Memory Loss and the Onset of Alzheimer’s Disease Could Be Under the Control of Extracellular Heat Shock Proteins

Abstract

Alzheimer’s disease (AD) is a major contemporary and escalating malady in which amyloid-β (Aβ) peptides are the most likely causative agent. Aβ peptides spontaneously tend to aggregate in extracellular fluids following a progression from a monomeric state, through intermediate forms, ending in amyloid fibers and plaques. It is generally accepted now that the neurotoxic agents leading to cellular death, memory loss, and other AD characteristics are the Aβ intermediate aggregated states. However, Aβ peptides are continuously produced, released into the extracellular space, and rapidly cleared from healthy brains. Coincidentally, members of the heat shock proteins (hsp) family are present in the extracellular medium of healthy cells and body fluids, opening the possibility that hsps and Aβ could meet and interact in the extracellular milieu of the brain. In this perspective and reflection article, we place our investigation showing that the presence of Hsp70s mitigate the formation of low molecular weight Aβ peptide oligomers resulting in a reduction of cellular toxicity, in context of the current understanding of the disease. We propose that it may be an inverse relationship between the presence of Hsp70, the stage of Aβ oligomers, neurotoxicity, and the incidence of AD, particularly since the expression and circulating levels of hsp decrease with aging. Combining these observations, we propose that changes in the dynamics of Hsp70s and Aβ concentrations in the circulating brain fluids during aging defines the control of the formation of Aβ toxic aggregates, thus determining the conditions for neuron degeneration and the incidence of AD.

Keywords

Aging Alzheimer’s disease heat shock proteins neurotoxicity protein oligomerization

INTRODUCTION

Alzheimer’s disease (AD) is a major contemporary disease product of progressive neurodegeneration culminating with dementia and death [1]. The etiology of this condition is multifactorial influenced by the genetic background, sex, environment, and injury, among other factors [2]. AD has been subjected to the most intense research because of its tremendous social and economic relevance. The application of the most advanced laboratory techniques has generated abundant information on the cellular and molecular characteristics of this disease. In spite of this intense basic and clinical research progress, no effective cure or prevention has come to light so far [3]. In addition, there are still many inconsistencies and critical basic questions unanswered about the etiology of AD. For instance, although the most likely causative agent of AD, the amyloid-β (Aβ) peptide, is enduringly present in the brain of healthy individuals, the genesis, timing, and basis for the late onset of the disease remain unanswered. Moreover, the triggers for the late selective cellular degeneration in localized brain regions and the inception of the disease are still in debate and subject to a variety of conjectures [4 –6]. AD symptoms are produced by progressive and selective degeneration of cells controlling neuronal circuits in the neocortex, hippocampus, and basal forebrain cholinergic system. The dysfunction of those regions is traduced into skills and memory loss, which is followed by the progressive degeneration of the brain, gradually incapacitating the individual until decease occurs by multiple related causes [1].

To date, despite intensive efforts, the mechanism(s) responsible for selective neuronal death and dysfunction in AD remain unclear. Currently, the predominant theory for AD is the “amyloid hypothesis,” which states that abnormally increased levels of Aβ peptides result in the production of a variety of aggregates that are neurotoxic [7, 8]. Aβ peptides are normally produced throughout the life of individuals after the sequential proteolysis of the amyloid-β protein precursor (AβPP) by β- and γ-secretases, resulting in the release of Aβ peptides into the extracellular space. The major β-secretase, named BACE-1, cleaves AβPP within the extracellular domain, resulting in the secretion of the large ectodomain of the protein leaving behind a membrane-tethered C-terminal fragment that serves as a substrate for γ-secretase. The γ-secretase cleaves at multiple sites within the transmembranous region of the remaining fragment of AβPP generating Aβ peptides ranging in size from 38 to 43 amino-acid residues in length [9]. These peptides are released into the extracellular space where they initiate a regular self-aggregation process mainly driven by hydrophobic effects and electrostatic forces, progressing, from a monomeric state, through intermediate forms, ending, allegedly in pathological conditions, in long unbranched amyloid fibers and plaques [10]. The Aβ peptides production is a normal physiological process and it is present in the extracellular brain fluid of healthy individuals. At low picomolar concentrations, Aβ peptides mediate various physiological mechanisms underlying learning and memory. In contrast, at high concentrations, such as those observed in AD, Aβ peptides exert a synaptotoxic effect [11]. The toxicity of the Aβ peptides in vitro, and seemingly in vivo, has been known for a long time, but the specific mechanism(s) for Aβ-induced cytotoxicity has not yet been completely elucidated. Since the majority of Aβ peptides is released in the extracellular milieu, it is reasonable to assume that toxicity begins outside the cells, when the toxic species of Aβ peptides are formed and interact with cell membranes [12], initiating events that disrupt basic cellular processes and ensuring cell death. One thing that is clear, and generally accepted now, is that the neurotoxic agent leading to cellular death, memory loss, and other AD characteristics is an intermediate aggregated state of the Aβ peptide [13]. Regrettably, intermediate structures of Aβ peptides, such as structured monomers and oligomers, have remained relatively unexplored, largely due to their transient nature and propensity to form insoluble aggregates.

A POTENTIAL MECHANISM FOR Aβ PEPTIDE TOXICITY

The mechanism by which oligomeric species of Aβ peptides can be neurotoxic remains elusive. Based on extensive investigations by our group, we have proposed a mechanism that involves the formation of ion channel-like pores [14, 15]. This potential mechanism has received wide acceptance in view of abundant and growing evidence demonstrating that Aβ peptides increase ion conductance in both artificial and natural membranes by forming conductive pores [16 –22]. These observations echo the long-standing assumption that the pathology of Aβ peptide neurotoxicity is associated with increasing levels of cytosolic Ca^2 + [7 , 24]. In fact, cells incubated with Aβ peptides showed the presence of the peptide on the cell surface and an increase in intracellular calcium [25, 26]. We proposed that aggregation states of Aβ peptides insert directly into the plasma membrane forming well-defined pores with specific ion selectively disturbing cell homeostasis. Channel-like annular structures of Aβ peptides oligomers have been observed by electron microscopy [27] and by atomic force microscopy [18, 28]. Theoretical models of these potential ion channel structures formed by Aβ peptides oligomers have been reported [29 –34]. We argue that preventing Aβ channel formation and other forms of aggregates that produce membrane disruption has the potential to reduce Aβ peptide-induced cytotoxicity and perhaps alleviate AD.

Extracellular heat shock proteins are systemic sensors for stress

Heat shock proteins (hsp) are a family of molecular chaperones some of which are involved in basic cellular processes, particularly protein folding. Other members of the hsp family participate in the repair of cellular damage after stress [35, 36]. The biological function of these chaperones occurs within the cytosol and other subcellular compartments. However, there is overwhelming evidence that hsps are also present in the extracellular environment. Pioneering studies by Hightower and Guidon (1989) [37] showed that Hsp70 was present in the extracellular medium of both healthy and mildly stressed cells, suggesting extracellular roles. Concomitantly, Tytell et al. [38] demonstrated that Hsp70 was transferred from glial cells to neurons under certain physiological conditions, suggesting a supplemental role for this protein in transcellular support of axonal functions. Hsp70 is indeed exported from cells by an active mechanism different from cell death [37, 39]. This protein is also found circulating in fluids [40, 41].

The fact that these proteins could also be found outside cells has opened a major discussion, particularly because these proteins lack the consensus secretory signal for their export via the classic ER-Golgi pathway, with the exception of ER-resident Grp78 and Grp94. Indeed, Hightower and Guidon [37] demonstrated that secretion of Hsp70 was not blocked by typical inhibitors of the ER-Golgi pathway, such as brefeldin A. This observation has been confirmed by others [39]. Many other proteins besides hsp, are secreted independently of the ER-Golgi pathway [42], which has been coined the non-classical secretory pathway [43]. The non-classical secretory pathway is not a unique mechanism, but rather a collection of alternative passageways, which have as a common denominator the exclusion from the ER/Golgi compartment. Several hsps have also been detected in extracellular vesicles or exosomes [42 , 45], particularly, we have shown that Hsp70 is present within the membrane of these vesicles [46]. The observation that Hsp70 is present within membranes is not surprising since we have previously reported their association with artificial membranes [46 –48]. Moreover, Hsp70 has been detected on the surface of tumor cells [49, 50]. Therefore, the possibility that hsps are exported in association with extracellular vesicles is a real possibility. However, hsps could also be released during lysis after cell death by necrosis [44, 51]. Extracellular hsps appear to act as signaling molecules [42 , 50]. In particular, Hsp70 has been reported to activate a variety of cells, including macrophages, monocytes, dendritic cells and natural killer cells [42, 44]. Extracellular Hsp have been associated with various disease conditions [42, 52]. Thus, extracellular hsps have been considered as part of a process named the “Stress Observation System (SOS),” which is the mechanism responsible for the sensing of stress conditions in the systemic environment [42]. Finally, there is extensive evidence that hsps protect cells from Aβ peptide toxicity [53 –59]. Hsps have been detected in several cells composing the nervous system [55]. Unfortunately, there are no reports of the presence of hsp within the extracellular space of the brain.

Fig.1

Aβ peptides oligomerization process in the absence or presence of Hsp70s. a) Without any interference, once released from neurons or glial cells, Aβ peptides initiate spontaneous oligomerization to form oligomers that will eventually end up building protofibrils, fibrils, and plaques. Depending on the size and configuration some oligomers could interact with the cell plasma membrane augmenting non-selective permeability and resulting in membrane disruption. Smaller oligomers could insert into the cell membrane forming ion-selective pathways or ion channels. The magnitude of these events could lead to cell dysfunction. b) When hsps are in the extracellular milieu, Aβ peptides could interact with Hsp70s mitigating the oligomerization process and reducing cell toxicity.

A novel mechanism for controlling circulating Aβ peptide toxicity

Classic information on the varying presence of Aβ peptides in brain fluids combined with recent findings on the properties of hsps have provided us with the basis to raise a novel view of how AD could be set and controlled in senior individuals. Considering that hsp and Aβ peptides are both present outside cells in vivo, opens the possibility that they could meet and interact in the extracellular milieu of the brain, providing an opportunity for hsp to control the aggregation process of Aβ peptides. Indeed, we recently reported that the formation of Aβ peptide intermediate states in vitro, particularly those that are presently considered to be cytotoxic, is restrained by the action of members of the Hsp-70 family: Hsp70 (HSPA1a), HSC70 (HSPA8), and Grp78 (BIP, HSPA5) [60]. These observations echo other studies on a Drosophila model of AD [61].

The hypothesis is that once Aβ peptide monomers are released from neurons or glial cells, Aβ peptides initiate a spontaneous oligomerization process to form oligomers which will eventually end up building protofibrils, fibrils, and plaques. Although all Aβ monomers will interact with the plasma membrane of cells with no significant harm, some oligomers could produce a non-selective lipid bilayer permeability resulting in membrane disruption. Thus, smaller oligomers could translocate within the membrane forming ion conductive pathways or ion channels that alter the physiological conditions of cells resulting in cell dysfunction and death. When Hsp70 is present in the extracellular milieu simultaneously with the released Aβ peptide monomers, their interaction reduces the capacity for Aβ peptide monomers to proceed with the oligomerization process, thus diminishing their ability to interact with cellular membranes and reducing toxicity (Fig. 1).

Our initial observations indicated that the interaction of Hsp70s with Aβ peptides is not stoichiometric and occurs in the absence of nucleotides [60], which is in diametrical contrast with the chaperone function of hsp. Indeed, intracellular Hsp70 interacts with a single polypeptide client at a time and in a process regulated by ATP and ADP, enhanced by the interaction with co-chaperones [36 , 63]. Thus, it is difficult to reconcile the chaperone function of hsps with our observations indicating that Hsp70 interacted with Aβ peptides at a 1:150 molar ratio between Hsp70 and Aβ peptides [60]. Another study showed a reduction in Aβ peptide fiber formation by substoichiometric levels of Hsp70 [64]. Small heat shock proteins have also been shown to interact with Aβ peptides affecting amyloid fibers formation and reducing cerebrovascular Aβ peptides toxicity [65 –67], which are working at a substoichiometric level as well [66]. Moreover, DNAJB6 was reported to affect the assembly of high molecular weight complexes of Aβ peptides in a nonstoichiometric fashion [68].

Fig.2

Dynamics of hsp and Aβ peptide levels during aging. The dotted area highlights the age of the individuals when the formation of toxic Aβ peptide oligomers is favored in view of simultaneous increases of Aβ and decreases of hsp in body fluids.

Based on our observations, we postulate that Hsp70 interacts with Aβ peptides (monomers to trimers) reducing their ability to form high-mass oligomers, particularly those that are cytotoxic. However, the nature of the interaction is unclear, but it may be trigger by a conformational change of Hsp70 mediated by an initial interaction with Aβ peptides. This new conformation stage of Hsp70 may interfere with an early event in the oligomerization process of Aβ peptides, perhaps by blocking the transition from a random coil structures to a β-sheet conformations [69 –71]. Other investigators have proposed that Aβ peptide oligomerization is initiated by a “seed” complex [72], which may be the target for an irreversible interaction with Hsp70 thus arresting further oligomerization. Therefore, the influence of Hsp70 on Aβ peptide oligomerization could be envisioned as a “domino” effect in which, by blocking the first step avoids the association of more peptide subunits into oligomers.

The dynamics of Hsp70 and Aβ peptides with aging

Aβ peptides are continuously produced and released by neurons and glial cells into the external medium of the brain. However, there is apparently no cytotoxicity of these peptides and their aggregates in healthy individuals. One possibility is that Aβ peptides could be cleared from the brain very rapidly and efficiently [73 –76]. However, another possibility is that Aβ peptides are not toxic because of the presence of Hsp70s restraining the formation of harmful intermediate aggregates. We propose that varying the balance between extracellular Hsp70s and Aβ peptides may increase the level of neurotoxicity contributing to the development of AD. If indeed Hsp70 is capable of regulating Aβ peptide oligomerization, the question that emerges is why the onset of AD occurs at a late stage of life. An answer to this question resides in the interpretation of independent studies of the fate of Hsp70 and Aβ peptides during aging.

The capacity of cells to respond to stress and synthesize hsps is reduced with age [77 –79], which is also reflected in changes in circulating levels of these proteins in body fluids [40, 41]. Indeed, Hsp70 levels in blood showed a linear decline from an average of 400 ng/ml in individuals of less than 40 years old, to about 20 ng/ml in subjects of more than 90 years old [40]. Although the specific mechanism for the decay of hsps expression with age is not clearly understood, it has been proposed to be associated with a decrease in the levels and activity of HSF1 that is the major transcriptional factor involved in the stress induction of these proteins [80]. Although, there is no direct evidence of decreased circulating levels of Hsp70 in AD, the levels of HSF-1 and Hsp70 within exosomes isolated from AD patients were lower than matched controls [81, 82]. In divergence, circulating levels of Aβ peptides increased with age [83], creating the scenario for the simultaneous reduction in hsp levels and the corresponding increase in Aβ peptides within the extracellular cerebral environment. The inference from these observations is that the possible anti-aggregation activity of extracellular Hsp70 is limited with aging, thus increasing the risk of accumulation of aggregation-prone Alzheimer’s Aβ peptides and the development of the disease (Fig. 2).

CONCLUSIONS

Herewith we place in context our previous investigations showing that the presence of Hsp70s mitigate the formation of low molecular weight Aβ peptide oligomers resulting in a reduction of cellular toxicity [60]. Since the production of Aβ peptides is relatively constant in healthy individuals, the appearance of Hsp70s in the extracellular brain fluid may control the aggregation of Aβ peptides reducing the chances that they will form toxic oligomers. However, the reduced expression of Hsp70s during aging and the relative increase in Aβ peptides production disrupts the balance between the two molecules and increases the probability of the appearance of potentially toxic oligomers, which will likely increase neuronal dysfunction, culminating in the development of a neurodegenerative disorder leading to dementia that characterizes AD.

The idea that we discussed here is appealing for serious consideration and seems plausible as endorsed by solid experimental results. However, our viewpoint does not ignore other proposed mechanisms for maintaining the normal levels of circulating Aβ peptides in a non-toxic conformation. For instance, the clearance of Aβ peptides from the cerebral spinal fluids into the circulating blood performed at the epithelial layer [73, 84] and microglia [85], is also likely to contribute to avoiding the incidence of AD. Consequently, much work remains to be done in order to provide a more molecular, cellular and physiological basis for the development of this devastating condition.

Footnotes

ACKNOWLEDGMENTS

We would like to thank Barbara Rho for editing the manuscript. This work was supported by grants from NIGMS (R01 GM11447) and the UC San Diego Academic Senate.

Authors’ disclosures available online ().

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