Assessing Neuroprotective Agents for Aβ-Induced Neurotoxicity

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Opinion

Assessing Neuroprotective Agents for Aβ-Induced Neurotoxicity Kelly H. Forest1,2 and Robert A. Nichols1,* Alzheimer’s disease (AD) is a relentlessly progressive neurodegenerative disease, currently incurable, which presents one of the largest unmet needs in medicine. AD is histologically characterized by the accumulation of extracellular amyloid-beta (Aβ), evident as senile plaques and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. However, the levels of diffusible extracellular Aβ, a neuropeptide largely present in oligomeric form, rise by orders of magnitude many years before evident pathology and subsequent AD diagnosis. The long delay in neurotoxicity and synaptic dysfunction triggered by Aβ and driven by abnormal tau indicates the presence of inherent neuroprotective systems in brain. Here, we propose that strategic approaches for the identification and implementation of neuroprotective agents could provide novel therapeutics for this devastating disease.

Highlights A rise in amyloid-beta (Aβ) occurs years before pathology leading to Alzheimer’s disease (AD), indicating the presence of neuroprotective mechanisms in the brain. Multiple targets for Aβ have been identified in diverse contexts, including receptors linked to neuromodulation by physiological levels of Aβ. Signaling through Aβ-interacting receptors mediating neuromodulation may only partially overlap with signaling linked to neurotoxicity, synaptic dysfunction, and/or behavioral deficits.

The Slow Progression of Alzheimer’s Disease (AD): Endogenous Neuroprotective Pathways versus Developing Pathology AD (see Clinician’s Corner) is the most common form of dementia (see Glossary), manifested as compromised short-term memory, dysfunctional language, and deficits in cognitive function, mood, and sleep that arise in parallel to progressive neuropathological changes, including pronounced neuronal loss in select regions of the brain. During the long prodromal period leading to AD, the slow development of neuropathology, including neurodegeneration, indicates the presence of endogenous elements and signaling pathways supporting synaptic, neuronal, and glial viability. This occurs in the face of a dramatic increase in the extracellular levels of the peptide beta amyloid-beta (Aβ), as much as 10 000-fold, in select regions of the brain early in the prodromal period [1,2], well prior to accumulation into plaques and the appearance of hyperphosphorylated tau leading to neurofibrillary tangles [3]. The prominence of Aβ in AD histopathology has made it a reasonable target for therapeutic interventions, and the majority of drug research and development to date has been centered around the amyloid cascade hypothesis [4–6], aiming to reduce Aβ formation and aggregation, inhibiting Aβ toxicity, and/or increasing Aβ clearance. Unfortunately, drugs developed to attenuate the impact of Aβ toxicity through reduction in the levels or aggregation state of Aβ have had little, if any, success in alleviating AD [7], raising questions in regard to the role of the amyloid cascade in AD pathogenesis [8]; specifically the lack of clinical improvement in the face of lowered Aβ or plaque burden in humans immunized with Aβ [9]. One caveat is the requirement that clinical trials focus on individuals with AD, whereas Aβ levels had already risen up to 10–20 years prior to disease diagnosis [10]. Moreover, neurodegeneration correlates best with neurofibrillary tangle burden [3]. Nonetheless, a large body of evidence demonstrates that soluble Aβ oligomers, not insoluble fibrils or plaques, contribute to the synaptic dysfunction, neuronal damage, and memory deficits exhibited in AD [11–13], each involving tau hyperphosphorylation (see [3]). Consequently, for individuals identified as at risk for AD, alternative approaches to Aβ, other than simply lowering levels of the peptide, are needed, particularly those that engage neuroprotective strategies to preserve and/or boost Trends in Molecular Medicine, Month 2019, Vol. xx, No. xx

A wide range of molecules, both natural and synthetic, with neuroprotective activity against Aβ-induced neurotoxicity have been reported, but most have been assessed in vitro in limited contexts. Recently, several promising, broadly acting neuroprotective agents for Aβ-induced neurotoxicity have been identified in preclinical studies for AD.

1

Department of Cell & Molecular Biology, John A. Burns School of Medicine, University of Hawai’i at Manoa, Honolulu, HI, USA 2 Current address: Department of Biology, Dartmouth College, Hanover, NH, USA

*Correspondence: [email protected] (R.A. Nichols).

https://doi.org/10.1016/j.molmed.2019.05.013 © 2019 Elsevier Ltd. All rights reserved.

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synaptic and neuronal viability. Here, we address potential novel neuroprotective strategies, focusing on neuronal targets for amyloid linked to cellular, synaptic, and behavior toxicity.

Current Therapeutic Strategies Most of the Aβ-based current strategies against AD to date have been directed towards reducing production, facilitating clearance, and/or preventing aggregation. Aβ is produced via sequential proteolytic cleavage of the amyloid precursor protein (APP) by the processing enzymes, γ-secretase and β-secretase (BACE) [14]. Reducing Aβ production by inhibiting the activities of γ-secretase or BACE has been shown to reduce the levels of plasma, brain, and cerebrospinal fluid Aβ in models overexpressing APP [15–18]. While able to reduce Aβ, γ-secretase inhibitors were unfortunately found to cause drug-related toxicities in the gastrointestinal tract [19], thymus [20], and spleen [17,21]. Moreover, a major issue for γ-secretase inhibitors from the outset has been lack of substrate specificity for APP, with numerous other substrates identified, most notably the developmental protein Notch [22]. As for β-secretase inhibition, higher specificity for Aβ was indicated; however, limited trials with β-secretase inhibitors were undertaken and later suspended, owing to unanticipated adverse side effects, consistent with discovery of multiple substrates for β-secretase [23]. As found for animal studies, vaccine-based or passive immunotherapy to lower Aβ in humans has also proven effective [24,25], however it is without significant clinical improvement and, worse, has resulted in sporadic inflammatory microhemorrhagic responses. Interventions focused on the clearance of Aβ have been aimed at stimulating central nervous system removal and/or degradation [26–28] or export of Aβ across the blood–brain barrier (BBB) [29–31] into the peripheral circulation [32]. The action of many of these drugs has been substantiated in animal models and they have undergone extensive clinical trials in humans, but have all failed to meet their primary clinical endpoints. As a separate approach, inhibition of Aβ aggregation has been attempted through the development of small molecules to prevent Aβ oligomerization into fibrillar forms [33], but also without clinical success. In sum, although much progress has been made in our understanding of AD pathophysiology, approaches aimed at reducing the levels or fibrillar state of Aβ as therapeutic applications have, as noted, fallen short in clinical trials.

Neuroprotection: Molecular, Synaptic, Neuronal, and Behavioral Without any success in developing a drug to treat the underlying conditions of AD, a shift towards the investigation of neuroprotective agents to slow down degeneration and to preserve the function of existing neurons has taken place. Agents claimed to have neuroprotective properties have been aimed at preventing the progression of the disease by halting and slowing down synaptic and/or neuronal dysfunction and/or loss, as a means to preserve neuronal population survival. Many of the current broadly acting neuroprotective treatments for neurodegeneration facilitate the reduction of cellular and synaptic oxidative stress through the application of antioxidants [34–36] or block of excitotoxicity through application of glutamate receptor antagonists [37–39]. Neuroprotective strategies specific for Aβ toxicity have also included targeting Aβ-induced oxidative stress [36,40] and excitotoxicity [41,42]. Additional neuroprotective strategies relevant to AD include regulating APP [43], neurotransmitter receptors (e.g., [44]), downregulating stress kinase signaling cascades [45–47], blocking activation of caspases (e.g., [48]), and/or upregulating cell survival pathways [49–52].

Strategies in Neuroprotection Ideally, a comprehensive neuroprotective agent against Aβ toxicity should slow or, better, halt disease progression by reducing degeneration and preserving existing function across four key 2

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Glossary Amylin: islet amyloid polypeptide, originally described as an endocrine peptide secreted with insulin from the pancreas, but later found in brain to regulate autonomic function, pain, and cognitive function. Amyloid: insoluble fibrils deposited extracellularly in various tissues. Amyloid-beta (Aβ): a 36–43 amino acid peptide that regulates synaptic signaling at physiological levels, but at pathological levels can aggregate into oligomers and fibrils often seen in Alzheimer’s disease. Amyloid precursor protein (APP): a transmembrane protein that produces Aβ when cleaved sequentially by β-secretase and γ-secretase. Blood–brain barrier (BBB): a highly selective vascular barrier that protects the brain against circulating toxins or pathogens. The barrier is formed by endothelial cells of the capillary wall forming tight junctions. Brain-derived neurotrophic factor (BDNF): a member of the neurotrophin family of growth factors, which plays a role in neuronal and synaptic support, growth, and differentiation. It also plays a role in synaptic plasticity to modulate long-term memory. cAMP response element-binding protein (CREB): a transcription factor that binds to the cAMP response element (CRE) on DNA. Relevant to Aβ neurotoxicity, CREB regulates genes involved in neuronal plasticity and long-term memory. Cellular prion protein (PrPc): glycosyl-phosphatidylinositol (GPI)-membrane anchored glycoprotein (also known as CD230), which, when misfolded, leads to prion disease (s) known as spongiform encephalopathies. PrPc has been identified as the major Aβ receptor in brain linked to Aβ neurotoxicity. Dementia: clinical symptomatic description of neurological impairment, which may include some or all of the following: memory loss, language difficulties, attentional deficits, and impaired judgment and reasoning. Excitotoxicity: cell damage or death primarily due to the overactivation of excitatory glutamate receptors leading to neuronal calcium dysregulation. Glia: non-neuronal brain cells (astrocytes, microglia, oligodendrocytes) having a range of supportive and regulatory functions, including maintenance

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categories: cellular, signaling, synaptic, and behavior (Box 1). Altered cellular function and intracellular signaling relates to progressive neurodegeneration in AD. However, early in the disease, synaptic dysfunction and loss are the dominant pathogenic changes, which leads, in turn, to altered circuitry underlying cognitive behavioral deficits [53]. In addition to showing that neuroprotective agents delay or arrest Aβ-induced neuronal death and enhance survival at a broad cellular level, agents that claim to have neuroprotective properties should also arrest or alter known apoptotic signaling cascades and/or enhance survival mechanisms and/or antiapoptotic pathways. Furthermore, protection at the cellular level may not necessarily translate to protection at the synaptic level against Aβ-linked dysfunction [53,54] and loss [55], thus neuroprotective agents should also be able to prevent and/or rescue synaptic dysfunction. Lastly, an effective agent should reverse cognitive and behavioral deficits brought about by the loss of neurons and synapses, and not just spatial memory. Behavioral deficits in AD extend to impaired attention, orientation (space and time), working memory, executive function and judgment, language function, mood (depression, anxiety, agitation), and sleep [56]. A compilation of citations reporting neuroprotective agents with noted activity against various aspects of Aβ toxicity under the four key categories, sampled across a 5-year period, is provided in Table S1 (see the supplemental information online. While the list only captured primary publications explicitly focusing on Aβ neurotoxicity (PubMed search: ‘Neuroprotection and Beta Amyloid’; Table S1), it offers a snapshot of the breadth of activity in neuroprotective agent discovery. The agents have been catalogued with regard to their origin: natural products/derivatives, biologics, or synthetics (Table 1). A majority of the agents are natural products that have been extracted or derived from various plants, herbs, fungi, or algae, including a wide range of polyphenols, flavonoids, quinones, terpenes, and anthocyanins. A subset of the agents falls under the category of biologics, consisting of peptides, proteins, sugars, or nucleic acids. Synthetics encompass a wide variety of small molecules and organic compounds. In a fair number of cases, only extracts have been examined, without identifying the active compound(s) or compound family. In a large number of studies, a toxic fragment of Aβ encompassing amino acids 25–35, with uncertain specificity, was tested rather than full-length Aβ (1–40; 1–42). Only a small subset of putative neuroprotective agents were assessed across all four noted categories (cellular, signaling, synaptic, and behavior) for which Aβ triggers frank toxicity, dysfunction, and deficits (Table 1). Although the compilation indicates that a considerable amount of research and time have been spent exploring and developing protective therapies against Aβ toxicity, no putative agent has yet been shown to provide neuroprotection in AD, namely rescuing or altering the course of the disease in humans. Consequently, the development of an effective neuroprotective compound with therapeutic potential for AD must satisfy a number of factors in addition to those already outlined. First, it is imperative that the neuroprotective agent be able to penetrate

Box 1. Summary of Strategies and Criteria for Neuroprotective Agents in Aβ Toxicity Strategies for Neuroprotection:

of the BBB, neuronal survival, regulation of extracellular neurotransmitter, response to injury, immune modulation, synaptic pruning, and myelination, among others. Hyperexcitability: excessive excitation of neural circuits, typically due to an imbalance of excitation and inhibition. Hyperphosphorylated tau: opportunistic phosphorylation of the microtubule-associated protein, tau, on multiple sites, driving formation of neurofibrillary tangles evident in AD. Metabotropic glutamate receptors: G protein-coupled neuronal surface receptors activated by the neurotransmitter glutamate. Neurofibrillary tangles: paired helical filaments of abnormally folded, hyperphosphorylated tau that are pathological hallmarks of AD. Neuroprotection: a means to slow down, stop, or rescue neuronal and/or synaptic dysfunction leading to neurotoxicity, and/or to preserve existing cellular and/or synaptic function in unaffected neurons. Nicotinic acetylcholine receptor (nAChRs): nonselective ligand-gated cation channel activated by acetylcholine, as the endogenous agonist, or nicotine, as a selective exogenous agonist. Subtypes of this receptor have been identified as high-affinity Aβ targets. NMDA-type glutamate receptor: nonselective ligand-gated cation channel activated by glutamate in the presence of the co-agonist D-serine binding to the ‘glycine’ site under depolarizing conditions to release Mg2+. The influx of Ca2+ through this receptor has been strongly linked to synaptic plasticity and long-term memory formation. Oxidative stress: net accumulation of reactive oxidative species in cells undergoing oxidative stress, which has been linked to cell damage and apoptosis. Plaques: clusters of fibrillar amyloid. Synaptic plasticity: the ability of synapses to strengthen and weaken in response to different patterns of nerve stimulation, affecting the strength and/or number of synaptic connections.

• Blocking identified Aβ-interacting target receptors linked to Aβ neurotoxicity by full or partial agonists • Targeting Aβ-interacting receptor-linked signaling pathways • Activation of alternative neuroprotective pathways, such as neurotrophic or antiapoptotic pathways Criteria for Neuroprotection: • Prevention and/or rescue of neurotoxicity at the molecular, cellular, synaptic, and behavioral levels in appropriate AD-related models • Accessibility and delivery to targets in brain; metabolic stability; lack of non-neural toxicity (e.g., cardiac, renal, or hepatic)

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Table 1. Analysis of Citations for Natural, Biological, or Synthetic Neuroprotective Agents Protecting against Aβ Toxicity for Inclusion of 1, 2, 3, or All 4 Key Categories: Cellular, Signaling, Synaptic, and Behavior Citations including 1, 2, 3, or all 4 of the key categories for neuroprotection against Aβ toxicity: 2012–2017 (Table S1)

a

Type of neuroprotective agent:

1 category

2 categories

3 categories

4 categories

Naturala

46

97

21

1

Biologics

19

21

4

2

Synthetics

25

44

14

3

Primarily extracts, as compared with defined biologics, such as BDNF.

into the brain and access essential targets at a therapeutic concentration, whether administered orally, systemically, or intranasally, at the right window of opportunity to ensure neuronal and synaptic protection against toxicity leading to any further damage. In some cases, the agent may also need to be able penetrate cells to act on intracellular targets, which presents an even greater challenge. Second, endogenous ‘physiological’ levels of Aβ (pM range), as determined in the mouse hippocampus under native (wild type) conditions [57], have important physiological functions [57,58], and disruption of this homeostasis may contribute to toxicity. The agent must therefore have minimal if any disruptive action on the physiological activity of Aβ. Third, neuroprotective therapeutics should not only preserve surviving neurons, but potentially augment their function, including plasticity and adaptability, as a means to compensate for dysfunction resulting from proximal neurodegeneration. Lastly, neuroprotection cannot be limited to one aspect of Aβ neurotoxic signaling. This limitation may explain, in part, the large number and diversity of alleged neuroprotective agents revealed in the literature survey, though in many cases the actual targets were not identified. Moreover, the impact on downstream signaling pathways linked to neurotoxicity (Figure 1, Key Figure), in particular hyperphosphorylation of tau, needs to be an important component to neuroprotective agent screens, and yet is typically overlooked.

Blocking Aβ Targets: Neuroprotection by Blocking Neurotoxicity One of the most compelling strategies for protection against Aβ toxicity is through blockade of Aβ interacting target receptors. Aβ has been shown to interact with various receptors to induce synaptic and neuronal dysfunction and death. Targets notably include cellular prion protein (PrPc) [59,60] coupled to the metabotropic glutamate receptor 5 (mGluR5) [59,61], amylin receptors [62–64], NMDA-type glutamate receptors (and perhaps AMPA glutamate receptors) [65,66], and nicotinic acetylcholine receptors (nAChRs) [67–69]. Though explanation for the wide diversity of putative receptors interacting with oligomeric Aβ remains to be articulated (Box 2), the complement of receptors involved in Aβ-linked synaptic dysfunction, while partially overlapping with those connected to neuronal loss, appears to involve a wide array of targets and cellular sites with distinct temporal, spatial, and affinity constraints (e.g., [59–71]), whether the impact is on synaptic plasticity or synapse structure (primarily dendritic spine morphology [72]). While the development of compounds that can block Aβ interaction to various targets have been extensively explored, multiple levels of Aβ toxicity (synaptic, cellular, memory dysfunction) appear to be mediated by combined activation of Aβ interacting targets, and thus identifying one agent that can prevent Aβ binding to all of the receptors has been challenging. Furthermore, putative neuroprotective compounds binding to these receptors (and other protein targets) may also affect their normal physiological functions [58]. Lastly, Aβ has impacts on brain behavior at levels beyond synaptic, neuronal, and memory processing, namely circuit hyperexcitability, sleep, and anxiety (Box 3), among others, which are nonetheless exceedingly important in regard to AD symptomology and even disease progression. 4

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Key Figure

Major Receptor Signaling Pathways Linked to Amyloid-Beta (Aβ) Neurotoxicity: Targets for Neuroprotective Agents

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Figure 1. Proposed pathways linked to Aβ neurotoxicity triggered by Aβ interaction with major target receptors [60,61,64–69,88,89], which display significant crossover. Details for downstream signaling linked to Aβ neurotoxicity for other identified receptors (e.g., amylin, RAGE, LilrB2, insulin [62,63,70,90,91]) remain to be determined, but likely intersect with many of these pathways. Neuroprotection may involve simple antagonism of these pathways and/or differential signaling. Abbreviations: ABAD, Aβbinding alcohol dehydrogenase; AC, adenyl cyclase; Apaf1, apoptosis protease activating factor 1; ATF, activating transcription factor; BDNF, brain-derived neurotrophic factor; CaM, calmodulin; CaMKII, calmodulin-dependent kinase II; CaN, calcineurin; Casp, caspase; CDK, cyclin-dependent kinase; CHOP, C/EBP homology protein; CREB, cAMP response element binding protein; CytC, cytochrome C; DAG, diacylglycerol; eIF2, eukaryotic initiation factor 2; ER, endoplasmic reticulum; GC, guanyl cyclase; IP3, inositol trisphosphate; IP3R, IP3 receptor; IRE1, inositol requiring enzyme; MAPK, mitogen-activated protein kinase; mGluR5, metabotropic glutamate receptor 5; nAChR; nicotinic acetylcholine receptor; NFAT, nuclear factor of activated T-cells; NMDA-R, NMDA-type glutamate receptor; NO, nitric oxide; NOS, nitric oxide synthase; PERK, PRK-like ER kinase; PI3K, PI3 kinase; PKA, cAMP-dependent protein kinase; PLC, phospholipase C; PP1, protein phosphatase 1; PrPc, cellular prion protein; ROS, reactive oxygen species; RyR, ryanodine receptor; UPR, unfolded protein response.

As one example of a successful strategic approach to selective targeting resulting in comprehensive neuroprotection has been to focus on the amylin (islet amyloid polypeptide) receptor, an unexpected target in brain for Aβ. Amylin was originally described as a pancreatic peptide linked to diabetes via its glycemic regulation and pancreatic β-cell dysregulation [73]; however, Trends in Molecular Medicine, Month 2019, Vol. xx, No. xx

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Box 2. Multiplicity of Aβ Targets Why have so many alleged targets for Aβ been reported? Going back several decades, putative targets number in the dozens and have been described primarily on neurons. While part of the basis for the multiplicity of targets lies with the large number of structural forms for Aβ (monomers, oligomers, prefibrillar forms, fibrillar forms, unstructured aggregates, plaques), compelling evidence for identification of bona fide receptors specifically binding to and regulated by Aβ has been limited. In addition, physiological levels of Aβ (pM range) play a neuromodulatory role in synaptic plasticity and spatial memory [57,58], whereas elevated levels (μM) contributes to AD pathology; therefore the Aβ-interacting targets that promote its neuromodulatory role may only partially overlap with the targets that trigger neurotoxicity. Target receptors for which there is evidence linking them to Aβ neurotoxicity include, first and foremost, PrPc [59,60], as well as amylin receptors [62,63,75], nicotinic receptors [89], receptor for advanced glycation end-products [90], paired immunoglobulinlike receptor B [91], and NMDA receptors [65]. There are also distinct targets that promote Aβ removal. The low-density lipoprotein receptor-related protein mediates Aβ transcytosis across the BBB to clear Aβ from the brain [92]. Other targets involved in Aβ clearance facilitate its cellular uptake and degradation [93]. Most of the identified Aβ-interacting target receptors also reside on glial cells, the most abundant cells in the brain, which function in brain plasticity, neuronal survival, and protection against injury. Hyperactivation and/or dysfunction of glial cells, particularly astrocytes and microglia, may promote neurodegeneration, synaptic dysfunction and loss, hyperexcitability, and learning, memory, and behavioral impairments. Reduced numbers of activated microglia lead to Aβ deposition and increased mortality [94] and high loads of Aβ lead to microglial dystrophy [95]. Preclinical studies in AD models have thus investigated activation of microglia for Aβ phagocytosis and clearance [96]. Limited studies have also examined the ability of activated astrocytes to reduce Aβ load [97,98]. Despite the clear but complex roles for glia in AD progression, very few studies aimed at identifying candidate neuroprotective agents have included consideration of Aβ impact on glia through identified Aβ-interacting target receptors. When identifying the Aβ-interacting receptor targets involved in Aβ toxicity or neuromodulation, the structural form as well as the endogenous concentration of Aβ, physiological or pathological, along with the range of cellular targets, should be delineated in the contexts of neuromodulation, neurotoxicity, neuroinflammation, synaptic dysfunction, and behavioral deficits.

receptors in the brain for amylin were later discovered [74], largely linked to autonomic regulation. Amylin receptors were found to be regulated by Aβ [62,64] and both peptides acting via amylin receptors trigger apoptotic neuronal death [75]. Recently, neutral amylin receptor antagonists were found to reverse synaptic and memory deficits in AD-like mouse models [63,76]. The precise mechanism by which amylin receptor antagonists reverses these deficits remains to be demonstrated, but appears to engage multiple cellular targets regulated by both amylin and Aβ. An alternative approach has been to screen for inhibitors of Aβ-interacting target receptors linked to Aβ neurotoxicity. A successful example of this approach was recently reported by Stritmatter and colleagues, who have been focusing on a primary Aβ-interacting target responsible for Aβtriggered toxicity, namely cellular prion, or PrPc, linked to mGluR5 [60,77]. An orally active, polymer-based inhibitor capable of rescuing the pathophysiology of transgenic AD-like mice, including high-affinity neutral block of Aβ binding to PrPc, attenuation of synaptic loss, and reversal of memory deficits, was identified through a cell-based screen [78]. It was proposed that the efficacy of the polymer inhibitor in blocking Aβ interaction with PrPc was the result of multivalent binding to the target receptor. Identification of these antagonists for defined receptors interacting with Aβ represent highly promising and notably effective approaches to the development of novel AD therapeutics, encompassing multiple categories of neuroprotection in AD model systems (Boxes 1 and 2).

Engaging Neuroprotection In addition to competitive antagonism of Aβ to protect against neuronal toxicity, a number of receptor-based neuroprotective pathways have been identified and utilized as a potential 6

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Box 3. Physiological Impact of Neuroprotective Agents: Hyperexcitability, Anxiety, and Sleep

Clinician’s Corner

In preclinical studies involving potential neuroprotective agents, initial assessment of the impact on Aβ-linked neurotoxicity in various forms under the four listed categories (cellular, signaling, synaptic, and behavior) is a start. However, several aspects of promising candidate neuroprotective agents also need to be considered, optimally in parallel, namely relative stability (in serum), cardiac, hepatic, and renal toxicity (initially via in vitro models), and potential for BBB penetrance/ transcytosis (for potential oral delivery). In addition, the impact on tau phosphorylation and localization, as a primary intracellular effector of Aβ-triggered neurotoxicity, should be assessed.

AD is the sixth leading cause of death. By 2050, the cost for care of individuals with AD and other dementias could reach over $1 trillion. Delaying disease progression or improving patient function will substantially reduce the economic and social impacts of AD on the family and on society as a whole.

As AD progresses, there are changes in attention, orientation, language (word retrieval, aphasia), mood (anxiety, agitation, depression), and sleep patterns (slow-wave sleep), and a small but significant increase in risk for seizures. Regarding the latter, there is much evidence to indicate that elevated levels of Aβ alter the balance of excitability, leading to more generalized hyperexcitability [99], and that the hyperexcitability is particularly acute in circuits near plaques [100] and involves local dendritic calcium dysregulation [101]. These changes may contribute to altered mood, but the latter likely involves complicated changes in circuitry. As for sleep, Aβ clearance is increased during the slow-wave phase [102], and a large number of studies have identified alterations in slow-wave sleep with elevated Aβ in AD (e.g., [103]). We propose, therefore, that after the initial screening of neuroprotective agents largely via cellular, synaptic, and learning memory models (for both Aβ- and tau-linked toxicity), subsequent assessment in models for hyperexcitability (e.g., [99]), anxiety (e.g., [104]), and sleep (e.g., [105]) should be performed.

strategy. In AD models, the cAMP response element-binding protein (CREB), a constitutively expressed nuclear transcription factor that regulates neuronal survival and function, has been shown to be downregulated in hippocampal neurons [50–52]. It has been shown that CREB is important in neurotrophin-mediated signaling linked to neuronal survival [79] and in neuroprotection via the PI3-kinase/Akt pathway [80]. Contributing to a feedforward mechanism, CREB is believed to upregulate neurotrophin expression and activity, importantly, brainderived neurotrophic factor (BDNF), which is important in modulating synaptic plasticity [81], and a neurotrophin target receptor, the tropomyosin receptor kinase receptor B (TrkB) [82]. The use of neurotrophins, such as nerve growth factor (NGF) or BDNF, or small-molecule neurotrophin mimetics [51], are attractive alternatives for neuroprotection against Aβ toxicity but, to date, neurotrophins used for AD treatment have not been successful but remain promising. As noted, another broad approach to neuroprotection has been to counter oxidative stress and/ or its sequelae. An example of an intriguing crossover to AD for an orally active free radical scavenger, edaravone, originally developed for acute ischemic stroke, was discovered wherein strong reductions in Aβ-induced oxidation, APP processing, Aβ deposition, neuroinflammation, neurite and dendritic loss, and synaptic and behavioral dysfunction were observed in in vitro and in vivo APP models [83]. These findings indicate the possibility of a highly effective, broad neuroprotective agent, which may have its most significant impact as a preventative in regard to oxidative stress and neurodegeneration.

Although the development of AD pathology is understood in great detail, attempts to alleviate symptoms and/or slow the course of disease based on approaches to reduce AD pathology in validated animal models, particularly amyloid burden, have been unsuccessful in humans diagnosed with AD. By the time patients are diagnosed with AD, there is already severe loss of synapses and neurons in key brain regions manifesting AD pathology. Imaging for Aβ reflects aggregates and plaques, not the total load of Aβ in susceptible regions driving synaptic dysregulation and neurodegeneration. Alternative approaches to assess risk for AD as well as prodromic development of AD are under development. Novel therapeutic approaches to AD are needed, independent of Aβ pathology and guided by clinical progress of the disease (memory deficits; altered sleep; attentional deficits; cognitive impairment, especially executive function; language dysfunction; seizure propensity; changes in mood; degradation in basic life skills). Translation of broadly acting, effective neuroprotective agents to AD therapy represents a promising alternative.

A newly developed avenue has focused on Aβ itself, which, at low physiological levels (pM range), acts as a positive neuromodulator [57,58]. The approach has been to exploit the endogenous neuronal regulatory activity of Aβ rather than simply lowering Aβ levels or blocking Aβ binding. Aβ, primarily in monomeric form, appears to be neuroprotective [84]; but it would be difficult, if not impossible, to control the concentration and structural form of Aβ in vivo [85]. Using mutational analysis together with physiological and behavioral assays in mouse models, it was determined the hydrophilic N terminal domain of Aβ is responsible for the neuromodulatory activity of full-length Aβ [86]. Consequently, a nontoxic fragment of Aβ (1–15/16) that is derived from the N terminal domain, naturally derived from Aβ by the action of α-secretase, and does not oligomerize [87], was shown to be more effective in regard to neuromodulatory activity compared with that observed for Aβ [86]. This heightened efficacy was evident as increased presynaptic Ca2+, augmented post-tetanic potentiation and long-term potentiation, and enhanced fear memory

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[86]. Moreover, the α-secretase-generated N terminal Aβ fragment, and a core hexapeptide (N-Aβcore) encompassing the key residues conferring neuromodulatory activity within the naturally occurring N terminal fragment [86], were found to be broadly neuroprotective against Aβ-induced toxicity. Specifically, the N terminal Aβ fragment and the N-Aβcore rescued fulllength Aβ-induced mitochondrial dysfunction, oxidative stress, and apoptotic neuronal death in vitro in rodent neuron models, whether added as cotreatment or, notably, post-Aβ treatment [88]. The Aβ-derived peptides were also shown to rescue Aβ-induced deficits in synaptic plasticity (long-term potentiation) and contextual fear conditioning (spatial memory) in APP model mice [86,88]. It was further found that the N terminal Aβ peptides protect against Aβ toxicity involving both low-affinity and high-affinity receptors (nAChRs) interacting with Aβ [88] as well as glutamate-induced excitotoxicity, suggesting that the N terminal Aβ peptides may be broadly neuroprotective through engagement with all key Aβ interacting target sites and/or activation of alternative pathways, such as the aforementioned neuroprotective pathways and perhaps antiapoptotic pathways. Collectively, the findings indicate the potential of the N terminal Aβ peptides as effective neuroprotective agents against Aβ-linked neuronal toxicity, synaptic inhibition, and behavioral deficits (Box 1). Moreover, the core hexapeptide is small enough to provide a platform for small molecule drug development.

Concluding Remarks Of the agents identified in the last 5 years, only a small subset (~2%) appear to have met all essential criteria for classification as comprehensive and effective neuroprotective agents (Table 1), which is only the first stage in developing a stable therapeutic able to reach and act on key targets in the brain, leading to prevention and/or alleviation of AD. Indeed, most have only been tested in in vitro assays (Table S1 in the supplemental information online). Very few of these agents have been shown to definitely alter Aβ interaction with identified receptors (e.g., [63,78,88]). There are additional issues regarding whether an agent is selective for neurons and/or must act intracellularly, and whether these agents only provide symptomatic relief rather than addressing underlying mechanisms (see Outstanding Questions). The consideration of the role of various glial cells in neuroprotection has also been rather limited (Box 2). Although a subset of these agents have been shown to lower cytosolic Aβ and/or block Aβinduced neuronal death (Table S1 in the supplemental information online), the mechanisms of neuroprotective action of most of these agents remain to be determined. Overall, whether these ‘neuroprotective’ agents are being comprehensively investigated with appropriate in vitro and in vivo models remains an open question. Nonetheless, progress is underway in identifying broadly acting agents that meet the standards of definition for neuroprotection in AD-like models and, ultimately, AD (Box 1). In view of the multiple signaling pathways affected in AD at multiple sites (synapses to circuits to behavior) neuroprotection leading to clinically effective therapy in AD may well require a combination of novel approaches. Acknowledgments R.A.N. is supported by funding from the National Institutes of Health and the University of Hawaii Foundation. We thank Ms Megan Lantz for critical reading of the manuscript.

Disclaimer Statement The authors declare they have no competing interests.

Supplemental Information Supplemental information associated with this article can be found online at https://doi.org/10.1016/j.molmed.2019.05.013.

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Outstanding Questions How do we rigorously define neuroprotection in the context of Aβ-triggered cellular toxicity leading to neurodegeneration in select brain regions, synaptic dysfunction leading to altered synaptic communication disrupting neural circuits, and behavioral deficits leading to symptomology in AD? What clues to the mechanisms of action of neuroprotective agents against Aβtriggered neurotoxicity can be derived from understanding the regulation of downstream signaling pathways (via extracellular versus intracellular targeting by the agents)? Does protection by neuroprotective agents against Aβ-triggered cellular toxicity involve targets and downstream signaling pathways separate from those involved in Aβ-triggered synaptic dysfunction and/or behavioral deficits, in particular, those leading to tau hyperphosphorylation and its impact on neuronal and synaptic function? What is the critical timeframe for administration of a neuroprotective agent? And how should long-term protection be assessed in AD-relevant models? What steps are necessary for translating promising neuroprotective agents in preclinical studies to therapeutic application?

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