and chemokines.

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The presence of asymptomatic inflammatory markers in body fluids before the emergence of

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overt dementia supports the concept that inflammation can be involved in initiating AD and promoting its progression. Studies in wild-type laboratory animals have demonstrated that

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immune processes are capable of triggering and driving the development of neuropathology

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characteristic of AD, together with activation of microglia and reactive gliosis. When microglia are activated they become sources of cytotoxic substances which were intended by nature to

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destroy invading pathogens but, in the aging body, may contribute to the cellular damage and loss of function found in AD dementia. Yet, it must be pointed out that some of the inflammatory

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modulators expressed in astrocytes may act on microglia to suppress inflammatory responses. In this way they are responsible for fine-tuning microglial activation.

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Although the evidence continues to increase that innate immunity-generated neuroinflammation plays a significant role in AD pathogenesis, details of that role remain unclear. Is chronic neuroinflammation necessary to the pathogenic process? Is it sufficient? Does the development of AD depend on some kind of interaction between aging and inflammation? Is redox dysregulation involved? It now seems possible to get better answers to some of these questions. As described above, it has been shown in mouse models that βOHB holds promise in alleviating illnesses mediated by the NLRP3 inflammasome. It has also been reported that βOHB is a specific inhibitor of class 1 histone deacetylases (HDAC). Administration of exogenous βOHB increased global acetylation in mouse tissues; moreover, substantial protection against oxidative stress was achieved when the mice were pretreated with βOHB. If βOHB is administered to aging mouse models of AD, it will be interesting to learn

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ACCEPTED MANUSCRIPT whether the reduction in neuroinflammation that presumably ensues will affect the pattern of the

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AD’s development.

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Acknowledgements

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Special thanks are due Bruce S. McEwen, PhD, A.E. Mirsky Professor, Rockefeller University,

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for critical examination of the manuscript, and for recommending special research reports for inclusion. The author is also grateful to Christina M. VanItallie, PhD, Staff Scientist, National

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Institutes of Health and Leslie A. Lewis, PhD, professor emeritus of biology, York College of CUNY, for helpful editorial comments and scientific advice. Useful editorial advice was

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provided by Ernst J. Schaefer, MD, distinguished professor of medicine, Tufts University School

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of Medicine, and Sami A. Hashim, MD, professor emeritus of nutritional medicine, Columbia

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University. Publication of this article was supported by the Collège International de Recherche Servier (CIRS). The author is a member of CIRS’s Scientific Committee.

Conflict of interests: None

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