REVIEW ARTICLE

Could Intracrine Biology Play a Role in the Pathogenesis of Transmissable Spongiform Encephalopathies, Alzheimer’s Disease and Other Neurodegenerative Diseases? Richard N. Re, MD

Abstract: Transmissible spongiform encephalopathies have been shown to result from the misfolding of normal cellular prion proteins in neurons caused by a transmissible abnormal form of the protein. In recent years, similar transmission of abnormal proteins capable of inducing abnormal folding of their normal homologues has been reported in other neurological disorders including Alzheimer’s disease, Parkinson’s disease and the so-called tauopathies. Thus, a new paradigm—the notion that some neurodegenerative disorders are protein “foldopathies”—has gained wide support. In addition, over recent years, the notion that some intercellular signaling proteins/peptides are intracrines—that is, they can in some instances act within their cells of synthesis or within target cells— has also gained currency. Tenets of this intracrine physiology/action have been developed. Here, it is argued that the protein functionalities demonstrated by foldopathy-related proteins are similar to intracrine actions and that these disorders could be intracrine in nature. If correct, this proposal would have therapeutic implications. Key Indexing Terms: Transmissible encephalopathies; Alzheimer’s disease; Intracrine. [Am J Med Sci 2014;347(4):312–320.]

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he causative factor of the transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease and scrapie was for some time difficult to elucidate. In the 1960s, radiation biologist Alper and his colleague John Griffith proposed that the responsible infectious agent consisted solely of protein, a speculation driven by the resistance of the agent to ionizing radiation.1 Subsequently, Stanley Prusiner not only confirmed this hypothesis but also identified the agent responsible for Creutzfeldt-Jakob disease as a protein infectious particle or prion.2 In so doing, he not only established the cause of the transmissible encephalopathies but also introduced a new paradigm by which pathologyinducing misfolded proteins propagate without benefit of nucleic acid biology by traveling to normal cells and inducing misfolding in their normal protein homologues. This process propagates protein misfolding and disease in the nervous system. The power of the Prusiner’s paradigm has become even more apparent recently given emerging data that tau protein, alpha-synuclein, and superoxide dismutase 1—proteins involved in the pathogenesis of Alzheimer’s disease (AD), Parkinson’s disease (PD) or amyotropic lateral sclerosis (ALS), respectively—display at least some elements of prion-like activity.2–5 This, in turn, suggested that a common therapeutic approach might be effective in all of these diseases. Although not as accepted as the prion paradigm, the intracrine hypothesis deals with the biology and spread of

From the Research Division, Ochsner Clinic Foundation, Ochsner Health System, New Orleans, Louisiana. Submitted April 27, 2013; accepted in revised form June 14, 2013. The author has no financial or other conflicts of interest to disclose. Correspondence: Richard N. Re, MD, Research Division, Ochsner Clinic Foundation, Ochsner Health System, 1514 Jefferson Highway, New Orleans, LA 70121 (E-mail: [email protected]).

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peptide signaling molecules in normal development and disease. The tenets of this view have been described in detail elsewhere.6–12 Briefly, the intracrine view holds that many extracellular signaling molecules, be they hormones, cytokines, DNA-binding proteins or enzymes, can function in the intracellular space after either internalization by target cells or retention in their cells of synthesis. Five modes of intracrine action have been described, and these involve intercellular trafficking via secretion, endosomes or nanotubes as outlined in Figure 1.10 In some circumstances, most easily seen in the case of homeodomain transcription factors, intracrines establish finite gain feedforward transcriptional loops in cells, thereby producing an active form of differentiation or altered hormonal responsiveness. In the present context, one could then ask if intracrine biology plays a role in the propagation and pathogenicity of prions and other foldopathy-related proteins. The goal of this manuscript is to describe 1 or more mechanisms by which intracrine biology could play a role in TSEs and related disorders so as to stimulate investigation in this area. In addressing the issue of intracrine participation, one must recognize that the TSE literature, similar to that dealing with AD and “tauopathies,” is replete with apparently contradictory results, and trying to construct a schema for explaining the pathogenesis of these disorders requires some judicious culling of the available data. This review, therefore, will not attempt to be exhaustive but will rather focus on what appear to be the observations most likely to be relevant. Nor will this review analyze the pathological variation between inhered, infectious and sporatic forms of TSEs and other disorders. Certainly, it must be noted that specific mutations in the PRNP gene, which encodes the human prion protein, PrPc, can result in human TSE.13,14 This points to abnormalities of PrPc as the causative factor in these disorders. The fact that the overproduction of amyloid precursor protein (APP), the precursor of the pathogenic abeta protein (AB) in Down’s syndrome, leads to AD in middle age similarly points to a causative role for AB in AD.15 But, the differences in pathology produced by each inherited protein variant are more complex than can be dealt with here. Finally, analogous to the analysis of established intracrine systems, it will be necessary to address the mechanisms of amplification, intercellular trafficking and intracellular actions of the purported causative proteins. The mechanisms of cell death must also be defined. Given the observation that, in both TSEs and AD, cell dropout is the result of apoptosis and autophagy rather than necrosis, the possibility that aberrant intracellular intracrine action produce apoptosis and is the cause of the pathology will be considered.16,17

OBSERVATIONS ON TSEs AND RELATED DISORDERS Characteristics of PrPc At first glance, the idea that TSEs are intracrine disorders seems absurd because normal cellular prion protein, PrPc, is not known to be an extracellular signaling molecule. However,

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FIGURE 1. Intracrines can act in any of the 5 ways: in the cell that synthesized them (type I), after secretion and reuptake (type II), after secretion and uptake by target cells (type III), after trafficking in endosomes to a target cell (type IV) and trafficking via nanotube (type V).10

there is evidence suggesting that PrPc functions as a growth/ differentiation factor and that it can be secreted and taken up by cells; therefore, it likely is indeed an extracellular signaling molecule.18–20 In addition, many intracrines traffick to nucleus and, in doing so, regulate various aspects of cell function, but they also, in many instances, upregulate their own synthesis, that of their receptors and/or elements of their signaling pathways. PrPc, on the other hand, is widely viewed as a cell surface membrane protein. However, there is good evidence for the presence of PrPc, and of its pathological scrapies variant, PrPsc, in nuclei of 1 or another cell type—including evidence for presence of PrPc in association with chromatin in uninfected cells.21–23 The native peptide has been detected on the inner side of the nuclear envelop of uninfected pancreatic beta cells, and other reports suggest a wider intranuclear distribution. Others have reported PrPc fragments in nucleus.24 An additional issue related to the relevance of intracrine biology in TSEs is how prions traffick between cells to act in an intracrine mode. The infectivity of the TSEs after the oral ingestion of infected material clearly demonstrates that such trafficking occurs at least for pathological prions. Moreover, PrPc has been detected in extracellular fluid, cerebrospinal fluid and endosomes released into the extracellular space that can be taken up by distant cells.25,26 Prion fragments and PrPsc have been shown to be internalized by target cells. A PrPc fragment, PrPc (173–195) can be internalized by target cells and then traffick to nucleus where it may affect transcription.24 Although this fragment has not been detected in normal or diseased nervous tissue, intact PrPc could mimic its action, especially given that murine PrPc and some prion fragments bind DNA.24 Parenthetically, there is evidence that tau protein, which has recently be shown to act in a prionlike fashion in several disorders and plays an important role in TSE pathology, also trafficks between neurons as does AB.2–5,25,27,28 In summary, there is a significant base of data indicating that normal and diseased prions can traffick between cell interiors and access nucleus. That is, the basic skeleton of intracrine action is present in the prion paradigm and, in particular, in the biology of the normal prion PrPc. To be more specific, PrPc has been reported in nucleus and can traffick between cells, and the entire protein or a fragment can Ó 2013 Lippincott Williams & Wilkins

be taken up by target cells, traffick to nucleus and potentially act in a biologically relevant fashion; if the PrPc (173–195) fragment discussed earlier occurs naturally or if intact PcPc shares its nuclear functionality, all the requirements to classify PrPc as an intracrine would be met. To be sure, the intracrine action of PrPc at best seems to occur at a low level, suggesting it is not an important aspect of its biology. But, at the least, it could serve as the basis for the more aggressive intracrine action of its related pathological prions that is being investigated here. Prions and Disease This raises the more determinative issues of how prions propagate and how they produce pathology—and, more to the point, do intracrine mechanisms play a role in these processes. It is customarily believed that pathological propagation of prions involves the interaction of misfolded pathological prions with normal prions either on the cell surface (in which case, the newly formed PrPsc rapidly undergoes endocytosis) or in the endosomes after cell surface PrPc had been internalized.29,30 Misfolded proteins are not returned to the cell surface. These proteins are resistant to enzymatic degradation by virtue of their newly acquired conformation; they form various filaments within the cell and form insoluble extracellular aggregates. In time, these aggregates accumulate to the point that they break down, thereby spreading pathological prions to nearby cells. Pathologic condition is felt to somehow be triggered by the presence of 1 species or another of intracellular filaments/ tangles, possibly a result of their disruption of cellular machinery.24 Several observations regarding this schema can be made. First, the propagation of prions according to this scenario is very slow, being dependent on the slow accumulation of misfolded normal proteins cell by cells. The long latent period of the TSEs is consistent with a slow reproduction rate, but more rapidly progressive infection has been reported.5,13,30 How does this occur? In addition, it is not clear how aggregated extracellular proteins or intracellular fibrils cause cell death; so, other mechanisms have been sought including diminution of the antioxidant properties of normal prion protein and impaired proteosomal function leading to the build up of proapoptotic proteins, among others.31–33

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Confronted with these facts, one could be tempted to seek out additional mechanisms by which prions could upregulate their own production—a hallmark of many intracrine systems. To be sure, current thinking about these disorders is consistent with intracrine principles in that a feedforward mechanism exists in the case of pathological prions. According to the standard hypothesis, prions do function in cells, can exit cells, traffick to target cells and then they, or fragments derived from them, can be taken up by and act in those target cells to produce disease and to amplify the original prion signal in a feedforward fashion. Arguably, this sequence of events constitutes a primitive intracrine system. But, this schema leaves several issues unaddressed. First, can chemical “seeding” produce the requisite prion amplification for disease propagation or is another mechanism required, one possibly involving genomic regulation. The observation that PrPsc cannot be transmitted to PrPc2/2 animals (which are relatively, but not completely, free of baseline pathology) clearly implicates PrPc synthesis in the transmission and production of disease. But does the mechanism simply involve misfolding of previously synthesized normal PrPc produced in normal amounts? The difficulty of producing significant amounts of pathological prions in vitro absent multiple rounds of sonication and reseeding suggests this is not the case. Moreover, it has been difficult to develop infectious prions in transgenic animals expressing 1 prion mutant or another; these models often produce agents capable of causing disease in transgenic animals overproducing abnormal or normal prions but not in normal animals. A transgenic animal overexpressing normal vole prion has produced infectivity that is active even in normal animals, but vole prion is very sensitive to the production of infectious particles after exposure to any of a variety of prions.2,29,30,34,35 Collectively, these observations suggest that the amplification and effective propagation of pathological prions requires the upregulation of normal prions to serve a substrate or requires the presence of a particularly alterable and, therefore, infective native (vole) prion. A more traditional mode of intracrine feedforward action could therefore be operative. Second, although the pathological action of aggregates is plausible, the intracrine view would suggest investigating normal prion functions, which, if distorted, produce neuropathology. Third, one could ask if normal PrPc acts in an intracrine mode, what intracrine functions does it serve and what, if any, amplification or feedforward loops does it participate in, given that seeding is not germane in the case of PrPc? In addressing the issue of prion amplification, it can be noted that PrPc is upregulated by copper, as is APP from which AB is generated in AD.36,37 PrPc binds copper at its N terminus and has long been thought to be a functionally important protein in the regulation of intracellular copper, either in the whole cell or in selected compartments.38 Of note, PrPsc does not bind copper.39 If PrPc is involved not just in copper transport but in buffering copper in a sensitive cellular compartment such as nucleus, its partial replacement in that compartment by PrPsc could lead to locally elevated copper concentrations and enhanced PrPc synthesis given that copper upregulates PRNP gene transcription.37,40 This increase in PrPc then would serve as a ready substrate for conversion into PrPsc, not only as a result of seeding but also to a lesser extent by spontaneous conversion. Indeed, copper facilitates such conversion. The importance of PrPc upregulation in the pathogenic process is illustrated by the fact that animal models in which PrPc is upregulated are more susceptible to disease following brain inoculation with TSE brain extract.2 At first blush, this notion of PrPc upregulation in TSEs seems wrong on its face—PrPc messenger RNA (mRNA) steady state levels do not appear to be appreciably altered in TSE brains

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nor in chronically prion-infected cultured cells.41,42 It is true that PrPc is upregulated in scrapie-infected Peyer’s patch lymph cells compared with noninfected cells, and in this model, higher PrPc synthesis in host animals was associated with increased susceptibility to infection.43 But, overall, there is no clear evidence in favor of continuous PrPc gene upregulation in TSE. PrPc synthesis is necessary for infection to occur, increased PrPc expression greatly facilitates infection, but steady state PrPc mRNA levels are not usually increased above normal. Can this be reconciled with an intracrine feedforward mechanism? The answer is yes if upregulation of PrPc is essential for successful initial infection by extracellular prions. Initial PrPsc molecules could enter a normal cell but do no harm because of their low numbers. If some migrated to nucleus, they could alter copper concentration leading to an upregulation of PrPc. This could provide a better substrate for protein conformational change by oncoming extracellular PrPsc molecules, which then convert newly synthesized PrPc to the PrPsc configuration likely at the cell membrane or in a contiguous compartment.2,30,34,44 If newly synthesized PrPc is the preferred substrate for conformational conversion by extracellular PrPsc, then once a critical mass of PrPsc is produced and internalized in endosomes, it could begin conversion of existing PrPc, as it cycles through the endosomal compartment. This, in turn, would decrease PrPc on cell surface, reduce copper influx and return PrPc transcription to normal even though PrPsc remained in nucleus—similarly, PrPc represses its own transcription, and this could also tend to reduce expression after an initial synthetic burst.45 At that point, sufficient PrPsc would have been produced to permit ongoing conversion of PrPc synthesized at baseline levels of PrPc expression to support PrPsc expansion; sufficient PrPsc would then be present to permit the endosomal conversion of PrPc after the latter is internalized. Under other circumstances, high levels of PrPsc in nucleus combined with less depletion of surface PrPc could lead to persistent PrPc upregulation. In this schema, the infectivity of native or synthetic prion involves (1) adoption of a conformation capable of inducing conformational change in native PrPc, (2) loss of copper binding capacity and (3) a conformation that facilitates trafficking to nucleus. If correct, this schema explains why synthetic prions (functionally deficient as regard upregulating PrPc) must be propagated in PrPc overexpressing cells (to give the extracellular prions the opportunity to generate daughter prions with the 3 required characteristics). It is also possible that after passage in an overexpressing host, daughter prions are transferred to host animals with sufficient tissue-derived copper to stimulate an initial burst of PrPc synthesis. Although high extracellular copper concentrations seem to result in the internalization of cell surface PrPc, a copper transport protein, and thereby protect the cell against PrPc-facilitated PrPsc internalization, or against PrPc conversion by PrPsc at the cell surface, somewhat lower but still elevated, copper concentrations could simply increase nuclear copper and PrPc synthesis and, therefore, facilitate infection.24,34,37,46 Similarity, sonication techniques could produce prions possessing all the 3 required characteristics stochastically given the large number of substrated molecules and the physical chemical stresses involved in these procedures (Figure 2).2 Tau is a microtubule-associated protein, which also binds copper. A second feature of TSEs and AD is the occurrence of tau filaments within affected cells. These structures contain hyperphosphorylated tau and in some neurodegenerative disorders tau itself trafficks between cells and induces the formation of abnormal tau in nearby cells after internalization—that is, tau can in some circumstances act in a prion-like fashion. In some disorders, tau appears to produce toxicity directly. Similar to PrPc, tau is essential for producing AB toxicity in neurons. AB Volume 347, Number 4, April 2014

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increases phosphorylated tau.47–60 Of note, PrPsc also appears to upregulate tau phosphorylation and result in the accumulation of intracellular tau and tau filaments.4 However, PrPsc can produce pathology in cultured tau2/2 cells.55 Collectively, these observations suggest a synergistic pathogenic relationship between PrPsc and tau. Finally, disordered autophagy appears to play a complex and poorly defined role in intracellular tau accumulation.61 PrPc Participates in Intracrine Trafficking Mechanisms and Feedforward Loops As for trafficking, PrPc can be secreted and apparently can be internalized by target cells as well. In addition, beta-amyloid and tau are released by cells into the extracellular space and taken up by cells, including neurons.62–68 For example, it recently has been shown that PrPc, in addition to AB, tau, beta-amyloid and synuclein (which forms inclusion bodies in PD), all traffick in exosomes.28,56–60,69–73 These are small membrane bound vesicles that are released by cells and can then fuse with target cells releasing their cargo into cytoplasm or various intracellular organelles (type IV intracrine trafficking). Because exosome release can be triggered at synapses by synaptic activity, exosome trafficking can lead to sequential infection in neurons involved in a given neural network; this could explain the pattern of infection in AD and other protein folding disorders. In addition, nanotubes—small cytoplasmic tubes linking adjacent cells and serving as a trafficking pathway for a variety of intracellular proteins and organelles (type V intracrine action)—are found linking brain neurons and likely serve as an additional path for infection spread.5,10,28,59,60,73 Collectively, these findings suggest that the simple inhibition of extracellular trafficking of free prions or prion-like proteins may not be adequate to halt progressive neural decline. The precise mechanism by which apoptosis is induced in TSEs or AD is unclear. Suggested mechanisms include excitotoxicity, damage from impaired buffering of reactive oxygen species and deranged axon transport as the result of intracellular tau fibrillatory tangles.4,16–18,20,31,65 As noted, the fact that both PrPc and tau are required for toxicity in AD (but not in related disorders) suggests a synergism between these factors in inducing apoptosis in AD. Given the likelihood that distinct, albeit related mechanisms are at work in TSEs, AD and other disorders, the focus here will be on TSE as an exemplar with allusions made to other disorders. It has been reported that PrPsc induces apoptosis in cultured cells.74 Moreover, there are also data to suggest that, in some circumstances, PrPc overexpression can upregulate p53 and induce or facilitate apoptosis.74–76 Parenthetically, there are data, albeit controversial, to indicate that amyloid intracellular domains, the fragments left in the cell after the cleavage and release of AB, can directly upregulated p53 by a direct transcriptional action at nucleus.77,78 Because p53 indirectly upregulates PrPc through an interaction with mdm2, the potential for a positive P53/PrPc feedforward loop in TSE and AD exists.79 There also is a report80 describing the ataxia-telangiectasia-mutated facilitation of the binding of p53 to the PRNP promoter in response to copper-induced oxidative stress; this in turn leads to upregulation of PrPc which, in the model reported, is associated with decreased oxidative stress and decreased cell mortality. However, it is possible that the described p53/PrPc feedforward loop could, in circumstances that permitted it to become sufficiently robust, lead to apoptosis. If correct, this mechanism would link disordered copper handling secondary to the presence of PrPsc (which does not bind copper) and a feedforward loop that produces toxicity. The loss of cells secondary to apoptotic death would mask PrPc upregulation consistent with near normal steady state PrPc mRNA levels in scrapie-infected cells and animals.41,42 Tau and PrPc are required for AB excitotoxicity, and although Ó 2013 Lippincott Williams & Wilkins

PrPc could lead to apoptosis, the role of tau is unclear.4 In 1 model of tauopathy, enhancing autophagy reduces cell toxicity, suggesting tau fibrils are toxic and lowering the intracellular concentration of these fibrils is beneficial (Figure 2).81

AN ILLUSTRATIVE PATHOPHYSIOLOGIC SCHEMA: NORMAL AND PATHOLOGIC FUNCTION OF DISEASE-MEDIATING PROTEINS PrPc expression appears during the development of the fetal brain, and there is evidence indicating that apoptosis plays an important role in normal brain development.82–84 It is possible that a normal function of PrPc during development is to sensitize appropriate neurons for apoptosis with upregulation of PrPc in 1 cell leading to trafficking by 1 or another intracrine trafficking mechanism to nearby cells. Excitation-stimulated exosome release at synapses seems a likely mechanism, but trafficking after secretion and by nanotubes could also occur. In target cells, the PrPc could upregulate p53 and secondarily increase PrPc leading to a PrPc/p53 feedforward loop that results in apoptosis. Presumably, with time, the upregulation of PrPc is countered by other mechanisms and apoptosis ceases. The preponderance of evidence indicates that PrPc2/2 mice are not normal but suffer only relatively mild neurological defects, suggesting a subtle or modulating role for the protein during development and afterward. PrPc functionality could then lay dormant until later in life when the system is pathologically reactivated by either an external insult (such as the introduction of PrPsc) or some form of cell autonomous dysfunction (such as the accumulation of mutated PrPc). TSE then results because abnormal PrPsc alters the copper concentration in nucleus leading to upregulation of PrPc synthesis, which serves as a substrate for misfolding on the PrPsc scaffold at or near the cell surface. Note also that this proposed mechanism would explain the fact that prion protein-induced pathology does not require protein aggregation as would be supposed if the toxic effect were simply related to the accumulation of an abnormal protein mass.24 Finally, in addition to this proposed intracrine-like functionality, PrPc plays a role in additional functions by acting as a growth regulator, copper transport protein and protector against oxidative stress.18 A similar argument can be made in the case of AB. It is known that the protein is released at synapses on synaptic excitation and plays a role in memory formation. APP, its precursor, is upregulated during development in at least some neurons.84–87 AB trafficks to nearby cells through 1 intracrine pathway or another including via secretion, nanotubes and synaptic exosome release.57,59,60,87 In target cells, AB, a copper-binding protein, could upregulate PrPc by altering nuclear copper concentration and secondarily, therefore, upregulate p53, which then establishes a feedforward loop with PrPc and upregulate tau phosphorylation.49,74–79,88 Indeed, in support of this scenario, there is evidence that p53 is upregulated in AD and that it secondarily upregulates tau phosphorylation.89 In addition APP, which similar to AB trafficks between cells in exosomes, not only binds copper but also, after cleavage, produces amyloid intracellular domains, which appears to directly upregulate p53 transcription and PrPc.25,58–60,77,78,90–92 Copper stimulates APP just as it does PrPc although it may decrease abeta secretion.36,49,93–95 Thus, the same intracellular conditions produced by abeta and APP could upregulated both APP and PrPc in tandem. This process could play a role in normal developmental neuronal apoptosis only to become harmful later in life secondary to environmental stress or cell autonomous damage.82,83 In this regard, it is interesting to note that lead exposure during early development leads to

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FIGURE 2. (A) PrPc on cell surface interacts with PrPsc at the cell surface or in a near-surface compartment and is converted to PrPsc, which does not return to the cell surface but can be released into cytoplasm and intracellular compartments, including nucleus where it could regulate PrPc expression. Cytoplasmic PrPsc can form filaments and aggregates. When cells die, aggregates are released into the extracellular space and these can shed PrPsc to infect other cells. PrPsc trafficking in exosomes also occurs. (B) In the absence of surface PrPc, less PrPsc may be internalized. More importantly, in the absence of PrPc at the cell surface site of protein conformational conversion, no PrPsc amplification takes place. Therefore, no pathology is produced.30,34,46

a transient increase in APP gene expression, which could build to establish an abnormally robust feedforward cycle leading to AD later in life, just as inherited overexpression of APP in Down’s syndrome leads to premature AD.96 This formulation suggests that PrPc is an important component of AD pathological conditions and is supported by multiple observations including that AB is not, or more likely is less, excitotoxic or neurotoxic toxic in a PrPc2/2 genetic background.51 A recent report, however, suggests that overexpression (5-fold) of PrPc can protect experimental animals from AB toxicity and that abeta toxicity can occur in PrPc2/2 animals.97 These findings suggest a dose effect for the AB/PrPc interaction with relatively low levels of PrPc overexpression facilitating cell death as described above, whereas higher levels are protective possibly by binding up abeta oligomers or through other mechanisms; the discordant findings regarding pathologic conditions in PrPc2/2 animals and cells suggests that, in some cases/models, other effector proteins could substitute for PrPc or that direct APP upregulation of p53 occurs in some models as described earlier. The intracrine view is what is being proposed here even if the specific role of PrPc upregulation in AD or TSEs is held in abeyance. Nonetheless, the preponderance of evidence supports an active role of PrPc in the genesis of AD pathologic findings consistent with the schema proposed here. Finally, this mechanism is consistent with evidence indicating that AB oligomers, not amyloid plaques, are associated with disease.51–54 In the case of tau protein, there is even less information available on which to base speculation regarding function. Tau, similar to PrPc and AB, binds both copper and zinc. It is present in nucleus where it could alter the concentrations of these metals. In some cases, it trafficks between cells via exosomes.36,48,53–58,98,99 Therefore, it presumably could act in a fashion similar to the one we have proposed for PrPc/PrPsc. In addition, if it were to lower nuclear zinc concentration, it could downregulate p73, upregulate p53 and thereby increase tau phosphorylation.97,100 p53 upregulation could lead to apoptosis as described for PrPc. In addition, the unfolded protein response to phosphorylated tau could lead to cell death.50 The similarities between many neurodegenerative disorders leads to the proposal that similar intracrine mechanisms are operative in TSEs, AD, ALS, Huntington’s disease, chronic traumatic encephalopathy (CTE) and tauopathies.3–5,14 This then raises the question of why the clinical presentation of these disorders is so different. The answer to this question is unclear but may involve the nature of the initiating insult and the relevant protein it upregulates, the cell type in which the initiating insult occurs/arises, and the protein background of the host neurons.

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Even if the schema proposed here is substantially correct, it likely is not complete. For example, the proposed role of p53 in producing apoptosis has been emphasized, but it must also be noted that excitotoxicity, and cell calcium overload secondary to, for example, AB-produced ion channels, among other mechanisms, could be operative.101,102 Depending on the specific diseaserelated protein involved, these effects could affect the clinical and pathological presentation.

THERAPEUTIC IMPLICATIONS

Based on findings to date, a variety of therapeutic approaches to these disorders have been developed. Antibodies designed to bind and clear extracellular abeta plaques in AD and the possibility of inhibiting tau phosphorylation with drugs such as lithium, for example.4,32,103 This therapeutic literature is too expansive to review here, and the approaches to treatment almost certainly will vary disease to disease. However, if intracrine functionality is operative in these disorders, then several therapeutic implications are suggested. First, to the extent that the trafficking of pathological intracrines occurs via secretion or passive release from cells, monoclonal antibodies could be effective in reducing disease spread. Indeed, this may be the dominant mode of trafficking of synuclein in PD. But, to the extent that trafficking occurs via exosome or nanotubes, they will not be. Because there are no available means of blocking this latter type of trafficking, the intracrine hypothesis has little to offer regarding inhibition of trafficking. However, the hypothesis also posits that active amplification is required for propagation. If correct, this notion may provide an opportunity for therapeusis. For example, if copper concentration in the nucleus is the driver of PrPc upregulation and of APP upregulation, then lowering copper concentrations at that site could reduce pathogen amplification and, therefore, disease spread. There are 2 implications to this idea. First, more research should be done on the regulation of copper and heavy metal concentrations at intracellular sites, and in addition, the mechanisms of upregulation of the normal analogs of pathological proteins should be investigated. Both these lines of attack could provider fruitful leads. It is proposed here that currently available information suggests that lowering total body copper could prove beneficial—not because of any toxic effects of copper but as a means of reducing amplification of the substrate for the initial production of altered prion proteins in target cells. This is counterintuitive because whole brain homogenates of scrapie-infected mice contain significantly less copper (and zinc) than are found in normal animals.31 But, cellular copper levels could very well vary Volume 347, Number 4, April 2014

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between (1) uninfected cells, (2) those in the early stages of infection and (3) those in an advanced state of infection—in the latter, decreased PrPc secondary to replacement by PrPsc could result in a global brain reduction in copper and an increase in oxidative stress and apoptosis secondary to lower superoxide dismutase activity resulting from lower levels of PrPc-bound copper.31 In cells in the earliest stage of infection, copper levels could be normal. In that case, copper reduction could help prevent the spread of infection, on the hypothesis that normal copper levels are necessary for the establishment of infection in normal cells. Conversely, there are also data suggesting that raising extracellular copper concentrations to supraphysiological levels can retard the spread of infection in part by downregulating surface PrPc and, thereby, reducing either PrPsc uptake by cells or PrPc conversion to PrPsc at the cell surface.30,34,46 If this is true, it could well be that copper plays a dual role in prion disease: upregulating PrPc transcription while downregulating cell surface PrPc by increasing its endocytosis. Because cell surface PrPc efficiently internalizes zinc, but zinc does not stimulate PrPc transcription, it may be that lowering ambient copper concentrations while raising zinc concentrations could limit the spread of TSE prion disease—although this likely is not the case in AD where AB, not PrPsc appears to be the major trafficking intracrine/copper-binding protein and where zinc may increase toxicity and there is evidence for both benefit and harm from increasing copper.99,104,105 Recently, it has been reported that glimepiride, a sulfonylurea approved for the treatment of diabetes, decreases cell surface PrPc, PrPc conversion to PrPsc and toxicity produced by prion fragment PrP (82–146). This suggests investigating the use of this agent in all stages of TSE and likely in AD as well.106 Although similar mechanisms seem to operate in TSEs, AD, PD, Huntington’s disease and other neurodegenerative disorders, the precise interplay of regulatory factors including metals such as copper differs among them.3–5 In TSE, a dual strategy could be considered. Increasing zinc levels and decreasing copper in early disease could be beneficial. Increasing both zinc and copper in late disease in the expectation that raising intracellular copper in severely infected cells could reduce oxidative stress and apoptosis might also be an effective. Glimepiride or similarly acting drugs could be beneficial in all disease stages. It may be that combination therapy with antibodies to reduce pathogenic extracellular protein trafficking, agents designed to reduce active amplification and inhibitors of specific intracellular reactions such as the inhibition of tau phosphorylation with lithium could prove more effective than any single therapy. Although for illustrative purposes the focus here has been on p53-mediated pathology, it is equally possible that other active mechanisms such as the unfolded response leading to glycogen synthase kinase-3 produced cell dysfunction and death are operative in these disorders. In that case, inhibitors directed at these mechanisms would also be beneficial.50 Similarly, various prions and prion fragments localize to nucleus and bind to DNA, whereas PrPsc has been associated with euchromatin.24 In addition, PrPc fragment PrPc (106–126), a moiety that has not been reported in normal or diseased brain, nonetheless, shares many of the physicochemical properties of PrPsc and is neurotoxic in vitro and in vivo; it is interesting that, although it has not been reported in nucleus, it does upregulate PrPc while producing apoptosis.107 Collectively, these observations raise the possibility that the pathogenic action of PrPsc could be the result of the inappropriate upregulation of specific genes. In that case, preventing this upregulation and/or blocking the downstream effects of the upregulated genes could be viewed as therapeutic targets. It also has been suggested that PrPc is a signaling cell surface protein and that PrPsc enhances that signaling leading to oxidative stress and Ó 2013 Lippincott Williams & Wilkins

apoptosis. In this view, PrPsc is not toxic per se but rather acts by amplifying PrPc signaling. In that case, decreasing cell surface PrPc by increasing extracellular zinc or by the use of glimepiride would still be beneficial. Irrespective of the precise mechanisms involved, the possible utility of PrPc depletion in TSE is borne out by several recent reports. Studies of scrapie-infected transgenic animals designed to become depleted of neuronal PrPc during the course of the infection clearly demonstrated that PrPc depletion during infection produces dramatic therapeutic benefit.106–108 Moreover, recent findings indicate that high concentrations of tacrolimus reduce cell surface PrPc on neuroblastoma cells by a nontranscriptional mechanism and block prion replication in the cells. Similarly, the allergy drug astemizole blocks prion replication in the cells. At high dose, the drug reduces cell surface PrPc, but it, nonetheless, inhibits prion replication at a low dose, one sufficient to only reduce cell surface PrPc by about 20%. Astemizole increases autophagy, and this apparently leads to clearing newly formed PrPsc; the drug also increases the lifespan of prion-infected mice, suggesting that regulation of autophagy may prove to be a therapeutic modality.109 Finally, given the importance of PrPc and tau in the pathogenesis of AD and the fact that PrPc serves as a cell surface receptor for AB and mediates some of its toxic effects, drugs that downregulate PrPc such as glimepiride, tacrolimus analogs, astemizole and/or drugs that block tau phosphorylation such as lithium seem worthy of investigation as prototype therapeutic agents for AD irrespective of the effects of copper and zinc in that disorder.

CONCLUSIONS Prions and the other proteins involved in the degenerative disorders discussed here are certainly not classical intracrines. However, they do exhibit some features of intracrine action, and to the extent these similarities with intracrines exist, they may point to a better understanding of the function and pathological actions of these proteins. Here, we have proposed scenarios by which intracrine action could play a role in these neurological disorders. Although, as already noted, other intracrine mechanisms could be operative, several have been proposed here as being illustrative. The absence of evidence supporting prion upregulation during infection is at odds with this proposal but only limited data exist related to this point. In fact, the definitive detection of PrPc upregulation during infection would serve as strong support for the proposed schema.107 But, in any case, the effort here is to expand the pathological paradigm associated with these disorders, irrespective of the correctness of the specifics suggested. In addition, although PrPc and AB have been the center of these speculations, this is likely because more work has been done on TSE and AD than on other disorders. It is clear there are many similarities in the functioning of these proteins and the proteins involved in other foldopathies—copper binding, exosome trafficking and cross regulation being a few of these. This in turn suggests that the basic principles of intracrine functioning in TSE and AD, if they are confirmed and better defined, will be applicable to PD, ALS, chronic traumatic encephalopathy and other related disorders. The view presented here differs from the standard formulation of these disease processes in several ways. First, the trafficking of the relevant proteins between cells is viewed as being active and to involve secretion, release of exosomes and trafficking through nanotubes instead of being solely the passive result of release on cell death or the accumulation of extracellular aggregates. Second, amplification of the relevant proteins is proposed to be an active process likely driven by the effects of

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altered copper and zinc concentrations in selected intracellular compartments and subsequent transcriptional regulation, instead of being solely the passive alteration of preexisting normal proteins; alteration of protein configuration occurs, but it occurs on the background of enhanced protein synthesis. Third, the intracellular actions of the relevant proteins are proposed to have limited but definite physiological function, which go awry late in life, possibly as the result of environmental exposure. This schema implies that the toxicity of these proteins is not solely the result of the cellular burden of abnormal proteins but rather is an active process involving mediators such as p53, a process that is a pathological reawakening of a normal function. In all these ways, the hypothesis proposed here, if correct, points to the possible intracrine nature of the relevant proteins and suggests novel approaches to limiting or halting disease progression. REFERENCES 1. Alper T, Cramp W, Haig DA, et al. Does the agent of scrapie replicate without nucleic acid? Nature 1967;214:764–6. 2. Colby DW, Prusiner SB. Prions. Cold Spring Harb Perspect Biol 2011;3:a006833. 3. Olanow CW, Prusiner SB. Is Parkinson’s disease a prion disorder? Proc Natl Acad Sci U S A 2009;106:12571–2. 4. Reiniger L, Lukic A, Linehan J, et al. Tau, prions and Ab: the triad of neurodegeneration. Acta Neuropathol 2011;121:5–20. 5. Goedert M, Clavaguera F, Tolnay M. The propagation of prion-like protein inclusions in neurodegenerative diseases. Trends Neurosci 2010;33:317–25. 6. Re RN. The origins of intracrine hormone action. Am J Med Sci 2002; 323:43–8. 7. Re RN. The intracrine hypothesis and intracellular peptide hormone action. Bioessays 2003;25:401–9. 8. Re RN, Cook JL. The basis of an intracrine pharmacology. J Clin Pharmacol 2008;48:344–50. 9. Re RN, Cook JL. The physiological basis of intracrine stem cell regulation. Am J Physiol Heart Circ Physiol 2008;295:H447–53. 10. Re RN, Cook JL. Senescence, apoptosis, and stem cell biology: the rationale for an expanded view of intracrine action. Am J Physiol Heart Circ Physiol 2009;297:H893–901. 11. Re RN, Cook JL. The mitochondrial component of intracrine action. Am J Physiol Heart Circ Physiol 2010;299:H577–83. 12. Re RN, Cook JL. Noncanonical intracrine action. J Am Soc Hypertens 2011;5:435–48. 13. Jansen C, Parchi P, Capellari S, et al. Human prion diseases in the Netherlands (1998-2009): clinical, genetic and molecular aspects. PLoS One 2012;7:e36333. 14. Shi Q, Chen C, Gao C, et al. Clinical and familial characteristics of ten Chinese patients with fatal family insomnia. Biomed Environ Sci 2012;25:471–5. 15. Ness S, Rafii M, Aisen P, et al. Down’s syndrome and Alzheimer’s disease: towards secondary prevention. Nat Rev Drug Discov 2012;11: 655–6. 16. Liberski PP, Gajdusek DC, Brown P. How do neurons degenerate in prion diseases or transmissible spongiform encephalopathies (TSEs): neuronal autophagy revisited. Acta Neurobiol Exp (Wars) 2002;62:141–7. 17. Liberski PP, Sikorska B, Bratosiewicz-Wasik J, et al. Neuronal cell death in transmissible spongiform encephalopathies (prion diseases) revisited: from apoptosis to autophagy. Int J Biochem Cell Biol 2004;36:2473–90.

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