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Review

Protein aggregation and prionopathies Agre´gation de prote´ines et prionopathies M. Renner a,*, R. Melki b a b

Biologie cellulaire de la synapse, institut de biologie de l’E´cole normale supe´rieure (IBENS), Inserm U1024 – CNRS 8197, 46, rue d’Ulm, 75005 Paris, France Laboratoire d’enzymologie et biochimie structurales, CNRS UPR 3082, baˆtiment 34, avenue de la Terrasse, 91198 Gif-sur-Yvette, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 December 2013 Accepted 28 January 2014 Available online xxx

Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are ‘‘transmissible’’ between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob’s disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays. ß 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Prion Alzheimer Parkinson Huntington Alpha-synucleinopathies Tauopathies Amyotrophic lateral sclerosis Protein aggregation

R E´ S U M E´

Mots cle´s : Prion Alzheimer Parkinson Huntington Alpha-synucle´inopathies Tauopathies Scle´rose late´rale amyotrophique Agre´gation des prote´ines

La prote´ine prion et les prote´ines qui ont des proprie´te´s de type prion partagent un certain nombre de caracte´ristiques. Du point de vue mole´culaire, ces prote´ines constitutives s’assemblent en particules prote´iques insolubles qui e´chappent a` la machinerie cellulaire de de´gradation des prote´ines a` la suite de changements conformationnels. Ces particules s’amplifient par le recrutement de la forme soluble des prote´ines qui les constituent. Ces agre´gats prote´iques sont responsables d’un certain nombre de maladies neurode´ge´ne´ratives comme les maladies de Creutzfeldt-Jacob, d’Alzheimer, de Parkinson et de Huntington. De nombreuses e´vidences soutiennent la propagation de ces agre´gats prote´iques et sugge`rent qu’ils sont « transmissibles » entre cellules. Il existe aussi des donne´es qui montrent que des homoge´nats de cerveaux d’individus de´veloppant les maladies d’Alzheimer ou de Parkinson propagent ces maladies dans des animaux mode`les tout comme les homoge´nats de cerveaux de patients de´veloppant la maladie de Creutzfeldt-Jacob. De ce fait, la propagation des agre´gats prote´iques de cellule a` cellule pourrait eˆtre un phe´nome`ne ge´ne´rique qui contribue a` l’e´volution des maladies neurode´ge´ne´ratives ce qui pourrait avoir des conse´quences importantes dans ces maladies. De plus, alors que la distribution des agre´gats prote´iques est caracte´ristique pour chaque maladie, de nouvelles e´vidences sugge`rent un chevauchement/interfe´rence de ces agre´gats prote´iques dans les diffe´rents de´sordres neurologiques. Malgre´ les nombreuses e´vidences qui soulignent l’importance de la propagation de type prion des agre´gats prote´iques implique´s dans les maladies neurode´ge´ne´ratives, il persiste de nombreuses questions sans re´ponses concernant les me´canismes a` l’origine la toxicite´ de ces particules qui constitue a` pre´sent un domaine de recherche intense. ß 2014 Elsevier Masson SAS. Tous droits re´serve´s.

* Corresponding author. E-mail address: [email protected] (M. Renner). http://dx.doi.org/10.1016/j.patbio.2014.01.003 0369-8114/ß 2014 Elsevier Masson SAS. All rights reserved.

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1. Introduction Several progressive neurodegenerative disorders are the consequence of protein misfolding and aggregation (reviewed in [1–3]). Indeed, Creutzfeldt-Jacob (CJD), Alzheimer (AD), Parkinson (PD) and Huntington diseases are associated with the aggregation of aberrantly folded proteins or imperfectly degraded peptides into insoluble amyloid polymers, which are highly ordered fibrilar aggregates with elevated b-sheet content. In a first step, these proteins aggregate into oligomers that are diffusible and nonfibrillar in nature. Further aggregation leads to larger polymers that compose the inclusion bodies or extracellular deposits (plaques) that characterize these disorders [4]. More importantly, once aggregated these proteins can grow and amplify by recruitment of their soluble counterparts [3–5]. According to the nucleated polymerization model, seeds composed of abnormally folded proteins template the conformational transition of soluble cognate proteins. This seeding capacity, together with the ability to grow indefinitely and the resistance to degradation, confer to amyloid fibrils the capacity to propagate and is central to the infectious protein (prion) concept. The correlation between protein aggregation and pathology is undeniable. Even if the mechanisms through which these aggregates damage cells remain elusive, the pathologies are most likely due to a gain of toxic properties associated to misfolding [6,7]. The production of these aberrant protein conformers is favoured by chemical or environmental stress and countered by molecular chaperones and cellular proteolytic activities (reviewed in [8,9]). The age-related decline in protein homeostasis challenges the capacity of cells to counteract the accumulation of misfolded proteins. This may explain in part the late onset of amyloidosis [9]. The continued accumulation of misfolded proteins probably defies the capacity of the lysosomal and proteasomal clearance systems, promoting further protein accumulation and the development of a self-propagating cycle [10]. It is not clear whether fibrils and plaques are the toxic species in all neurodegenerative diseases or if they result from a defensive response aimed at protecting cells from more toxic oligomeric species. Even if the aggregation is common to several neurodegenerative diseases, the different proteins involved and their subcellular location probably leads to differences in the toxicity of the different aggregated forms. Notably, some large-order aggregates are extracellular (plaques in AD) and some others, intracellular (inclusion bodies in PD). Intracellular aggregates could be more toxic because of their ability to:  sequester essential cellular components, in particular molecular chaperones;  generate oxidative species;  inhibit proteasomal activity [11].

The polypeptides responsible for AD, PD or Huntinton’s disease are well known. Despite the differences between these polypeptides in terms of sequence and biosynthesis, the three diseases share surprisingly similar pathological pathways. In particular,

neuronal degeneration spreads through interconnected brain regions [5,12–15]. This feature is compatible with a model where the misfolded proteins might spread from a site of onset to adjacent cells, recruiting their endogenously expressed counterparts to induce pathology throughout the nervous system [16]. The seeding of intracellular proteins assemblies requires seed export from affected cells and import by healthy cells. A few years back the idea that protein aggregates could penetrate inside cells was probably as heretic as the prion concept once was. However, there are increasing evidences that misfolded proteins are released in the extracellular medium and that they can enter cells (Table 1). Cells can release aggregates either by exocytic process or passively, either by local rupture of the membrane or after cell lysis [3]. Internalization mechanisms probably differ depending on the size and structure of the protein aggregate and on the cell-type. Tunnelling nanotubes (50–200 nm diameter hollow filaments linking cells) also constitute a passage route for protein aggregates [17,18]. The similarities between prions and other misfolded proteins responsible for neurodegenerative diseases make it tempting to consider all these proteins as prions. However, there is one crucial difference between prions and all other amyloids: prions are infectious agents that are transmissible between individuals. Other self-aggregating proteins have not been so far shown to propagate within communities and to cause macroepidemics such as Kuru and bovine spongiform encephalopathy. This is why they have been called ‘‘prionoids’’ [16] or prion-like [2,3,19] so far.

2. Prion diseases Prion diseases, also called transmissible spongiform encephalopathies, are rare fatal neurodegenerative diseases with genetic, sporadic and acquired forms. They include bovine encephalopathy, scrapie and Creutzfeldt – Jakob disease in humans. These diseases are characterized by neuronal loss, gliosis and spongiform changes in the brain leading to dementia and ataxia (reviewed in [20–22]). Prionopathies are tightly associated to the aggregation of a 23 kDa constitutive protein with unknown function named PrP. The key causative event in neurodegeneration is the conversion of the normal prion protein PrPC into a disease-associated form that resists limited proteolysis, PrPRes. The conversion process is thought to be autocatalytic and implies refolding and aggregation of PrPC to form PrPRes and/or the failure of quality control mechanisms for PrPRes suppression or degradation [20]. This conversion, which is rare and certainly stochastic, takes place at the plasma membrane. After formation, PrPRes is rapidly internalized [23] and either recycled to the plasma membrane or transported to the Golgi apparatus. During the early stages of prion infection PrPRes is mostly cleared within the lysosomes [23]. The fraction of PrPRes that escapes clearance persists in cells and templates PrPC polymerization into amyloid fibrils and coalescence into plaques [20–24]. Prion diseases can be genetic, sporadic or infectious. Sporadic and genetic forms develop endogenously following the spontaneous

Table 1 Propagation of misfolded proteins associated with neurodegenerative diseases. Pathology

Main implicated protein

Deposits

Localization of deposits

Trancellular propagation?

In vivo propagation?

Prion diseases Parkinson disease Alzheimer disease Tauopathies Amyotrophic lateral sclerosis Huntinton disease

PrPres a-synuclein Amyloid b peptide Tau SOD1 Htt with polyglutamine expansion

Amyloids Lewy bodies Amyloid plaques Neurofibrillary tangles Inclusion bodies Inclusion bodies

Extracellular Intracellular Extracellular Intracellular Intracellular Intracellular

Yes Yes Yes Yes Yes Yes

Yes [19,20] Yes [38,39,41] Yes [66,67,69–71] Yes [78,79] Not reported Not reported

[17,26,27] [38,41,45,49] [72] [76,80] [86] [93]

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Fig. 1. Natively unfolded a-synuclein (A), adopts mostly an a-helical conformation upon interaction with the cell membrane (B). The protein can also populate a b-strand rich conformation that pile into toxic fibrillar assemblies (C).

misfolding, aggregation and seeded assembly of PrP molecules. Genetic forms are associated with mutations of the prion protein gene, PRNP, which result in an increased likelihood of conversion to the pathogenic form. More importantly, prions are also infectious and transmit within or even between species. PrPRes propagates/recycles in cultured cells by direct cell-to-cell contact, for example by nanotubes [17,24] or by release of prion infectivity into the medium, likely by exosomes [25,26] and uptake through endocytosis [27–29]. 3. Parkinson’s and related diseases PD is a movement disorder primarily associated to the degeneration of dopaminergic neurons in substantia nigra and other brainstem regions. The neuropathological hallmark of PD is the formation of proteinaceous intraneuronal inclusions, named Lewy bodies (LBs) (reviewed in [30]). Fibrillar aggregates of asynuclein (a-syn) are the primary component of LBs, which are also found in many others neurological disorders (reviewed in [31]). Familial forms of early PD are associated with a-syn mutations and gene amplification (reviewed in [32]). a-syn is a small (14 kDa) protein found in cells throughout the nervous system that binds avidly to membranes. Its normal function appears to involve membrane interactions such as the binding to synaptic vesicle membranes [33] contributing to the function of presynaptic terminals (reviewed in [34]). a-syn displays a remarkable structural variety, from unfolded monomers to amyloid fibrils. When associated with membranes, a-syn adopts an a-helical conformation, but under pathologic conditions it populates b-sheet-rich protein folding intermediates that are prone to assemble into fibrils (Fig. 1, reviewed in [30]). The specific signal that triggers a-syn misfolding and aggregation is unknown. It is proposed that stochastic misfolding occurs at low frequency. This, together with decreased capacity of cells unfolding protein response with time could account for the agedependent onset of PD. The accumulation of aggregated a-syn affects various functional structures of the nervous system leading to serious cognitive and behavioural alterations. This accumulation of a-syn follows an ascending and predictable pattern of progression, spreading from the lower brainstem and olfactory bulb into the limbic systems and finally the neocortex [35]. Classically considered an intracellular protein, a-syn, in particular in its aggregated form, is capable of transfer between cells leading to current consideration of a prion-like mechanism operating in PD pathology spread (reviewed in [3,36,37]). Indeed, classical Lewy Bodies were shown to form in healthy human fetal midbrain tissue transplanted into the brains of PD patients [38,39]. a-syn deposition and aggregation appeared in the fetal grafts following implantation into the PD brain. The grafted cells manifested Lewy bodies, Lewy neurites and neuronal dysfunction after approximately 10–14 years. Studies on grafted

patients dying sooner (1–5 years after transplant surgery) did not reveal any protein aggregates in the grafted neurons. This surprising observation indicates that the environment of the PD patients’ brains induce normally soluble a-syn to convert to a highly aggregated pathologic form. A plausible mechanism for this would be if aggregated a-syn, released from diseased neurons could infect neighbouring healthy tissue [38,39]. Indeed, a-syn can be found extracellularly, for example, in the cerebro-spinal fluid [40]. Additional evidences for the ability of a-syn to propagate within the brain come from experiments in rodents, in which neuronal stem cells or dopamine neurons were grafted into the hippocampi or striata [41–43]. As observed in grafted Parkinson’s disease patients, animals sacrificed following transplantation showed human a-syn aggregates and inclusions in grafted embryonic cells. Further, a-syn transferred from host cells was demonstrated to seed the aggregation of wild-type a-syn in healthy implanted cells [42]. Thus, a-syn aggregates can spread and seed aggregation in the brain (reviewed in [3,10]). How can aggregated a-syn spread within the brain? a-syn can be secreted from cells by an endoplasmic reticulum/Golgiindependent exocytosis. The secreted a-syn, which is thought to be more prone to aggregation than the cytosolic protein, is found inside the lumen of vesicles [44] and could be released in the medium or exported within vesicles that can be uptaken by other cells. Exogenous a-syn was also demonstrated to be internalized by cells in vitro. Indeed, fluorescently-tagged a-syn added to the culture medium was found inside the cells after 24-48 hrs of incubation [38,41]. Altogether these observations strongly suggest that a-syn traffic between cells. In agreement with this, neuron-toneuron transmission of a-syn fibrils following axonal transport has been recently demonstrated [45]. The sequence of events that let internalized a-syn to reach the cytoplasm is not known. One possibility is the interaction of a-syn with membranes that could allow the crossing of the plasmamembrane directly and/or the escape from the lumen of vesicles [46]. Interestingly, some but not all types of a-syn oligomers derived from recombinant protein were capable of inducing intracellular wild-type and mutant a-syn inclusions [47]. Some types of oligomers induced cell death via disruption of cellular ion homeostasis at the level of the plasma-membrane whereas other oligomer types enter the cell and increased a-syn aggregation. The ability of a-syn fibrils to bind and permeabilize membrane is certainly associated to their toxicity, as their binding to the plasma membrane in cultured cells alters Ca2+ homeostasis inducing apoptosis in cultured cells [48]. Preformed fibrils of a-syn also can enter neurons, promoting recruitment of soluble endogenous asyn into insoluble PD-like LBs [49]. Most interesting is finally the finding that structurally distinct fibrillar a-syn assemblies exhibit different toxicity and seeding of endogenous a-syn properties [50]. The inoculation of aggregated a-syn-containing preparations into the brain induces or accelerates the formation of a-syn

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aggregates. Both aggregation and PD symptoms were transmitted from symptomatic to asymptomatic a-syn transgenic mice upon inoculation of brain homogenate containing aggregates [51,52]. Formation of these PD-like a-syn inclusions causes selective reductions in synaptic proteins, and progressive impairments in neuronal network function that culminate in neuron death [49]. Furthermore, a-syn fibrils assembled from recombinant protein were also shown to induce the formation of LBs following their injection in the brain of recipient mice [52]. Altogether these evidences strongly suggest that a-syn assemblies indeed traffic between cells within the central nervous system in a manner akin prions, thus certainly contributing to the evolution of PD.

4. Alzheimer’s disease Alzheimer’s disease (AD) is characterized by the loss of synapses and neurons in the cerebral cortex and certain subcortical regions. The classical neuropathological landmarks in AD are the aggregated forms of two different proteins Tau and b-amyloid peptide (Ab) into neurofibrillary tangles (see next section) and amyloid plaques, respectively [53]. Amyloid plaques are extracellular insoluble aggregates of Ab. Ab is produced by the cleavage of the extracellular portion and part of the transmembrane region of the amyloid precursor protein (APP). Once in solution, Ab oligomerizes into soluble globular species (3–4 up to 24 monomers). Further aggregation leads to the formation of fibrils and amyloid plaques (reviewed in [54]). Most cases of Alzheimer’s disease are idiopathic, although mutations in the gene encoding APP or in the enzymes that sequentially cleave APP cause inherited forms of Alzheimer’s disease (reviewed in [54]). As for PD, AD pathology propagates in a stepwise manner. Both plaques and tangles appear first in subcortical structures, spreading along cortico-cortical projections (reviewed in [55]). There are compelling evidences that Ab oligomers are responsible for the initial network failure that underlie the loss of memory and cognitive deficiencies [56–58]). Although their pathological capabilities are not fully understood, Ab oligomers have been shown to interact with particular synapses at the level of the plasma-membrane to perturb proper synaptic function, structure and maintenance ([58], reviewed in [59]). Interestingly, the same effects of Ab oligomers on the metabotropic receptor mGluR5 are observed in neurons and in glial cells [58,60]. Moreover, mGluR5 is overexpressed in reactive astrocytes surrounding Ab plaques in an Alzheimer’s disease mouse model [60]. Therefore glial cells may also be implicated in the early deleterious effects of Ab oligomers. The general consensus is that amyloid plaques are not toxic per se, however in Alzheimer mouse models, the neurons surrounding amyloid plaques display hyperactivity and altered calcium homeostasis [61,62], suggesting that the plaques can be a source of toxic oligomers and/or fibrils that can alter the neighboring cells. Despite the synaptotoxic effect of Ab oligomers, they may not be sufficient to lead to AD dementia. Cell death may depend on intracellular toxic effects such as mitochondrial dysfunction [63] and Tau protein aggregation [64,65]. Arguments in favor of considering Ab as a peptide with prionlike properties come from experiments in which human AD brain homogenates were inoculated intracerebrally into transgenic mice [66]. In mice overexpressing APP, injections of b-amyloid extracts derived from brains of Alzheimer’s disease patients accelerated the deposition of Ab [66]. The disease agents seem to be solely Ab, as the injection of synthetic Ab peptides also provoked a widespread amyloidosis [67,68]. Interestingly, synthetic peptides were less pathogenic, probably due to their lower resistance to proteolysis

by proteinase K [67]. Moreover, the transmission of the amyloid pathology was also observed after intraperitoneal inoculation of Ab-containing homogenates [69]. It is important to note that these experiments involved the extracellular deposition of b-amyloid and not intracellular seeding, making them different from the other neurodegenerative diseases discussed here. However the finding that variants of Ab seeds might govern the type of Ab aggregates further supports the concept of prion-like template misfolding of Ab [70]. This might also explain the heterogeneous morphology, pathogenicity and progression of Ab lesions and associated pathologies in AD. Recent evidences suggest trans-synaptic Ab propagation [71]. Indeed, the overexpression of mutant APP in the entorhinal cortex (one of the earliest targets of AD) caused cognitive and behavioral abnormalities as well as synaptic dysfunctions in the dentate gyrus and parietal cortex suggesting trans-synaptic propagation of neuronal dysfunction from entorhinal cortex through hippocampal and cortical networks. This view is supported by the demonstration of Ab assemblies’ propagation in cultured cell lines and neurons [45,72]. 5. Tauopathies Tauopathies are a group of neurodegenerative diseases, including AD and Picks disease, characterized by Tau protein aggregation in neurons and glial cells (reviewed in [73]. Normal Tau is a natively unfolded protein that modulates microtubules dynamics and stability in neuronal cells. It is thought that hyperphosphorylated Tau tends to aggregate into intracellular tangles of paired helical and straight filaments, named neurofibrillary tangles [73]. As for Ab plaques, Tau depositions follow stereotypical patterns. The development of the pathology seems to follow connections anterogradely [74], affecting the hippocampus, limbic structures, the basal nucleus of Meynert and the brainstem. The neocortex is not affected until the late stages of the disease [75]. In vitro, recombinant Tau microtubule binding region and full-length Tau fibrils seed endogenous Tau aggregation and the formation of filamentous inclusions resembling neurofibrillary tangles [76,77]. In addition, mouse brain wild-type Tau aggregation was triggered by the injection of mutant human Tau. The pathology spreads from the injection site to neighboring brain regions strongly suggesting that proteinmisfolding spreads within the recipient animal brain [78]. Using a different strategy, de Calignon and collaborators [79] described the propagation of Tau pathology from the entorhinal cortex, where mutant human Tau was overexpressed, to other brain regions connected synaptically. Finally, trans-cellular transmission was reported as intracellular Tau fibrils were shown to be released to the extracellular medium and to be taken up by co-cultured cells where they induced Tau aggregation [80]. All these observations, together with the finding that antibodies directed against Tau interfere with propagation suggests that Tau assemblies have prion-like properties. 6. Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is a member of a group of heterogeneous disorders that affect motor pathways. Just like AD and PD, only a proportion (10%) of ALS is dominantly inherited, with the remaining 90% sporadic [81]). Mutations in the Cu/Zn superoxide dismutase (SOD1), together with mutations affecting protein homeostasis are the main causes of ALS [82,83]). How mutant SOD1 leads to motor neuron degeneration remains unclear. However, SOD1-mediated toxicity is not due to loss of

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function but instead to a gain of toxic functions that are independent of the levels of SOD1 activity. Common to all ALS (as well as in mouse models of SOD1-mediated disease) are the prominent cytoplasmic inclusions in motor neurons and in some cases within the astrocytes surrounding them (reviewed in [82]). In ALS, motor neuron degeneration is focal and spreads to contiguous regions [84]. Furthermore, mutant SOD1 aggregates can be transferred from cell to cell by a process that depends on their extracellular release (reviewed in [85]). More importantly, once in the neuronal cytosol, mutant SOD1 aggregates seed the aggregation of the normally soluble, endogenous mutant SOD1. Aggregates have also been shown to self-propagate in cultured cells and in vitro [86,87] suggesting that SOD1 aggregates involved in ALS have prion-like properties. 7. Huntington’s disease The autosomal dominant inheritance of Huntington’s disease is determined by a mutation in the gene encoding the huntingtin protein (Htt). An expanded CAG triplet repeat leads to an expanded polyglutamine (polyQ) stretch in Htt which makes the protein prone to aggregate and to form intraneuronal inclusion bodies (reviewed in [88]). The length of the polyQ tract defines the aggregation propensity of Htt as well as the age of the onset of the disease. The formation of cytosolic or nucleoplasmic inclusions is drastically increased when Htt polyQ tract exceeds 37 Q [88]. Aggregates composed of fluorescent synthetic polyQ peptides are taken up by cultured cells [89] and recruit non-pathogenic soluble polyQ proteins into inclusions in mammalian cells [90,91]. PolyQ containing protein aggregates bind rapidly and tightly to the plasma membrane and enter the cells through a non-conventional mechanism. The binding to the membrane depends on the fibrilar structure of the assemblies [92] and as in the case of a-syn, Htt fibrils (and not oligomers) are highly toxic to cultured cells and induce apoptotic cell death, most probably by disrupting plasmamembrane integrity [48]. Electron micrographs of unroofed cultured cells shortly after their exposure to PolyQ aggregates revealed the presence of ‘‘naked’’ aggregates pointing toward the cytosol and suggesting that the aggregates do not cross the plasma membrane through vesicles but instead by breaching it [93]. Most interesting was the finding that Htt aggregates released within the medium upon cell death are taken up by cells expressing sub-pathologic Htt and that those aggregates seed the assembly of the soluble endogenous Htt. Thus, Htt assemblies also exhibit prion-like properties. These findings strongly suggest that protein aggregates associated to neurodegeneration are ‘‘transmissible’’ and that the propagation of protein aggregates from cell to cell may be a generic phenomenon at the origin of neurodegenerative diseases which has important consequences on human health issues. 8. Crosstalk between protein aggregates associated to neurodegeneration Although the location of protein aggregates involved in neurodegeneration is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Interactions between Ab, Tau and a-syn may account for overlapping AD, PD and tauopathies. Thus, these diseases could be triggered by a common stimulus, with outcomes depending on genetic background or environmental factors (reviewed in [94]). Indeed, Ab and Tau could act synergistically with Ab oligomers formation driving AD pathogenesis and triggering the chain of events leading to tauopathy, neuronal dysfunction and dementia (reviewed in [94]). AD and prions

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pathologies seem also to overlap. The inoculation of prions in an AD mice model that develop amyloid plaques significantly accelerated and exacerbated both pathologies [95]. Intriguingly, PrPC is a high-affinity receptor for Ab oligomers mediating their toxicity on synaptic plasticity [96–98] and the metabotropic glutamate receptor mGluR5 is implicated in the synaptotoxicity of Ab oligomers [58] as well as in PrPC-Ab oligomers interaction [98]. Other studies suggest that Ab is likely to promote the aggregation of a-syn enhancing its damaging effect on synapses [99,100]. Some protein aggregates may act as heterologous templates for the misfolding and aggregation of dissimilar proteins. Poly-Q aggregates, for example, have been reported to seed the aggregation of transactivation-responsive DNA-binding protein 43 (TDP43), a component of pathological inclusions in ALS, in cultured cells [101] and these two protein co-localize in patients with Huntington’s disease [102] or spinocerebellar ataxia [103]. However, cross seeding is not a generic rule and the cross talk between protein aggregates involved in neurodegeneration appears to involve other players.

9. Therapeutic implications The prion protein and those proteins that exhibit prion-like properties share a number of characteristics. These are all constitutive proteins that aggregate into insoluble protein particles that resist the cellular clearance machinery. They all amplify by breakage of the aggregates and recruitment of the precursor proteins from the cellular, soluble, pool of proteins. Those protein aggregates traffic between cells and possess toxic properties leading to neurodegeneration. The aggregation of those proteins is spontaneous with a strong dependence on polymorphism and ageing. Finally and most interesting, strains with distinct elongation/persistence properties have been reported. While the aggregated form of PrP (PrPRes) is infectious with reported contaminations cases through surgery, tissue transplant and food, no evidence for propagation of AD, PD HD and ALS through the same routes have been brought so far. Nonetheless, the ability of those proteins that exhibit prion-like properties to propagate between cells in a manner similar to prions raises concerns. In addition, the fact that these protein assemblies propagate within the central nervous system certainly contributes to neuronal loss and pathology. Thus, tools that interfere and/or halt protein aggregates intercellular spread within the central nervous system have potential therapeutic properties. Several strategies may be implemented to block the propagation of protein assemblies involved in neurodegeneration. The first consists of identifying the receptor of those protein assemblies so that they are made unavailable for protein aggregates anchoring to the cell membrane. A second strategy may consist of affecting endocytosis and autophagy rates. In both cases, pleiotropic effects are expected. A third strategy consists of sequestering outside the cells the protein aggregates during their transit between cells. This can be achieved by changing their surface properties in such a way that they are no more capable of binding to and being internalized by neurons. This strategy has the advantage of targeting specifically the toxic protein aggregates. One possible approach is to change the surface properties of the protein aggregates using specific antibodies [80,104]. The fact that the permeability of blood-brain barrier to large macromolecules such as antibodies is limited, constitutes a serious drawback. A related approach consists of using smaller macromolecules that bind toxic aggregates. Molecular chaperones have been shown to bind tightly fibrillar protein assemblies and to diminish their toxicity [105]. As such protein may not reach the target tissue an alternative consists of using smaller polypeptides derived from

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macromolecules that bind and affect the surface properties of toxic protein aggregates. Identification of the surface interfaces involved in ligand-toxic protein aggregates is a prerequisite for the design of such molecules. Such approaches have been initiated and should yield promising molecules [106]. Last, as cell-to-cell transfer implicates the presence of the misfolded proteins outside the cell, the clearance of these species in the extracellular space might be an effective way to reduce cytotoxicity. Plasmin is an extracellular serine protease derived from its inactive form plasminogen. Plasmin, which is present in the central nervous system, cleaves a variety of extracellular substrates such as fibrin, fibronectin, laminin, and matrix metalloproteinases (MMPs) [107]. Interestingly, plasmin degrade Ab [108] as well as a-syn [109] and strategies aimed at using this protease to clear extracellular toxic aggregates may have therapeutic potential. In the case of Ab aggregates, three strategies based on reducing the amount of aggregates have reached phase II clinical trials [110]. One aims the inhibition of Ab aggregation through small molecules binding to monomeric Ab (Neurochem’s ‘Alzhmed’ [tramiprosate]). This approach led to a reduction in amyloid-plaque formation in mouse models and rescued some AD-like phenotypes. Another strategy involves the reduction of Ab production by modulating the enzymes responsible for the cleavage of APP. Finally, Elan’s Ab vaccine (inoculation with an Ab-derived immunogen) also resulted in the reduction of amyloid plaques and rescue of some pathological phenotypes. Despite the increasing evidences that support prion-like propagation of protein aggregates involved in neurodegenerative diseases, there are many unanswered questions regarding the mechanisms of toxicity. In order to develop more efficient therapeutic strategies, it is necessary to identify the aggregation state of the misfolded proteins that are released. Another important issue is whether the amount of secreted proteins present in interstitial fluid or cerebrospinal fluid is enough to induce pathology under physiological levels of protein expression, or whether there is another key factor for toxicity such as neuroinflammation. Finally, it is important to investigate the origin for neuronal vulnerability. A multi-hit paradigm has been proposed to explain the age dependence and regional specificity of neurodegeneration for a specific disorder. In this scheme, the dysfunction of cell-cell interactions may act synergistically to precipitate neuronal death. In other words, protein misfolding and aggregation results in cell type specific dysfunctions, which individually cannot explain the full spectrum of disease symptoms, but in concert with network failure over time, will result in the distinct patterns of neurological dysfunction and/or neurodegeneration that characterize a given disorder [111].

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Please cite this article in press as: Renner M, Melki R. Protein aggregation and prionopathies. Pathol Biol (Paris) (2014), http:// dx.doi.org/10.1016/j.patbio.2014.01.003