A New Interpretative Paradigm for Conformational ...

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A New Interpretative Paradigm for Conformational Protein Diseases Luigi Francesco Agnati1,*,$, Diego Guidolin2,*,$, Amina S. Woods3, Francisco Ciruela4, Chiara Carone5, Annamaria Vallelunga1, Dasiel Oscar Borroto Escuela6, Susanna Genedani5 and Kjell Fuxe6 1

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; IRCCS San Camillo, Lido Venezia, Italy; Department of Molecular Medicine, University of Padova, Padova, Italy; 3NIDA IRP Structural Biology Unit Cellular Neurobiology; 4Departament de Patologia i Terapèutica Experimental, Universitat de Barcelona, Barcelona, Spain; 5 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy; 6Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 2

Abstract: Conformational Protein Diseases (CPDs) comprise over forty clinically and pathologically diverse disorders in which specific altered proteins accumulate in cells or tissues of the body. The most studied are Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, inclusion body myopathy, and the systemic amyloidoses. They are characterised by three dimensional conformational alterations, which are often rich in structure. Proteins in this non-native conformation are highly stable, resistant to degradation, and have an enhanced tendency to aggregate with like protein molecules. The misfolded proteins can impart their anomalous properties to soluble, monomeric proteins with the same amino acid sequence by a process that has been likened to seeded crystallization. However, these potentially pathogenic proteins also have important physiological actions, which have not completely characterized. This opens up the question of what process transforms physiological actions into pathological actions and most intriguing, is why potentially dangerous proteins have been maintained during evolution and are present from yeasts to humans. In the present paper, we introduce the concept of mis-exaptation and of mis-tinkering since they may help in clarifying some of the double edged sword aspects of these proteins. Against this background an original interpretative paradigm for CPDs will be given in the frame of the previously proposed Red Queen Theory of Aging.

Keywords: Mis-exaptation, mis-tinkering, neurodegenerative diseases, prions, protein conformations, Red Queen Theory of aging, Russian Doll model of brain circuit organisation. 1. GENERAL PREMISES From a theoretical analysis on evolution the concepts of exaptation and tinkering were proposed by Gould and Jacob, respectively [1-3]. In the present paper these concepts will be applied to the Central Nervous System (CNS) to propose a new model of the cellular networks in the brain and to explore their integrative actions [4]. Furthermore, the derived concepts of mis-exaptation [5] and mis-tinkering will be introduced to suggest a possible new conceptual frame to analyse some CNS pathologies, in particular Conformational Protein Diseases (CPDs). Let us briefly describe Gould’s and Jacob’s concepts: • Exaptation: an important distinction should be made between ‘adaptation’ and ‘exaptation’ [3]. Adaptation refers to a feature of natural selection for its current function (such as echolocation in bats), while exaptation is defined as an available feature that a living organism employs to perform a new function. In other words, such a feature was not produced by natural selection for its current use(such as feathers that might have originally arisen in *Address correspondence to this author at the Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; IRCCS San Camillo, via Alberoni 70, Lido Venezia, Italy; Tel: ??????????; Fax: ??????????; Emails: [email protected] and [email protected] $These Authors have equally contributed to the manuscript. 1389-2037/13 $58.00+.00

the context of selection for thermal insulation [6]). The concept was already used to study evolutionary aspects of the functions of some brain regions. In particular, Anderson has introduced the similar concept of redeployment (or reuse) of a neural structure for a new function [7,8]. • According to Jacob [1] from a structural standpoint, living organisms (and organs) are organized as complex “Russian Matryoshka Dolls” hence, smaller structures are buried within larger ones. It may be surmised that the functions of an organism (as well as of its organs) can be examined as the result of the integrated actions of the structures forming the Russian Doll. Our group has also proposed that the functional modules of the CNS are organized according to a “nested” (hierarchic) criterion [911]. It should be noted that the plasticity of such a hierarchic organisation, in which elements at each level are interconnected and carry out a task (i.e., the “building of the Russian doll”) is not a constant state, but is often a transient arrangement, that occurs as needed [12]. Some relevant aspects mentioned above for CPDs can be analysed according to the present CNS model and the concepts of exaptation/mis-exaptation and tinkering/mistinkering. This approach can help in finding a new perspective for analysing CPDs. © 2013 Bentham Science Publishers

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Against this background a new interpretative paradigm for CPDs will be given in the frame of the previously proposed Red Queen Theory of Aging [13]. 2. INTRODUCTION 2.1. Peculiar Features of the Conformational Protein Diseases Biology is facing a subject that has crucial theoretical and medical implications, namely the problem of CPDs. They usually occur when soluble proteins undergo conformational re-arrangements becoming capable of aggregate into -sheets conformations leading to the production of insoluble complexes known as amyloid deposits, that accumulate extracellularly or in the cytoplasm, in the nucleus and at the cell membrane depending on the disease. It is well known that potentially toxic molecules, such as Reactive Oxygen Species (ROS; [14]) and excitotoxic amino acids [15], can be synthesised by living organisms when they are needed for some basically important tasks, e.g., in learning processes [16-23]. The actions of proteins which can cause CPDs, however, have some similarities and marked differences in comparisons with, e.g., excitotoxic amino acids. In this respect, as demonstrated by the intense investigations on prion protein, the most dramatic difference is the possibility for a conformationally altered protein to act as a template for the steric change of normal proteins into conformationally altered proteins. In other words, one of the most intriguing crucial aspects is the possibility for a living organism to produce ex novo not simply a toxic molecule but a molecule, which can act as an infective-like agent [24-26]. To describe such a process the descriptive term of “Nosferatu’s effect” has been introduced (see below and [27]). In some instances the pathogenic protein can not only diffuse inside an organ [28] but also between organs of one and the same living being [29,30], and in few instances it is possible that the altered protein can have a real “infective character” spreading from one living being to another one of the same species [31] or, as in the case of the prions involved in scrapie (a spongiform encephalitis of the cattle), between living beings of different species [32,33]. This body of evidence poses problems that have not been solved and even if the pathogenic mechanisms will be described in detail in the next years the implications of such an evidence will require new theoretical approaches to have some logical hints to afford this fascinating and fundamental subject that has also blurred some well established definitions such as the one of infective agents [24,34,35] and has even opened up a new discussion on the origin of life [36,37]. The best investigated field of the CPDs is the one related to the neurobiological implications of these proteins, which actually behave as Doctor Jekyll and Mister Hyde since in the proper conformation (Doctor Jekyll) they play physiological functions while in the altered conformation (Mister Hyde) they have pathological effects [38-39]. It should be noted that both type of actions are not always clearly and univocally identified. However, an intriguing suggestion comes from bioinformatics studies. Thus, it has been reported [40] that proteins, such as A, -Syn and Tau, involved in CPDs are characterized by a quite high degree of

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intrinsic disorder. Disordered proteins are functionally useful (Dr. Jekyll), since they are very ductile and have a great propensity to interact with other proteins, but disordered proteins are also potentially dangerous (Mr. Hyde) [23], since they can easily form unwanted interactions leading to aggregates and/or pathologic PMs (see below). The present paper tries to contribute to the fundamental discussion on the theoretical biological basis to give a consistent picture of the pathogenic actions of CPDs in particular at the CNS level. The main focus will be on Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) that are characterised by abnormal conformation of -Amyloid (A) and Synuclein (-Syn), respectively. These proteins like several other proteins involved in the CPDs are capable of rearranging into -sheet conformation and these aggregates accumulate in the nervous tissue. Deregulated protein homeostasis could initiate and certainly enhance such protein failure. A plausible generic explanation for the toxicity of intracellular aggregates involves the sequestration of crucial proteins together with amyloid causing cellular dysfunction and death. In Alzheimer’s disease, for instance, Tau aggregates occur intracellularly and could indeed theoretically trap functional proteins, such as Tau itself, possibly leading to microtubule destabilization or other cellular dysfunction and death [41]. Other mechanisms, however, certainly play a role in the pathogenic actions of CPDs. Even if the main focus of the present paper will be on AD and PD other CPDs will be also briefly considered. The following logical scheme will be adopted for the presentation of the new paradigm of the pathogenic actions caused by CPDs: 1. Peculiar characteristics of proteins playing a role in CPDs. Actually, two interrelated chemical features play an important role for protein functions: their three-dimensional conformation and their intrinsic tendency to interact to build up macro-molecular complexes. These two features are also involved in their possible pathogenic actions. Bio-informatics data exploring the characteristics of some proteins playing a role in CPDs will be briefly reviewed. 2. Data on the actions, especially of A and -Syn, on neuronal networks will be mentioned. It should be noted that these proteins have important even if not completely demonstrated physiological actions beside pathological actions hence a Doctor Jekyll (physiological actions) and Mister Hyde (pathological actions) behaviour [38,39]. This opens up the question of what process transforms Doctor Jekyll (physiological actions) into Mister Hyde (pathological actions) and why potentially dangerous proteins have been maintained during evolution and are present from yeasts to humans [42-46]. 3. We will briefly discuss the role of the “Russian Doll” model on the morpho-functional organisation of brain circuits’ concepts and the related concepts of Functional Modules (FMs), Trophic Units (TUs) and Global Molecular Network (GMN). It will be pointed out that the hub structure within each FM is formed by aggregates of synapses, the socalled “Synaptic Clusters” [11,47,48] (see also below). The proposed model of the morpho-functional organisation of

A New Interpretative Paradigm for Conformational Protein Diseases

brain circuits should be thought as a heuristic hypothesis allowing a more precise investigation of the brain integrative actions and hence of the cognitive impairment observed in CPDs. 3.1. Recent discoveries on new modes for the intercellular communication in the brain, have allowed a better understanding of Volume Transmission (VT) and Wiring Transmission (WT). This is an important aspect since it could explain the spreading of prions, A and -Syn (and other pathogenic proteins) in the CNS. 3.2. Plasma membrane micro-domains in particular the Lipid Rafts and associated Horizontal Molecular Networks [49] will be analysed as special “plasma membrane interfaces” connecting extra- and intra-cellular molecular networks. The physiological and pathological role of Amyloid Precursor Protein (APP) will be considered in the frame of such plasma membrane interfaces. 4. Synaptic Clusters (SCs) will be proposed as crucial targets for the pathogenic actions of conformational altered proteins. Data will be presented demonstrating that there is a preferential targeting by conformational altered proteins of synaptic contacts and this can be an important component of their pathogenic mechanisms. 5. As mentioned above, the recently introduced concept of mis-exaptation [5,50] versus the classical concept of exaptation [3] and also the new concept of mis-tinkering versus the classical concept of tinkering [2] will be used since they may help in clarifying the enigma of Dr. Jekyll/Mr. Hyde [38,39]. 6. The conceptual frame discussed in points 1-4 and the previously proposed Red Queen Theory of Aging [13,38] will be used to suggest an original interpretative paradigm for CPDs. 3. PECULIAR CHARACTERISTICS OF PROTEINS PLAYING A ROLE IN CPDS In the famous Faraday’s Seminar (1952), Pauling stated that life depends on the tridimensional structure and plasticity of proteins. One of the properties of proteins is their stickiness, thus, proteins have the intrinsic tendency to interact (i.e. a “Lego property” [51]) and this feature allows proteins to build up macro-molecular complexes the so called Protein Mosaics (PMs), which are devices showing high plasticity, further enhanced by the fact that the energy landscape, i.e. the chemical-physical influences exerted by the micro-environment (ions, pH, temperature), affects the threedimensional conformation of single monomers and hence also their assembling into a PM [51, 52]. The term mosaic conveys the important aspect of the spatial arrangement (topology) of the monomers (i.e., the “tesserae” of the mosaic) in the high-order molecular complexes that can allow or prevent some of the allosteric interactions between monomers [53]. Unwanted protein–protein interactions can be formed as the most frequent consequence of alterations in the three dimensional structures of proteins [27,54,55] which may lead to the formation of potentially pathogenic protein aggregates. Thus, PMs can perform physiological actions but in some instances they can operate as pathogenic triggers (see the

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metaphor Dr Jekyll/Mr. Hyde [38,39]). A well investigated case of a pathogenic PM is that of AD where cross–-sheet units of A peptides that are arranged to form amyloid fibrils may be considered inert sinks of aberrantly folded proteins, lipids and free metals. Amyloid plaques are not inert aggregates since likely they are in equilibrium with oligomeric forms of A as suggested by neurons in the vicinity of plaques that display dystrophic neurites and synaptic loss, and elevated resting calcium levels [56]. It is also possible that the presence of these hydrophobic deposits triggers inflammatory processes that result ultimately in neuronal damage [41]. The relevance of different types of low molecular weight A homomers is underlined by the demonstration that A40 dimers, trimers and tetramers are 3-, 8- and 13-fold more cytotoxic, respectively, than A40 monomers [57]. Furthermore, it has been demonstrated that the -sheet dimers interact in a much more heterogeneous fashion than the parallel or antiparallel orientation observed in amyloid fibrils, providing a glimmer of insight into the heterogeneity of oligomers and a possibility of defining more precisely the nature of the oligomers present in vivo and on those tested in various biological assays [41]. The pathologic actions of a PM can be investigated on the basis of its molecular weight (a.) and biochemical mechanisms (b.). a). Relevance of the molecular weight for the pathological actions of PMs: • High molecular weight PMs can cause distortion of cellular structures due to the spatial encumbrance of the aggregate which could be followed by activation of cellular defence mechanisms and/or possible sequestration of functionally important proteins [58,59]. • Low molecular weight PMs (hence diffusible oligomers) could result in toxic actions by spreading in brain tissue [60,61]. It has been shown that the so called ADDLs (i.e. A derived diffusible ligands) affect learning and memory [62,63] and subsequently induce neuronal apoptosis [64,65]. Thus, a critical role could be played by low molecular weight intermediates termed “A soluble oligomers” that cause synaptic dysfunction, while large A insoluble deposits might function as reservoirs of the toxic bioactive oligomers. b). Relevance of some biochemical mechanisms that likely are involved in the pathological actions of PMs: • Loss of physiological functions, which can be due either to the improper conformation of monomers/oligomers or to the improper topology of the protein mosaic or, finally, to the improper allosteric interactions among monomers in a PM. The final result is the incapability of the mosaic as a whole to fulfil its task. • Gain of pathological functions, which can be due either to the formation of an improper output signal (a qualitatively or quantitatively altered signal), or to the formation of abnormal molecular devices as, for example, the formation of improper plasma membrane ion channels in AD (see below and [58,66]) or to alterations in receptor trafficking, as recently emphasized by studies showing that neuronal internalization of A involves lipid rafts


and various lipid raft-associated receptor proteins like NMDA and AMPA receptors that are closely associated with lipid rafts and GM1 [67]. However, AMPA recycling to and from the synaptic surface is fast compared to that of NMDA, suggesting that AMPA may act as a more efficient carrier of A than NMDA receptors [68]. In a study using primary hippocampal neurons, Zhao et al. [68] showed that AMPA receptor trafficking is regulated by A oligomers. The Nosferatu’s property [27] is a completely unexpected biochemical feature of some pathogenic PMs. In fact, the Nosferatu’s property has been defined as the ability of a protein with a pathological conformation to act as a template to transform the physiological conformation of another protein with the same (or similar?) amino acid sequence into a pathological protein. Thus, this property can induce a “Domino effect” in the pathogenic actions of some PMs by sequentially altering the conformation of other proteins. As mentioned above, a well investigated case is the one of Prion Protein (PrPc) which is a cell surface glycoprotein that has a half-life of about 6 hours, found especially in neuronal cells where it may play a role in copper metabolism and/or signal transduction [69]. It has been demonstrated that the physiological protein (PrPc) can convert to the lethal scrapie prion protein isoform (PrPSc), which is rich in -structure [70] and forms aggregates with other PrP molecules causing apoptosis of neurons and glial cells. The critical event, that is the “Conversion”, can be induced by mutations (genetic or spontaneous), low pH environments and exogenous PrPSc. Even if current evidence suggests that common misfolding diseases are not transmitted between individuals, the intercellular transfer of inclusions made of Tau, -Syn, Huntingtin and Superoxide Dismutase 1 (SOD 1) has been demonstrated, revealing the existence of mechanisms reminiscent of those by which prions spread through the nervous system [71,72]. Like the growth of crystals, PrPSc propagates by recruiting monomeric PrPc into its aggregates a process that has been replicated in vitro and in transgenic mice. The breakage of PrPSc aggregates represents the actual replicative event, as it multiplies and spreads the number of active seeds. The ability to form self-templating amyloid is not unique to prion proteins. Diverse polypeptides that tend to populate intrinsically unfolded states also form self-templating amyloid conformers that are associated with devastating neurodegenerative disorders. Moreover, fused in sarcoma (FUS) and Tar-DNA binding protein 43 (TDP-43), RNA-binding proteins, which form cytoplasmic aggregates in amyotrophic lateral sclerosis, harbour a ‘prion domain’ similar to that found in several yeast prion proteins [73,74]. An important theoretical and clinical question is whether pathogenic similarities exist between AD and PD (and other CPDs) with prion disease. Hence whether the formation and spread of these proteinaceous lesions might involve a common molecular mechanism: the corruptive protein templating caused and/or potentiated by the seeding phenomenon which is then amplified by intercellular transfer of the pathogenic protein (see also below and [72, 75-77]). In this respect, however, even if a self-

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templating phenomenon can be described in the case of A peptides and intra-cerebral diffusion of the pathogenic protein, there is no evidence for a direct transmission from patient to patient or from mouse to mouse; therefore, ‘infection’ should not be used to describe this phenomenon. Thus, an important aspect that differentiates prionopathies from common neurodegenerative diseases is “infectivity”. As a matter of fact, notwithstanding decades of investigations, no evidence is available of true, spontaneous infectivity of sporadic diseases such as AD, frontotemporal dementia or PD [78]. Pathogenic conformations can be caused or favoured not only by genetic changes, but also by aberrant behaviour of chaperones, deficits of proteasome protein-quality control function, overproduction of ROS and interactions of small molecules with proteins, leading to alterations of protein structure [66]. This may be the case of Homocysteine (Hcy) since high Hcy levels have been reported as an important risk factor for AD and PD [79] and capable of favouring -sheet conformation [28]. Furthermore, disordered proteins [80] are particularly subjected to acquiring unwanted conformations. Thus, studies on the conformations and assemblage of disordered proteins should be a major target for neuro-pathological investigations at the molecular level also by means of bioinformatics approaches (see below). Finally, the so called moonlighting proteins [81, 82] should be scrutinized since they are characterized by a high conformational capability making possible for the same strand of amino acids to carry out different functions (see, e.g., turtle -crystallin can also be the glycolytic enzyme enolase [81]). The moonlighting capability of a protein makes it potentially dangerous, since by binding to other proteins or small molecules like peptides, or following alterations of the physico-chemical conditions of the microenvironment it can acquire an altered conformation. Thus, a certain strand of amino acids of a moonlighting protein may favour the emergence of toxic functions leading to neurodegeneration. To address some of these relevant questions concerning the characteristics of proteins involved in CPDs, bioinformatics methods might provide an important contribution. Thus, in the next section a brief review of the main results obtained by means of bioinformatics studies of some proteins involved in human CPDs will be discussed. 3.1. Bioinformatic Analysis of Proteins Involved in CPDs The identification of protein sequences potentially involved in aggregate formation, the type of aggregates they could induce (whether fibrillar, or amorphous) and their capability to act as seeds triggering conformational changes in chemically similar proteins are the main questions investigated by bioinformatics. Several bioinformatics methods (see [83] for a recent review) have been proposed to predict the potentially amyloidogenic regions within a given protein. They are usually divided into two main classes: phenomenological and structure-based.


Phenomenological methods try to make predictions by using appropriate physicochemical properties of the single amino acids composing the protein chain. By contrast, structure-based tools try to identify the determinants of protein aggregation by observing the existing three-dimensional structure of peptides that adopt a known aggregated conformation. Phenomenological methods are in general easier to implement from a computational point of view. However, it should be emphasized that despite much higher computational demand with respect to the phenomenological tools, the structure-based approaches can provide more insight on the molecular rules underlying the aggregation event. The methodological foundations of the different methods were quite extensively discussed in previous papers (see [83]). Thus, only some main results will be briefly considered. The bioinformatics approach has been proved useful to predict differences in the physicochemical behavior exhibited by very similar proteins. Methods were devised [84,85] capable of correctly predicting the dramatically increased aggregation propensity of the A1-42 peptide in comparison with the A1-40 peptide. As a matter of fact, the additional isoleucine and alanine induced a much higher aggregation propensity by recruiting a bigger part of the C terminus into aggregation. A similar approach was also followed (see [85]) to compare -synuclein with respect to -synuclein and three-repeat Tau to four-repeat Tau, with results in a good correlation with experimental data, as well as with observed pathogenic propensities [86-88]. In a clinical context these results open the possibility that a bioinformatics approach could also be used to gain some understanding of the relationships between biochemical features of pathogenic proteins and the features of a neurodegenerative disease. For example bioinformatics approaches can predict disease severity from the mutations associated with familial forms of a CPD. In this respect, a study by Wang et al. [89] showed that disease duration in patients with familial ALS, which is associated with mutations of SOD1, was negatively correlated with the overall aggregation propensity of SOD1 after mutation. This result is remarkable for two reasons: first it demonstrated that a computational method based on physicochemical factors was helpful to predict amyloid formation in humans; second, the prediction allowed gaining information on the phenotypic effect of protein aggregation. From the biochemical point of view, predictors contributed in locating potential regions of interest for further structural studies. As far as proteins involved in neurodegenerative diseases are concerned, some examples are already present in the available literature. In quite recent investigations (see [23]) the predictions provided by the predictor AGGRESCAN [90] allowed the observation that the ‘hot spots’ for aggregation of -Amyloid and -Synuclein were mainly localised in the so-called leucine rich repeats, groups of repeats of a 24-amino-acid motif usually with cysteine-rich capping structures at the amino and carboxyl terminal of each block [91]. Leucine rich repeats domains are found in a very large and diverse group of proteins and have been inferred to be responsible for molecular recognition and protein-protein interactions [92]. Frousios et al. [93] by utilizing


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molecular graphics programs found that nearly all experimentally verified amyloidogenic determinants (short peptide stretches favouring aggregation and subsequent amyloid formation), and several ones predicted with the aid of a consensus algorithm (combining five different methods) called AMYLPRED [93,94], but not experimentally verified amyloidogenic determinants, are located on the surface of the relevant amyloidogenic proteins (as, for instance, SOD1). This finding may also be important in efforts directed towards therapeutic interventions aimed to inhibiting amyloid fibril formation. In this respect, a further potentially interesting question concerns the possibility to classify the identified amyloidogenic stretches within a protein in terms of the main mechanism for aggregation they can trigger. A possible bioinformatics approach combining different methods was very recently proposed [83]. The study was focused on the following set of particularly important proteins involved in human CPDs: • A associated to Alzheimer’s disease (AD). • Tau protein associated to Alzheimer’s disease, frontotemporal dementia, and other dementias. • SOD1, TDP-43 and FUS that are involved in amyotrophic lateral sclerosis. • -Syn associated to Parkinson’s disease. • PrP inducing Creutzfeldt-Jacob Sträussler-Scheinker diseases.

and

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• DISC1 associated to schizophrenia. Thus, it has not only been confirmed that all the sequences that have been so far experimentally found to be involved in the formation of protein aggregates were correctly identified, but also it has been suggested a possible procedure to classify the identified sequences in terms of the following aggregation mechanisms: • -aggregation, i.e. the ability to trigger the formation of ordered aggregates (fibrils). • This property should be somehow distinguished from the hidden -propensity, i.e. the tendency of a given sequence to switch from a native -helical to a -strand configuration depending on the energy landscape involved. These ‘chameleon sequences’ [95] can act as ‘conformational switches’ [94] playing the role of templates initiating amyloid formation. • The formation of -strands, however, is not the only mechanism for aggregates formation, since a generic propensity to aggregate [96] can also lead to the formation of amorphous (non fibrillar) aggregates. In particular as far as the ability of triggering the formation of -aggregates is concerned, this bioinformatics analysis correctly identified amino acid stretches experimentally found to -aggregate into fibrils. Examples are the Cterminal region of Amyloid- containing the sequence NKGAII or the region 623-627 of the Tau protein, closely corresponding to the sequence VQIVYK. Both these sequences were recently shown [97] to participate in the formation of steric-zippers leading to ordered -strands. It is


also noteworthy that a number of the predicted amyloidogenic stretches were classified as ‘chameleon sequences’, able to undergo local conformational changes, potentially triggering protein aggregation, only when suitable physicochemical conditions were realized. The majority of the predicted sequences, however, were classified as characterized by a generic aggregation propensity, suggesting the formation of amorphous aggregates as a significant mechanism exploited by the considered proteins to aggregate. This point could be of particular interest in the context of available experimental information. In fact, all the proteins here considered have been extensively studied from a biochemical-biophysical standpoint and for their neurobiological physiological and pathological implications. An important finding has been the demonstration that pathogenesis is not so much due to the insoluble amyloid fibrils as rather to the soluble oligomeric aggregates for several amyloid proteins, which were found to be neurotoxic [98]. Thus, it has been shown that PD-associated mutations in -Syn promote the formation of oligomers rather than fibrillar species, suggesting the oligomeric species, not the fibrils, are toxic. In cell culture and in some in vivo models [99], toxicity is seen without heavily aggregated -Syn, supporting the postulation that the soluble species of -Syn mediate toxicity [100]. In fact, over expression of wild-type -Syn in animal models led to neuronal loss and Lewy Body-like inclusion formation [100]. After biosynthesis in the neuronal cell body, -Syn is transported along the axons to the presynaptic terminals. Impairment of axonal transport contributes to abnormal accumulation and localized concentration of -Syn in perikarya and axons [100]. As in the case for infectious prion protein, it is also clear that A-derived oligomers are both synapto- and cytotoxic [101-103], and amyloid fibrils may also possess aspects of that toxicity [104]. Furthermore, brain homogenates containing A from human AD, or mouse model AD, are capable of “seeding” AD pathology in naive mouse models of AD [105107]. Experimentally cerebral -amyloidosis can be exogenously induced by exposure to dilute brain extracts containing aggregated A seeds. The amyloid-inducing agent probably is A itself, in a conformation generated most effectively in the living brain [108]. This suggests that the templating agents of AD in “infected” mouse models are structured but soluble species, consistent with small A oligomers as opposed to insoluble fibrils or plaques [105,106,109]. These seeding experiments are demonstrations of a limited interpretation of the amyloid cascade hypothesis [110] whereby nucleation-polymerization of A monomers into oligomers and plaques accounts for AD pathology. However, it should be noted that Eisele [107,109] does not provide direct evidence that peripherally injected A (independent of other A-associated cofactors) in the brain extracts is necessary and sufficient to trigger cerebral amyloid deposition. Induction of amyloid formation in vivo with synthetic A has been unsuccessful so far, leading to the hypothesis that an amyloid-enhancing factor or a particular conformation of A aggregates is required to trigger A deposition in vivo. Similarly, numerous attempts to trigger prion diseases with synthetic prion proteins alone have not

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been successful [111]. Accordingly, a recent study demonstrated the critical role of lipid molecules and RNAs as cofactors in producing recombinant infectious prions. It is possible that cerebral A amyloidosis also requires brainspecific endogenous amyloid enhancing factors [112]. Also, plaque-associated proteins and lipids, such as apolipoprotein E, proteoglycan, and ganglioside, might serve as cofactors and facilitate A aggregation. However, the chemical and physical features of the amyloid-inducing factor remain to be determined. Beyond these aspects concerning the structural biology of pathogenic proteins, more questions have been stated by Jucker [108] and especially the following two of these questions will be considered in the sections that follow: -

What accounts for the selective vulnerability of neurons in the proteopathies?

-

How do protein assemblies move from cell to cell and from region to region?

4. SOME DATA ON THE ACTIONS OF A AND SYN ON NEURONS From a general standpoint it can be surmised that two main mechanisms at the cellular level can be of high importance in the pathogenesis of CPDs. These two mechanisms are not mutually exclusive, since the first one may cause or favour the second one: • A possible general unspecific pathogenic mechanism due to the wearing off the resilience of neurons leading to the shift of a mild stress into a toxic stressor. This pathogenic mechanism can be triggered by calcium intracellular overload, mitochondrial failure, ROS overproduction, accumulations of potentially toxic metals such as Zn, Fe, Cu [113-115]. All these pathogenic mechanisms favour macro-molecular alterations hence proteosomal failure with the formation of abnormal PMs, which could be both a consequence of the above mentioned agents but also favour their production [116-120]. For example, it has been shown that the pre-synaptic protein -Syn, that plays a physiological function in the assembly of the protein complexes required for chemical neurotransmission, plays also an important role in PD, and one possible causal link with the pathogenesis of this disease is its capability of binding Cu2+. This metal can accelerate the aggregation of -Syn to form various toxic aggregates in vitro. Copper is also a redox active metal whose complexes with amyloidogenic proteins/peptides have been linked to oxidative stress in major neurodegenerative diseases [121]. Also the iron-related oxidative damage is mediated by -Syn oligomerisation during the development of PD. It should be noted that seeding induced by Syn oligomers, the toxicity of which has been demonstrated in vivo, can induce intracellular -Syn aggregation, providing evidence for spreading of -Syn pathology similar to that of prions [122,123]. • Possible synergistic interactions among conformational altered proteins each of which has not by itself overcome the threshold to act as a pathogenic trigger. Thus, it has been described synergistic pathogenic effects of -Syn, hyper-phosphorylated Tau, amyloid-, with accumulation


and cross-seeding of each other, the induction and spread of toxic protein aggregates and acceleration of cognitive dysfunction [124]. In particular, accumulation of -Syn alone can significantly disrupt cognition but -Syninduced synapse damage is enhanced by A 1-42 and Syn can in turn promote A accumulation [124-126]. It should be noted that A not only may interact directly with -Syn to form cation channels that contribute to neurodegeneration [127] but also, especially A42, is very effective at promoting oligomerization of -Syn in vitro. Furthermore, it has been shown that -Syn can enhance Tau aggregation and phosphorylation both in vitro and in vivo [128-130] on the other hand -Syn aggregates in a concentration-dependent manner but fails to form fibrils at lower concentrations unless co-incubated with Tau [128]. These complex reciprocal pathogenic interactions are further underlined by genetic, pathologic, and biochemical evidence demonstrating that aggregation of A is a critical, early trigger in the chain of events that leads to tauopathy, neuronal dysfunction, and dementia [108]. Also epidemiological data indicate an A, -Syn interaction since up to 50% of AD cases exhibit the aggregation of -Syn into Lewy bodies. Importantly, the presence of Lewy body pathology in AD is associated with a more aggressive disease course and accelerated cognitive dysfunction. Taken together, these data provide compelling evidence that A, Tau, and -Syn synergize to promote the aggregation, phosphorylation, and accumulation of each other and to accelerate cognitive decline. Thus, it could be of paramount importance to have a heuristic hypothesis of the main structural and functional targets of these potentially pathogenic proteins. To this aim a hierarchical model of the CNS morpho-functional organisation will be briefly illustrated. The main focus will be on SCs and the molecular networks which allow them to carry out their integrative actions. 5. ON THE “RUSSIAN DOLL” MORPHOFUNCTIONAL ORGANISATION OF BRAIN CIRCUITS The proposed paradigm to investigate CPDs is based on a model of CNS morpho-functional organisation, which relies upon the concepts of Functional Module (FM), Trophic Unit (TU) and Global Molecular Network (GMN). The concept of “Functional Modules” and “Trophic Units” [9,38,54,131,132] are strictly interconnected and such a functional link can shed some light on both the decline in cognitive capabilities and degeneration of neurons caused by CPDs and more generally during aging. These concepts can be briefly described as follows: • FMs are formed by networks of different degrees of miniaturisation nested within each other from the cell network level down to the molecular network level (Fig. (1)). Hence each FM has a ‘Russian Doll’ structural organization and at each miniaturisation level computing elements (e.g., cells, dendritic spines, or proteins) form a “mosaic” [9,133]. The term “mosaic” has been intro-


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duced to convey the concept of “spatial topology” (i.e., location of the elements in the computing device) and/or that of “functional topology” (i.e., order of activation of the elements in the computing device). The term mosaic may therefore have a potentially great heuristic impact in the analysis of the integrative functions of the assembly since it underlines the possibility that with the same set of “tesserae” (i.e., elements of a computing device) markedly different mosaics can be assembled at a certain level of miniaturization, which becomes capable of different handling information. A key mosaic is likely represented by the Synaptic Clusters (SCs), in which multiple synapses act cooperatively to modulate their efficacies [47,48]. It should be noted that the functional topology can have a peculiar relevance (i.e., the order of activation of the single synaptic contacts) for the SCs. Each FM can have one or more SC, often organised around the dendritic spines and partially isolated from the surrounding environment by glial cells and, as discussed below, by Extra-Cellular Matrix (ECM) [11,48,134]. • Trophic Units (TUs) are formed by Complex Cellular Networks (CCNs), i.e., by neurons; glia cells, pericytes, ependymal cells that functionally interact and support the physiological functions and each other’s survival. Each TU usually contains more than one FM and the crucial computational device of the FM (i.e., the SCs) is very sensitive to alterations in the ECM composition and geometry (see below and [135]). As a matter of fact, ECM plays a role in both the cells’ scaffolding and synaptic contacts, and in filtering and elaborating information, this latter function not just due to its protein constituents but also to hyaluronate [55]. In agreement with the early Agnati and Fuxe proposal, TUs are interconnected via WT and VT (see below), hence they tend to survive or to die according to their more or less strict interconnections [55,131]. This may explain both the patchy and the rather stereotypical degeneration that can be often observed in CPDs [120,136-138]. Against this background we will be put forward the hypothesis that TU’s main action is aimed at the trophic support of the SCs. Thus, it may be surmised that the SC demand of high energy and/or the reduced capability of energy production by the respective mitochondria can cause an “Energide/Mitochondria Divorce”. This is a pathogenic phenomenon that has been defined as the break down of the endo-symbiotic cooperation between two basic components of the eukaryotic cell [77]. • Global Molecular Network (GMN). It has been proposed that the ECM is part of a GMN enmeshing the entire CNS and plays a fundamental role in the structural and functional organisation of the CNS [54]. The GMN is formed by the ECM; the Horizontal Molecular Networks at the plasma membrane level and the Vertical Molecular Networks that can project from the plasma membrane level towards the extra-cellular and/or the intra-cellular side of the cell [139-141]. Proteins, carbohydrates, lipids build up the GMN, often with physical interactions which lead to high weight molecular complexes. From a functional standpoint, proteins are likely to be the most important building blocks for all the molecular networks, especially for the extra-cellular part of GMN,


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Fig. (1). Schematic representation of the concept of Functional Module (FM). Each FM is formed by networks of different degrees of miniaturisation nested within each other from the cell network level down to the molecular network level. It has been proposed that brain circuits are organized according to a “Russian Doll Model” and at each miniaturisation level computing elements (e.g., cells, dendritic spines, or proteins) form a “mosaic” [9,133]. Thus, four main miniaturisation levels have been indicated moving from the macro-scale down to the mesoscale, and the micro-scale to end up with the nano-scale. For further details see text.

glycosilated proteins which play also an important scaffolding role [54]. The proposal of an extra-cellular GMN has important physiological and pathological implications. With regard to the physiological aspects, the GMN plays a role in cell migration, formation of synaptic contacts (and hence of WT) and the perviousness, that is the permeability of the diffusion pathways for chemical signals in the extra-cellular space of the brain (and hence of VT). With regard to the pathological implications, alterations in the GMN could alter the communication processes mentioned above and favour the formation of pathological protein aggregates (e.g., amyloid plaques and neurofibrillary tangles) which affect cell function and survival.

in SCs integrative actions. Thus, it has been shown that brevican, an abundant extracellular chondroitin sulfatebearing proteoglycan, forms brevican-containing dense networks of ECM that wrap around synapses forming compartments. These ECM compartments act as a barrier to lateral diffusion of AMPA (-amino-3-hydroxyl-5methyl-4-isoxazole-propionate) receptors preventing them from clustering at the active synaptic site [142,143]. Thus, ECM also fulfils the important task of allowing the proper topological and functional regulation of SCs. As a matter of fact, proteoglycans, and more generally, ECM stabilize synaptic structure and may control the actions of signals promoting neural plasticity by, e.g., regulating lateral diffusion of AMPA receptors [142,143].

In agreement with such a view, Ajmo and collaborators [135] have pointed out that aggregation of amyloid- in the forebrain of Alzheimer's disease subjects may disturb the molecular organization of the extracellular microenvironment that modulates neural and synaptic plasticity. Proteoglycans are among the major components of this extracellular environment and may play important roles

This ECM role is impaired in AD since a possible link between brevican and AD has been suggested by recent data demonstrating that amyloid-, or another amyloid precursor protein dependent mechanism modifies the accumulation and/or turnover of brevican [135]. As a matter of fact, the molecular size of chondroitin sulfate chains attached to brevican was smaller in hippocampal


tissue from APPsw mice bearing amyloid- deposits compared to nontransgenic mice, likely due to changes in the chondroitin sulfate chains. The major proteolytic fragment of brevican was markedly diminished in extracts from several telencephalic regions of APPsw mice compared to non-transgenic mice and appeared to accumulate adjacent to the plaque edges. These results suggest that amyloid- or amyloid precursor protein exert inhibitory effects on proteolytic cleavage mechanisms responsible for synthesis and turnover of proteoglycans. 5.1. Recent Acquisitions on Modes for the Intercellular Communication in the Brain A new area of investigation for CPDs is the diffusion of potentially harmful proteins which is dependent on the communication modes in the brain. Thus, it is an important issue to evaluate the possible role of the newly discovered modes of intercellular communication in the brain. The proposal on the existence of two main modes of intercellular communication in the CNS was introduced in 1986 and called Wiring Transmission (WT) and Volume Transmission (VT) [144]. The major criterion for this classification was the different characteristics of the communication channel with physical boundaries well delineated in the case of WT (axons and their synapses; gap junctions) but not in the case of VT (the extracellular fluid filled tortuous channels of the extracellular space and the cerebrospinal fluid filled ventricular space and sub-arachnoidal space). The basic classification is binary, with two distinct alternatives (dichotomies) at each step of intercellular communication in the brain is still considered valid, but recent evidence on the existence of other modes for intercellular communication, such as microvesicles (exosomes and shedding vesicles) and tunnelling nanotubes (TNTs), has implied a refinement of the original classification. Thus, new sub-classes of WT and VT have been introduced, namely the “TNT type of WT” and the “Roamer type of VT.” In the TNT type of WT proteins, mtDNA and RNA can migrate as well as entire organelles such as mitochondria [77,145,146]. In the Roamer type of VT microvesicles are safe vesicular carriers for targeted intercellular communication of proteins, mtDNA and RNA in the CNS flowing in the extracellular fluid along energy gradients to reach target cells [145,146]. Recently it has been demonstrated that both classical VT as well the Roamer Type of VT are involved in the propagation of CPDs. As a matter of fact, aggregates composed of an ALS-causing SOD1 mutant penetrate inside cells by macropinocytosis and rapidly exit the macro-pinocytic compartment to nucleate aggregation of the cytosolic, otherwise soluble, mutant SOD1 protein [147]. Hence, mutant SOD1 aggregates transfer from cell to cell with remarkable efficiency, a process that does not require contacts between cells but depends on the extracellular release and diffusion in the Extra-Cellular Space (ECS) of aggregates hence of VT. Also the Roamer Type of VT plays a role since cell-produced -Syn is secreted via an exosomal, calcium-dependent mechanism and such an exosomal -Syn secretion may amplify and propagate PD pathology [148].


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Furthermore, it has also been shown that a minute fraction of A peptides can be secreted from the cells in association with exosomes [149]. Consistent with this finding, the presence of exosomal proteins has been observed in plaques from AD patient brains [149], supporting a potential role for exosomes in the pathogenesis of AD [75]. As far as the possible pathological role of exosomes is concerned, exosomes were found in a study by Vella et al. [150] in Cerebro-Spinal Fluid (CSF) from sheep and in these CSF-derived exosomes the authors were able to show an enrichment of prion protein over unfractionated CSF, suggesting a link between these vesicles and prion disease pathogenesis. These studies not only reveal that pathogenic monomers and/or oligomers and/or PMs can propagate in a prion-like manner in neuronal cells, but also the role of the classical and Roamer Type of VT in the mechanisms underlying aggregate uptake and cell-to-cell transfer [71,150]. Actually, increasingly detailed data indicate that prions and the following proteins causing CPDs may diffuse via the Roamer Type of VT [72]: – Amyloid- (A) in Azheimer’s disease (AD). – Tau protein in AD, frontotemporal dementia, and other dementias. – Superoxide dismutase 1 (SOD1), Tar-DNA binding protein 43 (TDP-43) and fused in sarcoma (FUS) in amyotrophic lateral sclerosis (ALS). – -Syn in Parkinson’s disease (PD). – Huntingtin in Huntington’s disease (HD). – Disrupted in schizophrenia 1 (DISC1) in schizophrenia. The crucial aspect of the interactions between pathogenic monomers and/or oligomers and/or PMs and target cells is subjected to intense investigations [41]. In the case of soluble A species in Alzheimer’s disease several investigations suggest that it is more likely that A peptides will exert multiple effects by binding to membrane proteins, targeting membrane lipids, causing oxidative stress and changing membrane dielectric properties and ion permeability instead of interacting with specific receptors [41]. In agreement with such a view it was confirmed that A1–42 oligomers affinity for PrPc, is nanomolar however it was demonstrated that PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that A1–42 oligomers are responsible for cognitive impairment in AD and that PrPc is not required [151]. This new perspective for neurodegenerative processes focuses on the effects of potentially toxic stressors on SCs, as the most sensitive component of the FMs. Alterations in SCs can propagate within the multi-level components of the FMs of a TU and, thereafter, between TUs both via WT and/or VT. This last aspect is related to the Domino effect concept that describes the progression of disease within the various miniaturisation levels of FMs as well as between TUs. In agreement with such a view, in AD patients a gradual evolution of the disease has been described possibly following TU interconnections since A plaques begin first in the


basal temporal neocortex. From there, the alterations spread to adjoining neocortical areas, initially sparing the cortical regions running along the corpus callosum and primary motor and sensory cortices. The perforant pathway then becomes studded with A deposits as it extends through the hippocampal formation [61]. 5.2. Plasma Membrane Micro-Domains in Particular the Lipid Rafts as Special “Plasma Membrane Windows” Connecting Extra- and Intra-Cellular Molecular Networks As mentioned above, in a previous paper it has been hypothesized that three-dimensional molecular networks fill up the intra- and extra-cellular space of the CNS and interact with each other at the boundaries of compartments, such as the plasma membranes, to form a Global Molecular Network [54]. The relative independence and the more or less stable interconnections of specific circuits of the GMN are fundamental problems that still await careful investigation. Thus, the extracellular part of the GMN finds its counterpart in the intra-cellular molecular networks, with an intense cross-talk which takes place at specialized “plasma membrane windows” such as the lipid rafts where likely crucial Horizontal Molecular Networks are located. Some membrane-associated proteins affect the plasma membrane micro-domain organization. In fact, it has been shown that integrin clustering induces the plasma membrane to organize into tightly packed, highly ordered lipid rafts [152] which can concentrate signalling proteins such as heterotrimeric G proteins and GPCRs [153-155]. Even a small increase in local concentration of signalling molecules as a result of partitioning into a lipid raft can cause a dramatic amplification of signalling cascades [156]. Integrins can have also a trophic role as suggested by their cooperation with growth factor receptors which interact with integrinmediated signalling on multiple levels [157]. One of the means by which neurons accumulate intracellular A is through uptake of extracellular A peptides, and this process may be a potential link between A generation, synaptic dysfunction, and AD pathology. Recent studies have found that neuronal internalization of A involves lipid rafts and various lipid raft-associated receptor proteins [67]. Thus, plasma membrane windows may play an important role in AD as well as in other CPDs. As a matter of fact, while most of the work has been focused on the interactions between lipid rafts and A, compelling evidence has also been obtained for htt, -Syn, PrP, and calcitonin [98]. In agreement with our proposal of the special role of plasma membrane windows where at least some CPD related proteins affect intra-cellular/extra-cellular cross talk, it has been demonstrated that APP is part of a class of proteins such as integrins capable of delivering input/output signals across the plasma membrane [157]. Furthermore, a special role for APP can be surmised at these plasma membrane windows since it has been shown that the extracellular sequence of APP can interact with various extracellular matrix components, such as heparin [158160], collagen type I [161], and laminin [162], indicating the

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role of APP in cell-matrix adhesion. Both Dahms et al. [163] and Gralle et al. [164] reported that heparin binding to the extracellular E1 or E2 domain induces APP/APP dimerization. Using a primary neuron/HEK293 mixed culture assay, Wang et al. [165] reported that transcellular APP/APP interaction induces pre-synaptic specializations in co-cultured neurons. These studies identify APP proteins as a novel class of synaptic adhesion molecules located in lipid rafts hence at special membrane windows level. A large body of evidence supports a trophic function of APP in neurons and synapses since the deletion or reduction of APP is associated with impaired neuronal viability in vitro and reduced synaptic activity in vivo. Again these data support the hypothesis that alterations in the SCs can be a primary event in neurodegeneration and APP plays an important regulatory and protective role in synaptic integrity. The interplay between lipid rafts and APP is also underlined by experimental data suggesting an involvement of lipid rafts in the interactions between amyloid proteins and cell membranes. As a matter of fact, multiple lines of evidence implicate lipid rafts in amyloidogenic processing of APP into A. A subset of BACE1 and full length APP (APP FL) associates with lipid raft domains and hence with cholesterol which is one of the major constituent of lipid rafts. Targeting the BACE1 lumenal domain to lipid rafts by the addition of a glycophosphatidylinositol anchor, increases APP processing at the -cleavage site [166,167]. These data support the epidemiological studies that correlated cellular cholesterol and AD pathogenesis [168]. Spatial segregation of -secretase processing of APP from that of other substrates suggested a special targeting of APP processing. Summing up, it can be surmised that lipid rafts (i.e., special “plasma windows”) participate in the control of A synthesis and likely also modulate extra-cellular/intra-cellular communication processes, which may be altered in AD. 6. SYNAPTIC CLUSTERS AS CRUCIAL TARGETS FOR THE PATHOGENIC ACTIONS OF CONFORMATIONALY ALTERED PROTEINS In line with the classical Hebb hypothesis on the possible existence of cell assemblies interconnected via reverberating circuits [169,170], it has been suggested that different FMs can be transiently interconnected to form a higher-order mosaic; hence, a “3-dimensional elaboration of the information” is in operation in the CNS [11]. As a matter of fact, a “horizontal elaboration” occurs, based on mosaics of FMs, while a “vertical elaboration” is simultaneously occurring inside each FM (Fig. (1) [12]). Thus, the proposed model of CNS as a system of FMs organised as Russian Dolls (i.e., as nested networks [9,12,132,171]) implies that each FM processes information at different levels of miniaturization from the cell network level down to the molecular network level and vice versa. It follows that the final, integrated, activity of the FM would emerge from the complex dynamics of this hierarchical system of relations. As already pointed out, it has been surmised that within each FM SCs play the role of integrating elaborations carried out at higher levels of miniaturisation. The integrated elaboration will be used by the higher hierarchic levels. As a matter of fact, structural and


functional plasticity are markedly affected by intra-SC biochemical mechanisms since plastic changes induced by Long Term Potentiation (LTP) at one synaptic contact lowers the threshold for the induction of LTP at neighbouring synapses at a stimulation strength that did not cause any plastic changes under control conditions [172,173]. These data are in agreement with our FM model where SCs act as a sort of ‘intelligent layer’ between the activity at cellular level and the integrative functions performed at molecular level by supra-molecular complexes such as those formed at the cell membrane by G protein-coupled receptors, owing to direct receptor-receptor interactions ([174-176] for reviews). Thus, physiological and pathological influences on SCs can have dramatic effects not only on the integrative activity of FMs but can also strain the respective TU. In agreement with such a view it has been demonstrated that practically all the pathogenic proteins affect the synaptic contacts and, in several instances, can potentiate each other actions (see above and [177,178]). Accordingly, it has been shown that A oligomers show synaptic toxicity as measured by changes in dendritic spine morphology, altered LTP and long-term depression (LTD) in hippocampal slices; effects on neurotransmission in cell culture; or defects in memory and cognition in rodents [41]. Thus, subtle disruption of synaptic activity, induced by A oligomers, precedes synapse loss that is followed by neurodegeneration. In particular, it has been described as the A induced neurodegenerative triad: spine loss, dendritic changes, and neuritic dystrophies through calcineurin activation [124]. Recently, it has been proposed that amyloid oligomers may act at two steps, separated in time, a first, very rapid step, where Ca2+ increases due to glutamate receptor stimulation by the oligomers, followed by a second, delayed step, where oligomers permeabilize nonspecifically the cell membrane, via the formation of amyloid pores [98]. As a matter of fact, a dramatic and prompt neurodegenerative action caused by A is the rapid and potent perforating property in neuronal membranes that increases intracellular calcium leading to synaptic transmission failure [98,179]. This phenomenon, originally described for A, has also been described for other mis-folded proteins and has been proposed as a common property of the amyloid protein family (see above and [180]). Several pieces of evidence sustain this hypothesis; Cribbs et al. [181] showed that both D- and Lstereoisomers of truncated form of A were neurotoxic in vitro. This observation argues against a role for specific ligand-receptor interaction in the mechanism of toxicity. The formation of pores in the plasma membrane by amyloid oligomers cause disruption of Ca2+ ions control with alterations in neurotransmitter release, generation of action potential, gene expression, synaptic plasticity, and neurite growth. The data indirectly support the present hypothesis of the crucial role of synaptic plasticity and hence the hub role of SCs in the integrative actions of the brain as well as of SCs as a possible crucial target of CPDs. As mentioned above, it has been surmised that SCs play a special role by integrating the elaborations carried out at molecular levels [11]. In view of their high energy demanding task, SCs need a continuous and large energy supply, hence an efficient Energide-Mitochondrial symbiosis that is


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also affected by the proper control of blood supply that is one of the main goals of the TUs. In agreement with such a proposal, available data demonstrate that energy failure causes are also the result of accumulation of proteins with altered conformations. Thus, pathogenic protein aggregates develop in parallel with a deficit of energy supply and hence of a reduced trophic support to SCs that cannot any longer carry out properly their integrative actions. The final result is a cognitive decline and neurodegeneration. The main aspects of the present proposal are summarised in the scheme (Fig. (2)), where also some of the main references that support the proposal are reported (see legend to the figure). The interconnections between mitochondrial alterations, pathogenic protein conformations and energy failure should be noted. Actually, it can be surmised that in some instances these phenomena can operate as a pathogenic positive feedback. Thus, according to the present hypothesis it may be surmised that a possible pathogenesis of CPDs involves firstly a toxic stressor that affects susceptible synaptic contacts altering SCs and its integrative actions in a sub-clinical way. Thereafter, if the toxic stressor overcomes the resilience of that level, the detrimental change propagates not only between the different components of that level, but also to lower and higher levels altering the TU action and diffusing to other TUs. When several TUs are disrupted the ‘‘toxic influence’’ can propagate from a neuronal system to others and the clinical condition can be clearly detected. Indirect indications for such process can be obtained by the Braak’s studies [120,136,137], which demonstrated for PD a progression of the diseases from the caudal brain stem towards the mesoand telencephalic regions as well as from studies on epilepsy [182]. 7. EXAPTATION VERSUS MIS-EXAPTATION AND TINKERING VERSUS MIS-TINKERING As mentioned in the “General Premises” the new concepts of “mis-exaptation” and of mis-tinkering may help in shedding some light on the conundrum of the Dr. Jekyll / Mr. Hyde possible actions of some proteins. As a matter of fact, the mis-exaptation concept has been introduced to describe a new feature which, although may confer new capabilities to carry out some tasks in one field of activity, may have negative or deleterious effects in another area, or it may reach such a degree of specialisation and/or an overwhelming presence, so as to have deleterious effects in this or in another field leading to a decrease in fitness of the individual [5]. The concept of exaptation/mis-exaptation can suggest a new point of view for discussing genesis and effects of altered protein conformations that is a consequence of their sequence, the micro-environment, the folding enzymes and chaperone proteins they encounter. As a matter of fact, the exaptation points to the data indicating that organisms have evolved to take advantage of the fact that many polypeptides


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Fig. (2). Schematic representation of the possible key role played by Synaptic Clusters (meso-scale level of miniaturisation) to integrate elaborations carried out at higher miniaturization level with elaborations at lower miniaturization level (i.e., at a higher hierarchical level). It should be noted the positive feed-back loop connecting alterations in protein conformations, mitochondrial deficits and energy failure as a critical trigger of Synaptic Cluster impairments and degenerations. References: 1=[58]; 2=[212]; 3=[77]; 4=[212]; 5=[214]; 6=[215]; 7=[216]; 8=[217]; 9=[218]; 10=[219]. For further details see text.

can form amyloid, despite the fact that amyloid can also be toxic. The discovery of functional amyloid in humans was is particularly interesting because of the compelling links between human pathogenic amyloid formation and disease [183].

during repetitive stimulations of the synapse. As pointed out above, mis-exaptation can simply occur following excessive A and/or A altered conformations which can cause, as discussed above, synaptic dysfunction and synapse loss [190].

Thus, the conundrum of why evolution has maintained from yeast to humans the potentially toxic amyloids can be solved by considering that these proteins have been exaptated and, although A was originally regarded as an abnormal and toxic species restricted to the brains of aged or demented humans, recent work has linked amyloid to physiological actions [183]. In agreement with the view of a physiological function for A (Dr Jekyll’s action), there is the demonstration of soluble A species in the bodily fluids of various species and in the conditioned medium of cell cultures [184,185]. Moreover, experimental data have demonstrated that low levels of A increase hippocampal longterm potentiation and enhance memory, indicating a novel positive, modulatory role on neurotransmission and memory [186]. Picomolar levels of A can also rescue neuronal cell death induced by inhibition of A generation (by exposure to inhibitors of - or -scretases) [187], possibly through regulating the potassium ion channel expression, hence affecting neuronal excitability [188]. Furthermore, the requirement of a lasting change to the synapse after a single signalling event could be fulfilled by an amyloid, a structural entity whose soluble-to-aggregate transition can be tightly regulated [189]. Because the formation of the ApCPEB amyloid results in a gain of function that leads to new protein expression, the temporal regulation of the amyloid (i.e., increase or decrease in levels) may also allow for ‘‘strengthening’’ the memory

On the other hand, the concept of mis-tinkering can be described as either a process by which a component of a contraption is not working properly (too much or too little) or it is wrongly located in the assembly or, finally, it can even “divorce” from the other components acquiring functional independence that leads to the failure of the tinkering process. A paradigmatic case may be the so called “Divorce between Energide and Mitochondria” [77] that is the failure of a fundamental cooperation that evolution has discovered in the tinkering together of different types of primordial cells to assemble eukaryote cells (evolutionary new contraption). As a matter of fact, from the early Margulis’ proposal (in a 1967 paper, The Origin of Mitosing Eukaryotic Cells [191]) evidence has been provided, that certain organelles, in particular mitochondria, originated as free-living bacteria that were taken inside another cell as endosymbionts. This has been a tinkering solution for utilization of oxygen and the high production of energy by mitochondria. However, according to our proposal, a mis-tinkering can occur either for a failure (e.g., over production of ROS by mitochondria) or for mitochondrial “divorce” from the Energide becoming independent entities involved in their own independent survival. One of the basic assumptions of the present hypothesis of mitochondrial secession is that mitochondria can revert towards


their original state as endo-parasites and thereby disharmonise the symbiosis of the eukaryotic cell. It has also been shown that altered mitochondria as well as potentially harmful proteins can diffuse from one cell to others via TNTs or micro-vesicles [192]. It should be noticed that mitochondria susceptible to divorce can be those of aged people that, in view of the reduced resilience of the symbiotic cells, in presence of a pathogenic allostatic overload can have mitochondria which revert to an independent state hence to a mistinkering condition. Obviously, the supervening deficit of energy production favours the accumulation of misfolded proteins. Thus, the concept of tinkering/mis-tinkering may help in explaining the genesis of altered proteins and also their pathogenic actions and can be at the basis of the Red Queen Theory of Aging [13]. 8. RED QUEEN THEORY OF AGING Some signals, which play an important role in neuroplasticity hence in learning, also affect neuronal survival. As pointed out above, a conundrum is why some signals have a Dr. Jekyll action which can be mis-exaptated becoming a Mr. Hyde action. A possible explanation is based on the hypothesis that neurodegenerative processes are the negative consequence of a maladapted boosting of the plasticity mechanisms triggered by these key-signals [23]. Thus, an exaptation/mis-exaptation phenomenon may be in operation and this view is in agreement with the Red Queen Theory of Aging [13] that hypothesises that during aging a shortage of neuronal plasticity occurs [13,38,133,193,194], which not only does not allow new learning, but also is insufficient for compensating brain wear-out. On this basis it is supposed that neurodegeneration is, at least in part, the consequence of the exaggerated boosting of biochemical mechanisms, which are triggered by potentially dangerous key-signals attempting to maximize neuronal plasticity [23]. It is well established that glutamate and dopamine are involved in neural plasticity hence in learning and memory, while A and -Syn not only affect synaptic plasticity (hence learning processes), but also play an important role in the control of glutamate and dopamine synaptic function, respectively [23]. As a matter of fact, it has been shown that AMPA antagonists inhibit A internalization [195] and glutamate receptor 2/3 (GluR2/3) subunit of AMPA receptors coimmunoprecipitated with A oligomers [195], implicating a role for AMPA receptors as an A carrier. Other data have also demonstrated endocytosis of AMPA receptors induced by A oligomers [196,197], leading to the speculation that endogenous A at physiological levels may have essential roles in the maintenance at the proper level the synaptic decoding mechanisms for excitatory signals [28]. Hence it is possible that APP at synaptic membranes promotes surface expression and stabilization of glutamate receptors, while cleavage of APP and synaptic release of A would promote opposite effects. In any case, the uptake of A by glutamate receptors may serve as a regulatory mechanism that prevents A-induced synaptic depression. Another neurotransmitter receptor implicated in the uptake of A is the 7 nicotinic cholinergic receptor (7nChR) for the neurotransmitter acetylcholine. The role of acetylcho-


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line and its receptors have received continuous attention in the AD field due to the high susceptibility of cholinergic neurons to degenerate in AD pathology [198]. Several hypotheses have been proposed to explain this regional transmitter-specific vulnerability based either on a modulation by cholinergic signalling of APP processing or, conversely, that A can affect acetylcholine release from the pre-synaptic terminal as well as signalling of nicotinic receptors in the post-synaptic compartment [198]. Also prions can have similar actions on neurotransmission as pointed out by Khosravani [199] discussing the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. It has been shown that PrP-null mouse neurons exhibit enhanced and drastically prolonged Nmethyl-D-aspartate (NMDA) – evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the over-expression of exogenous PrPc. The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrPc mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits. Consistent with this finding, in vitro and in vivo excitotoxicity assays reveal increased neuronal cell death in PrPnull cultures and animals upon transient exposure to NMDA. The ultimate pathogenic mechanism due to prions is not well clarified and only three recent experimental studies on such a subject will be reported: -

Prion infection causes iron imbalance in diseased human, hamster, and mouse brains as revealed by a phenotype of iron deficiency in the presence of increased total iron. An important underlying cause of iron imbalance is sequestration of cellular iron in detergent insoluble PrPScferritin complexes, thus rendering it bio-unavailable and creating a phenotype of apparent iron deficiency. Consequent up-regulation of iron uptake protein Tf and TfR (transferrin receptor) result in increased cellular iron uptake, creating a state of cellular iron imbalance. Since iron is potentially toxic due to its redox-active nature, these observations have significant implications for prion disease associated neurotoxicity [200].

-

Prion-related disorders can be caused by alterations in protein folding and quality control mechanisms at the endoplasmic reticulum (ER). It has been reported that alterations in ER calcium homeostasis are common pathological events observed in both infectious and familial Prion disease models. Perturbation in calcium homeostasis directly correlates with the occurrence of ER stress and higher susceptibility to protein folding stress. Torres and collaborators envision a model where alterations in ER function are central and common events underlying prion pathogenesis, leading to general alterations on protein homeostasis networks [201].

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A protective cellular mechanism triggered by rising levels of misfolded proteins is the transient shutdown of protein translation, through phosphorylation of the -subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased


eIF2-P levels are seen not only in prion diseases but also in patients with AD, PD. Moreno and collaborators [202] have shown that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2-P, associated with synaptic failure and neuronal loss in priondiseased mice. Furthermore, they have shown that promoting translational recovery in hippocampi of prioninfected mice is neuroprotective. Thus, a common pathogenic mechanism in several CPDs is the resulting chronic blockade of protein synthesis, which leads to synaptic failure, spongiosis and neuronal loss. Their important findings suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders [202]. Summing up, as shown in the present paper and discussed in the literature, CPD involved proteins play a multifunctional role and should be considered, inter alia, as ‘‘essential synaptic proteins’’ [39,203-207]. However, during aging, these substances, while becoming less effective in mediating synaptic control and hence learning (i.e. their physiologic actions), seem to play an increasing role in neurodegeneration [39,208-210]. Therefore, these proteins because of their altered conformation and/or because of their concentration and metabolic fate (i.e., mis-exaptated) can trigger the following pathogenic mechanisms: • Alterations in “plasma membrane windows” and hence in both intra- extra-cellular exchange of signals and in

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GMN geometry and function which can affect VT and WT; • Deregulation of synaptic structure and function due to mis-exaptation of key-signals involved in synaptic plasticity and learning [23]. In particular, boosting the actions of excitotoxic amino acids and potentially dangerous neurotransmitters (such DA), and disturbing the cholinergic neurotransmission; • Mis-tinkering of the Energide/Mitochondria endosymbiotic complex that may be cause or consequence of the two previous ones pathogenic mechanisms. According to the present hypothesis a final outcome of all these pathogenic mechanisms are: • Alterations in the integrative actions of SCs that are of basic importance for the FM integrative actions. These alterations in a first phase reduce the cognitive capabilities while in a more advanced phase cause neurodegenerations. Many aspects of these features can be integrated in a scheme on CPDs genesis as discussed in the following Section and illustrated in Fig. (3). CONCLUSIONS The most influential theory to explain the pathogenesis of AD has been the “Amyloid Cascade Hypothesis” (ACH) first formulated in 1992 [110]. The ACH proposes that the deposition of -amyloid (A) is the initial pathological event in AD leading to the cascade of pathogenic events: formation of

Fig. (3). Schematic overview of the present hypothesis that is based on the new concepts of mis-exaptation and mis-tinkering and it refers to the “Red Queen of Aging” [13]. For further details see text.


senile plaques (SPs) and then of neurofibrillary tangles (NFTs), death of neurons, and ultimately dementia.

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There are, however, two limitations of the ACH:

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SP and NFT may develop independently from each other

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SPs and NFTs may be the products rather than the causes of neurodegeneration in AD hence the cause-effect link is not clear and it is possible that another pathogenic phenomenon is behind both SPs/NFTs genesis and neurodegeneration.

[10]

A modification to the ACH has proposed which may better explain the pathogenesis of AD, especially in late-onset cases of the disease and suggests a number of predictions which could be usefully investigated [211]. The present paper points to a further modification of the ACH that has for starting assumptions the Armstrong’s scheme and takes into account Armostrong’s comment [211] and Arendt careful historical panorama [38] that points to a well established datum: there is an association between neurodegeneration and an overwhelming presence of conformationally altered proteins, but the real cause-effect link is still not completely elucidated.

[11]

The proposed hypothesis is illustrated in Fig. (3) and introduces the main aspects of our proposal, i.e., the exaptation/mis-exaptation concept that could explain the Dr. Jekyll/Mr. Hyde conundrum of the potentially dangerous proteins and the mis-tinkering of crucially important contraptions, in particular of the Energide/Mitochondria symbiotic contraption. These phenomena could be due to different pathogenic mechanisms such as overloads due to abnormally high energy requests, exogenous and/or endogenous toxic influences or aging. The final outcome of all these phenomena are more or less marked alterations in TUs and FMs, especially in SCs, leading to cognitive decline and neurodegeneration.

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CONFLICT OF INTEREST

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[13] [14]

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[17] [18] [19] [20] [21]

The author(s) confirm that this article content has no conflicts of interest.

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ACKNOWLEDGEMENTS

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This work was supported by a grant from the Swedish Research Council (04X-715) to KF. REFERENCES [1] [2] [3] [4]

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