Does intracrine amplification provide a unifying principle for the progression of common neurodegenerative disorders?

Many extracellular signaling proteins, such as hormones and growth factors, can act within their cells of synthesis or after secretion and internalization by target cells. These factors are called intracrines; aspects of their biology have been defined. The actions of these factors are mirrored in the cell-to-cell transmission of prions and prion-like proteins in common neurodegenerative disorders such as Alzheimer’s disease. This suggests the possibility that intracrine-like functionality is associated with these pathogenic proteins and that principles of intracrine biology—and in particular the intracellular amplification of intracrines—can point to therapeutic strategies for the treatment of these diseases. Here, possible modes of amplification of prion-like particles in neurodegenerative disorders are explored.

The pathogenesis of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease, kuru, scrapie, and bovine spongiform encephalopathy (mad cow disease) was defined by Prusiner who demonstrated that misfolded prion proteins could traffic between cells and induce misfolding in normal prion proteins in target cells. As he predicted, other misfolded protein neurodegenerative disorders (NDDs)—which, unlike TSEs, are not naturally infectious—involve trafficking of abnormal proteins between cells with subsequent induction of misfolding and pathology in those cells. This likely occurs in Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), tauopathies, chronic traumatic encephalopathy (CTE), and other NDDs. These diseases result from the trafficking of disordered proteins, and, in their inherited forms, also from the cell-autonomous production of mutant proteins susceptible to misfolding (1).

In recent years, considerable evidence has been developed to show that many extracellular signaling proteins can act within their cells of synthesis or in target cells after secretion and internalization by target cells. These factors are called intracrines; aspects of their biology have been defined (2,3,4). Many intracrines generate intracellular feed-forward loops that result in target cells being placed in altered (differentiated) states. The evidence for this kind of action is strong and the intracrine homeodomain transcription factors provide a clear example. Intracrines can, as in angiogenesis, interact in the intracellular space to form intracellular regulatory networks. Intracrines traffic between cells following secretion and uptake by target cells, but also via trafficking in exosomes and possibly trafficking via nanotubes (2,3,4).

There are parallels between the actions of intracrines and proteins involved in neurodegenerative disease; both involve protein trafficking between cells and the establishment of an altered state in target cells with subsequent propagation. In the case of physiological intracrine action, this process generally results in altered hormonal responsiveness or differentiation. In the case of the misfolded neuropeptides, it results in toxicity. These neurodegenative disorders display a primitive intracrine mechanism: proteins traffic from cell to cell, cause disease, and then re-capitulate themselves to affect other cells. Mechanisms of cell trafficking employed by intracrines have been proposed to be the operative modes of trafficking for misfolded peptides as well. Exosomes are released at synapses and exosomal trafficking of misfolded proteins complements, or even substitutes for, the secretion of misfolded peptides and/or their release from extracellular plaques or dying cells. The trafficking of misfolded proteins in nanotubes has been suggested. So there are commonalities between intracrine action and the actions of misfolded neural proteins.

A frequent feature of intracrine networks which potentially informs the understanding of TSEs, and therefore potentially of other NDDs, is the notion that intracrine systems often are self-sustaining through the mechanism of feed-forward regulatory loops. This implies an active amplification step, which, if present in NDD, could provide a therapeutic target. Similarities between normal and abnormal intracrine and prion biology, including the potential physiologic intracrine functioning of normal forms of NDD-associated proteins, have been reviewed elsewhere (5). From this, comes the general notion that some normal homologues of pathological prions at times act as intracrines and employ feed-forward loops (amplification). Once prion transformation occurs, this intracrine functionality is coopted, or aberrant intracrine functionality is developed, to spread pathology in the nervous system.

It is proposed that the neurodegenerative diseases recently suggested to develop from the propagation of transmissible proteins between brain cells—diseases such as Creutzfeldt-Jakob disease and other transmissible spongiform encephalopathies, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (Lou Gehrig disease), and chronic traumatic encephalopathy—result from distorted intracrine physiology. That is, the same actions characteristic of intracrines (function within the cell, trafficking by one means or another to adjacent cells, internalization by target cells, up-regulation of intracrine protein in target cells, action within target cells) are observed in the actions of prion-like proteins in the neurodegenerative disorders mentioned above. The differences are that in the neurodegenerative diseases, the primary trafficking protein is a misfolded form of a normal cellular protein rather than a normal intracrine, and rather than producing a physiologic effect in target cells, the misfolded protein produces a pathological effect. Nonetheless, the basics of the processes are similar and this suggests that focusing on common intracrine functionality could lead to a new understanding of not only the nature of TSEs but also of other common neurodegenerative disorders. In particular, the hypothesis that these neuroencephalopathies are intracrine in nature implies that transmissible prion-like protein up-regulation (amplification) occurs in target cells.  If the amplification of intracrines in target cells is mirrored in the amplification of prion-like proteins in target cells as is hypothesized here, this would present a new therapeutic target. The notion that intracrine biology plays a role in these common neurodegenerative disorders has been suggested elsewhere in the case of the transmissible spongiform encephalopathies (TSEs) (5). The TSEs serve as a paradigm for intracrine-like action in neurodegeneration because they are transmissible not only between cells but between organisms. That is, they are infectious (e.g., mad cow disease). Here, the hypothesis is applied more widely to the other common neurodegenerative disorders, which have not been shown to be infectious, and the implication of the hypothesis that amplification is characteristic of these disorders is similarly widely applied to neurodegenerative disorders. Moreover, possible mechanisms of amplification of the relevant proteins in these disorders are proposed. To do this, the nature of TSEs is briefly reviewed and the illustrated principles then applied more widely. Potential intracrine pathways in selected neurodegenerative disorders are proposed.

Supporting Information
It is proposed that the up-regulation, by any of a variety of mechanisms, of the normal forms of the common NDD-associated proteins is essential for disease progression. In the case of the TSEs, one can note that synthesis of normal cellular prion protein PrPc is essential to the propagation of abnormal scrapie prion PrPsc (19). Suppression of PrPc synthesis at any point stops disease progression (8). PrPc, but not PrPsc, binds and transports copper (as well as iron and zinc) into cells. Copper up-regulates PrPc synthesis (6). PrPc is found in the nucleus and could serve to buffer copper there. Were a significant amount of the normal prion protein to be replaced by scrapie prion, copper buffering would be reduced and PrPc synthesis increased. Because it appears that newly synthesized PrPc is the preferred substrate for conversion to PrPsc by the abnormal scrapie prion, this would increase the cellular load of abnormal prion (1,5,6).

Toxicity, and trafficking of PrPsc via endosomes to nearby cells, could then result. Although increased synthesis of PrPc in scrapie infected neurons (as opposed to Peyer’s patch lymphocytes) has not been detected, no detailed exploration of this issue has been undertaken and interpretation of the available data is complicated by neuronal cell loss during disease (5). Also, it is possible that up-regulation of PrPc synthesis need only be required in the initial phase of infection so as to produce sufficient abnormal prion protein to permit ongoing production with a more normal rate of PrPc production.

Similarly, the progression of AD requires the production of amyloid precursor protein (APP), which is cleaved to form the toxic extracellular plaque-forming abeta protein, while in vitro neurotoxicity produced by abeta protein requires the presence of both PrPc and the microtubule-associated protein tau (which forms intracellular tangles that correlate with disease severity in AD) (5,6,7). Copper up-regulates the synthesis of both PrPc and APP suggesting that disordered copper handling could lead to amplification of APP synthesis and secondarily to increased abeta protein synthesis (5). Abeta1-40 can in turn up-regulate tau (7). The requirement for PrPc in abeta protein in vitro neurotoxicity could result from the protein’s role in providing an efficient mode of copper influx and trafficking in neurons, in addition to any role played by direct PrPc binding of abeta protein. APP and abeta protein bind copper, and their arrival via exosomes in target cells could disrupt copper regulation in those cells, enhancing disease propagation.

In amyotrophic lateral sclerosis, two misfolded proteins have been associated with disease: copper-zinc superoxide dismutase 1 (SOD1) and TDP43 (1,1013). For brevity, TDP43 will be discussed here. TDP43 is a multifunctional DNA and RNA binding protein that is involved in RNA splicing. Interestingly, it controls its own synthesis by binding its own mRNA. Decreased TDP43 protein leads to increased translation of its message and increased synthesis of the protein; that is, it leads to up-regulation. In ALS, TDP43 aggregates in the cytoplasm with a near absence of TDP43 in the nucleus. Toxicity likely results from the loss of TDP43-mediated regulation of cellular mRNAs as a result of TDP43 sequestration in cytoplasmic aggregates. Thus, if an abnormal TDP43 fragment is taken up by a target cell and seeds cytoplasmic aggregation of endogenous TDP43, then cellular TDP43 will be up-regulated and the disease will progress. It is possible that another form of amplification occurs in ALS. APP and cellular copper are increased in ALS. There is some indirect evidence to suggest modest TDP43 up-regulation by copper compounds (13). Tissue iron also is increased suggesting impaired APP ferroxidase activity as may occur in AD secondary to extracellular zinc release from plaques (12). If the increased cellular iron in ALS does in fact result from increased extracellular zinc (whether through ferroxidase inhibition or some other effect on APP iron export), this could be important. Extracellular zinc causes intracellular TDP43 aggregation, thereby down-regulating functional TDP43 and up-regulating TDP43 synthesis (11). This would be expected to enhance production of TD43 prion-like particles and disease spread. Because copper, zinc, and iron are transported into cells by PrPc, these data suggest participation of PrPc in the disease (5,6,9). However, whereas PrPc down-regulation by zinc (secondary to internalization of PrPc by zinc) may be beneficial in TSEs, as noted in the case of TDP43 aggregation, the effects of zinc appear to be more complex in other NDDs.

Several NDDs, while expressing one prominent misfolded protein, express variable levels of others, suggesting some commonality of regulation and/or intracrine-like regulatory networking. One protein commonly involved is tau, which also appears to be the primary protein in disorders such as progressive supranuclear palsy. Possible amplification mechanisms for tau, in addition to up-regulation by abeta1-40, are only beginning to emerge. The transcription factor STOX1A in some circumstances can up-regulate tau. STOX1A down-regulates CNTNAP2, a member of the neurexin family. In the hippocampus of AD patients, STOX1A expression is up-regulated, while CNTNAP2 is down-regulated (14,15,16). Thus, in the case of STOX1A, a mechanism which up-regulates tau may directly participate in neurodegeneration in AD through the down-regulation of CNTNAP2. Like tau, up-regulation by abeta1-40 and the common up-regulation of PrPc, and APP by copper, STOX1A may represent another case of interaction between the pathologies of the NDDs. Although tau tangles are not seen in ALS, they are prominent in the related disorder chronic traumatic encephalopathy (CTE) where abeta protein, TDP43, and alpha-synuclein inclusions can also be found in some cases (17). Trauma likely produces a wide-ranging disorder, possibly because cytoplasmic RNA/protein stress granules are formed after trauma to protect the brain, and these lead to the aggregation of the mRNAs of many disease-related proteins. Alterations in the amounts of these proteins could lead to disordered homeostasis, including disordered transitional metal homeostasis. Amplification of each factor could then occur. For example, TDP43 RNA is sequestered in stress granules and this should up-regulate TDP43 gene expression. Up-regulated TDP43 interacts with tau mRNA and, depending on the details of this interaction and the rate at which TDP43 is aggregated, the result could be up-regulated tau mRNA translation.

Similarly, the mRNA binding protein HuD is sequestered in stress granules and this relieves the protein’s suppression of tau synthesis. Intracrine-like regulatory networking could up-regulate other substrate proteins. Up-regulation of substrate proteins could then lead to the stochastic development of misfolded proteins which, in turn, serve as seeds for the formation of aggregates and disease propagation (18,19,20).

The hypothesis has several implications. First, evidence of substrate protein amplification should be sought in all NDDs—and confirmed in Parkinson’s disease where amplification has already been reported (21). Second, prevention of amplification is a disease-controlling strategy; complete knock-down of substrate protein is unnecessary. Third, intracrine-like networks linking the regulation of various misfolded proteins should be sought; their interruption would be therapeutically beneficial in disease modification. These networks, usually weak and indolent, can, as is likely in some cases of CTE and AD, be more robust. They are likely mediated by misfolded protein-driven regulation of transition metal transport. Fourth, PrPc likely plays an important role in the non-heritable forms of many of these disorders, not only in TSEs, by virtue of its ability to transport copper, zinc, and iron with secondary regulation of substrate proteins (this need not be the case in genetic forms of the disorders where amplification can be unnecessary). For example, reducing CNS copper concentrations by dietary or other means would be expected to decrease PrPc synthesis and disease progression in TSEs and possibly in other NDDs. Although an abeta protein preparation recently has been reported to cause disease somewhat resembling AD pathology in PrPc-/- knockout animals (while a five-fold over-expression of PrPc in transgenic animals was actually protective), and although PrPc levels have been reported to be reduced in sporadic (but not heritable) AD frontal cortex, PrPc expression is required for neurotoxicity in vitro and in at least one in vivo model suggesting a complex relationship between PrPc and AD (5,2224). Indeed, there is evidence that at least some of the likely pathological interaction between PrPc and abeta protein involves the direct binding of the two. But PrPc accounts for only about 50% of the cell binding of abeta protein and so it is likely that other cell proteins can at least partially substitute for it (22). Additional research will be required to define the role of PrPc in AD, but it must be noted that abeta protein is the major prion-like protein involved in AD, and PrPc likely plays only a permissive or supplemental role (5,22). Nonetheless, it is likely that PrPc knock-down in AD is beneficial. In ALS, PrPc likely facilitates pathogenic zinc entry into cells and PrPc is therefore a possible therapeutic target in that disorder as well as in TSEs (5,11). Several drugs such as glimepiride, all-trans-retinoic acid, astemizole, and tacrolimis have been shown to lower cell surface PrPc expression at one concentration or another; they could serve as prototypes for the development of common therapies for NDDs and in particular for TSEs, ALS, and CTE (2527). Glimepiride, approved for the treatment of diabetes mellitus, may be a particularly useful prototype not only in TSEs where it lowers cell surface PrPc, but also in AD, both because it lowers cell surface PrPc (and therefore disease-enhancing PrPc pathologic effects) and because it induces shedding of PrPc into the extracellular space where it can bind abeta protein—a mechanism that has been proposed to be protective in the PrPc over-expression transgenic model mentioned above (22,23,26). Zinc, because it internalizes with PrPc without up-regulating PrPc synthesis, could act beneficially in TSEs (but likely not in other NDDs) by internalizing cell-surface PrPc (5,9). Intrathecal administration of liposomes containing PrPc antisense or anti-PrPc siRNA also could be considered. PrPc knock-down is not likely to be harmful given the fact that transgenic PrPc-/- animals demonstrate minimal phenotype changes and knocking down PrPc in PrPsc infected mice produces definite therapeutic benefit (8,25). The thrust of all these observations is the suggestion that because of interactions between prion-like proteins, it is possible that certain interventions such as PrPc knock-down or metal chelation could prove beneficial in more than one disease. That is, just as physiological intracrines can form interacting networks, so too may pathological prion-like proteins. If so, this would present therapeutic opportunities. Finally, in addition to their established roles in the aggregation of misfolded proteins, it is argued here that transition metals play important roles in protein amplification. Interrupting that amplification likely also is a common therapeutic strategy in these neurodegenerative disorders.

It is hypothesized here that there are clear parallels between physiologic intracrine action and the pathological action of prion-like proteins. At the same time, there are clear differences between normal intracrine function and the action of prion-like proteins in neurodegenerative diseases. For example, intracrine action is generally physiological, whereas misfolded prion-like proteins are pathological; also the mechanisms of protein amplification in the neurodegenerative disorders differ from those involved in the normal physiologic up-regulation of the respective proteins.

Therefore, these neurodegenerative disorders properly should be considered intracrine-like to distinguish them from physiological intracrine systems. They utilize an aberrant intracrine physiology. This has a variety of implications including potential therapeutic implications. Currently, a great deal of effort is directed to devising methods for limiting the spread of prion-like proteins between cells. The hypothesis presented here suggests, among other things, that aberrant protein amplification should be considered a therapeutic target in all of these disorders. More importantly, although TSEs serve as the paradigm for intracrine involvement in neurodegenerative disorders, it is proposed here that common intracrine mechanisms are operative in all these neurodegenerative diseases.H

This work was funded by the Ochsner Clinic Foundation.

Author declares no conflicts of interest.

About the author

The author is a physician who conducts research on the cellular biology of vasoactive proteins. He currently serves as Scientific Director, Ochsner Clinic Foundation, and is on the faculty of Tulane University School of Medicine.


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