Molecular mechanisms of neurodegeneration mediated by dysfunctional subcellular organelles in transmissible spongiform encephalopathies

Abstract

Transmissible spongiform encephalopathies refer to a group of infectious neurodegenerative diseases with an entirely novel mechanism of transmission and pathophysiology including synaptic damage, dendritic atrophy, vacuolization, and microglial activation. Extensive neuronal loss is the main cause of chronic brain deterioration and fatal outcome of prion diseases. As the final outcome of pathological alterations, neuronal death is a prominent feature of all prion diseases. The mechanisms responsible for prion diseases are not well understood. A more comprehensive understanding of the molecular basis of neuronal damage is essential for the development of an effective therapy for transmissible spongiform encephalopathies and other neurodegenerative diseases sharing similar features. Here, we review the molecular mechanisms of mitochondrial dysfunction and endoplasmic reticulum stress-mediated neuronal death, which play crucial roles in the pathogenisis of prion diseases.

prion disease, autophagy, neuronal cell death, endoplasmic reticulum stress, mitochondrial dysfunction

Introduction

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of neurodegenerative disorders including kuru [1,2], Creutzfeldt-Jakob disease (CJD) [3], Gerstmann-Sträussler-Scheinker (GSS) disease [4], and fatal familial insomnia (FFI) in humans [5], natural scrapie in sheep/goats [6–9], and mouflons [10], transmissible mink encephalopathy in ranch-reared mink [11], chronic wasting disease of mule deer and elk in North America [12,13], bovine spongiform encephalopathy or ‘mad cow’ disease [14], and its analogues in several exotic species of antelopes [15–17] and wild felids in zoos [18], and feline spongiform encephalopathy in domestic cats [18]. No treatment is available to halt the progressing of neurodegeneration once PrP^Sc have reached the brain. PrP^Sc, an abnormally structured and aggregated form of the host prion protein (PrP) [19,20], is thought to be the causative agent of TSEs [21–25].

Although the clinical profiles differ among the distinct prion diseases, the characteristics of brain damage are similar, including extensive spongiform degeneration, widespread neuronal loss, synaptic alterations, atypical brain inflammation, and the accumulation of protein aggregates [26,27]. In some cases, GSS patient can be with or without spongiform changes that are associated with the presence of specific prion protein species, while the most striking neuropathology of FFI is neuronal loss and reactive gliosis without abundant PrP^Sc aggregate and widespread spongiform degeneration. Synaptic dysfunction and dendritic loss seem to be responsible for the onset of clinical symptoms. Spongiform degeneration and PrP^Sc accumulation are the most specific neuropathological alterations, and extensive neuronal loss is the main cause of chronic brain deterioration and the fatal outcome of prion disease. Neuronal death is a prominent feature of all prion diseases and there is a good correlation between the type of clinical symptoms observed in each disease and the brain regions exhibiting the greatest extent of cell death. The mechanisms responsible for neuronal death in prion diseases are not well defined and the mechanism of subcellular organelle-mediated cell death is largely unknown. Neuronal survival is critically dependent on mitochondrial and endoplasmic reticulum (ER) integrity based on specific morphological, biochemical, and physiological features. Meanwhile, multiple mechanisms for cell death have been proposed for prion diseases including oxidative stress and mitochondrial dysfunction, accumulation of misfolded aggregates, and ER stress. Consequently, it is not surprising that mitochondrial and ER alterations can promote neuronal degeneration. We will review the roles of subcellular organelles in the induction of neuronal death. Currently, there is no available therapy and the diseases are inevitably fatal. Treatment of patients with established symptoms of prion disease would need to attack the cellular pathways implicated in brain damage. Therefore, a complete understanding of the underlying mechanisms of neurodegeneration in TSE is extremely important.

Neuronal Cell Death in Neurodegeneration of TSEs

The viability of a cell strictly depends on the functional and structural integration of a number of subcellular organelles such as the nucleus, mitochondria, lysosomes, and ER [28]. Each organelle can sense stressful cellular conditions and initiate cellular responses either to adapt or to activate specific cell death signaling pathways if a critical threshold of damage has been reached [29]. In general the process of cell death has two phases: an activator phase mediated by initiator molecules that are up-stream of executor molecules, and are associated with particular cell death stimuli, and a terminal phase that is mediated by executer molecules to which different apoptotic signals converge. These complex processes involve cross-talk among many signaling pathways and include different molecular components that regulate the cell death response. According to a previous review [30], it is now possible to discriminate 11 pathways of cell death occurring in mammals. These types of cell death include (i) necrosis, (ii) apoptosis, (iii) anoikis, (iv) caspase-independent apoptosis, (v) autophagy, (vi) Wallerian degeneration, (vii) excitotoxicity, (viii) erythropoiesis, (ix) platelet cell death, (x) cornification, and (xi) lens cell death. Only some of them can occur in neurons and thus play a role in TSEs.

The cellular pathways leading to neuronal loss in prion diseases are not entirely clear. Researches thus far have indicated that at least two main pathways are implicated in neuronal loss: autophagy and apoptosis. Similar to many other neurodegenerative diseases such as Alzheimer's disease caused by the accumulation of ‘toxic’ proteins, neurons in TSEs die via programed cell death (PCD), of which only the apoptotic process is relatively well characterized. It is understood that autophage plays two distinctively different roles in the central nervous system: protection against apoptosis and detrimental role in cell death, but the mechanisms are largely unknown [31]. There is increasing evidence that autophagy and apoptosis share several common regulatory elements and mechanisms [32,33].

Neuronal Autophagy in Prion Disease

Autophagy is an intracellular degradation system that digests organelles and most long-lived proteins and is also the primary form of nonapoptotic cellular demise to maintain neuronal homeostasis [34]. Cellular quality control through autophagy is particularly relevant in neurons, where the total content of altered proteins and damaged organelles cannot be reduced by redistribution to daughter cells by means of cell division. Neuronal surveillance mechanisms must identify these malfunctioning structures and assure their autophagic degradation before their intracellular buildup gives rise to neurotoxicity [35,36]. Besides neuronal homeostasis, autophagy is also responsible for the continuous remodeling of neuronal terminals that is required to support neuronal plasticity [37–39]. Indeed, induction of autophagy by trehalose or lithium significantly reduced PrP^Sc in a dose- and time-dependent manner in persistently prion-infected neuronal cells [40,41]. Conversely, inhibition of autophagy counteracted the antiprion effect of trehalose. These findings provided evidence that the induction of autophagy enhances the cellular degradation of prions [40]. Nevertheless, as a two-edged sword, neuronal autophagy also results in neuronal accumulation of autophagosomes, which have been described in multiple brain disorders [35,36]. Moreover, neurodegeneration and protein inclusions have been described in mouse models incompetent to perform autophagy in neuronal tissues [42,43]. Failure to induce autophagosome formation results in cytosolic persistence of unsequestered cargo, which could promote aggregation of other intracellular components (acting as an aggregation ‘seed’) or become a source of toxic products (for example, damaged mitochondria may produce reactive oxygen species). Accumulation of protein aggregates, higher content of abnormal [44], nonfunctional mitochondria, deformities of the ER, and an increase in the number and size of lipid droplets have been described in different conditional autophagy-related genes (ATG) knockout mice [37,42,43]. In circumstances of autophagic defects with poor clearance of autophagosomes, accumulation of autophagosomes inside cells can be detrimental for neurons. Data on autophagy in TSEs are very limited [45–51]. Experimentally infected animal models are widely used because of their relatively short incubation periods ranging from 16 to 18 weeks in mice, and 9 to 10 weeks in hamsters for the 263K strain and 24–26 weeks for the 22C-H strain. Initial experiments in hamsters and mice were subsequently extended by studies of human brain biopsies from patients with sporadic CJD, variant CJD, and FFI [48]. Based on the presence of large autophagic vacuoles in the cytoplasm of neurons, Liberski et al. [52,53] proposed that autophagy plays an important role in prion disease neurodegeneration and might participate in the spongiform lesions apparent in TSEs.

Interactions between the autophagic and endocytic pathways could be especially important in the case of prion diseases because endocytosis is a principal route of cellular entry for PrP^Sc [54]. Furthermore, endocytic compartments, specifically multivesicular bodies, can also mediate transmission of the pathogenic protein between cells. Upon fusion of endosomes and plasma membrane, PrP^Sc, located in the luminal vesicles of multi-vesicular bodies, gains access to the extracellular medium in the form of exosomes [54]. In theory, conditions that favor endosomal degradation over endosomal recycling should facilitate elimination of the pathogenic proteins by the lysosomal system. In this scenario, enhanced fusion of autophagosomes with endosomes may reroute the endosomal compartments toward lysosomes. Further investigation is necessary to determine whether the upregulation of macroautophagy with trehalose and lithium is the mechanism behind the lower intracellular levels of PrP^Sc and reduced PrP^Sc propagation [55]. In summary, autophagy certainly does occur in TSE, but its pathogenetic role as a cause of cell death is uncertain. In particular, more research will be necessary to determine the connection, if any, between PCD and the formation of spongiform lesions.

Apoptosis-mediated Neuronal Loss

Studies in humans and in mouse models of prion diseases (infectious models, hereditary models with mutated PrP^C and transgenic models overexpressing wild-type PrP) indicate that neuronal dysfunction and death occur via apoptosis [56–66]. Interestingly, some studies have shown that in post mortem samples of FFI [59] and CJD [60,67] patients, apoptotic cell death of neurons does not correlate well with the deposition of PrP [64]. It was proposed that the dissociation between neuronal damage and the amount of prion deposition reflects variations in the selective neuronal vulnerability to PrP^Sc toxicity [64]. However, it is also possible that neuronal loss associated with PrP^Sc might be more complex than a direct effect of prion on the integrity of neuronal cells.

Apoptosis is a programed form of cell death that plays a central role during development and homeostasis of multicellular organisms and is also implicated in pathological conditions. It can be induced by the activation of plasma membrane death receptors, which constitutes the ‘extrinsic’ pathway, or by the perturbation of intracellular homeostasis known as the ‘intrinsic’ pathway [68,69]. The central executioner molecules of apoptosis are a large family of cysteine proteases known as caspases [70]. Based on structural similarities, substrate preference and their role in the apoptotic pathway, caspases have been divided into initiators (such as caspase-8 and caspase-9), downstream executors (such as caspase-3) and inflammatory caspases (such as caspase-1). Caspase-dependent apoptosis can be initiated by the activation of death receptors or by mitochondrial stress. Another apoptotic-regulatory pathway has been described, in which the induction of ER stress, owing to the alteration of calcium homeostasis or the accumulation of misfolded proteins, triggers the activation of ER-resident caspase-12 [71]. In recent years, many reports have linked the ER-stress apoptosis pathway to diverse neurodegenerative diseases as a result of protein misfolding and aggregation [72].

Apoptosis – Mitochondrial Pathway

Apoptosis induced by aggregated PrP peptide fragments or mutant PrP variants (such as truncated or cytosolic PrP) proceeds via the mitochondrial pathway [73]. The central event in the regulation of apoptosis by this pathway is the release of mitochondrial proteins, such as cytochrome c, which triggers the activation of caspases through the formation of an ‘apoptosome’ complex [74]. This protein complex is formed when cytosolic cytochrome c binds to the adaptor protein apoptotic protease activating factor-1 (Apaf-1), recruiting the inactive form of caspase-9 and triggering its self-proteolytic activation [75]. Cytochrome c release depends upon the opening of a mitochondrial pore termed ‘permeability transition pore’ or PTP [76]. The opening of the PTP is strictly regulated by the Bcl-2 (B-cell lymphoma protein 2) family proteins, representing a critical intracellular checkpoint upstream of the caspase cascade [65]. Indeed, important roles of Bcl-2 and Bax (Bcl-2-associated X protein) as the well-known regulators of the mitochondrial apoptosis pathway have been reported [73,77]. Activation of alternative caspases including caspase-2 has been noted in certain apoptotic paradigms, which do not appear to require Apaf-1 [78]. Caspase-2 activity may represent an upstream initiating event or may be part of a downstream amplification loop.

There also exists a caspase-independent pathway [79,80], such as metabolic deaths resulting from irreversible damage to organelles including mitochondria and ER, other apoptogenic factors released from mitochondria (OMI, endonuclease G, or apoptosis-inducing factor) that cause caspase-independent deaths, or a novel path to activate alternative caspases. OMI relies both on its inhibitor of apoptosis protein (IAP) binding and serine/threonine protease activity (Fig. 1). Neither the release nor the proapoptotic activity of OMI is caspase dependent. The release of OMI may cause caspase-independent mitochondrial dysfunction as well as inhibition of IAPs. Notably, one study suggests that the serine protease activity of OMI plays a role in the maintenance of mitochondrial homeostasis under nonapoptotic conditions, as its loss is associated with mitochondrial dysfunction and neurodegeneration [85]. In general, the mitochondrial apoptotic pathway has been shown to be activated in neurons by growth factor deprivation, oxidative stress, and DNA damage or by changes in the expression levels of Bcl-2 family proteins [86].

Figure 1

Possible apoptosis mechanisms via mitochondria in prion disease Several intermembrane space (IMS) proteins are pro-apoptotic if released into the cytosol. Cytochrome c (C) activates caspase-9. In conjunction with apoptotic protease activating factor 1 (Apaf-1), pro-caspase-9 and cytochrome c form the apoptosome [81,82]. The apoptosome is a complex consisting of adaptor proteins, which mediate the activation of initiator caspases at the onset of apoptosis. SMAC (second mitochondrial activator of caspases) and HTRA2 inhibit cytosolic inhibitor of apoptosis proteins (IAPs). Both the inhibition of HTRA2's normal quality control function or enhancement of its IAP-degrading activity could promote cell death. Downstream of mitochondria are both caspase-dependent (cytochrome c)-dependent and caspase-independent cell death mechanisms. These include release of endonuclease G (endo G) and apoptosis-inducing factor (AIF), inducers of genomic digestion, and ROS from disrupted electron-chain transport. The released proteins are translocated to the nucleus and induce chromatin condensation and DNA fragmentation. Release of these proteins into the cell is modulated by recruitment of Bax (it is proapoptotic) or Bcl-2 (anti-apoptotic) to the outer mitochondrial membrane (OMM). Numerous extracellular and intracellular signals converge to regulate mitochondrial apoptosis [83,84].

ER Stress and Unfolding Protein Response-mediated Apoptosis

Two studies showed that neither Bax deletion nor Bcl-2 overexpression decrease neuronal death induced by prion infection or alter the progression of prion disease in animals [87,88]. The analysis of signaling pathways involved in neuronal apoptosis in neurodegenerative diseases associated with the misfolding and accumulation of protein aggregates in the brain has provided data supporting a novel apoptosis pathway implicating ER stress and the unfolding protein response (UPR) process.

Neurons exposed to purified PrP^Sc exhibit extensive ER stress, resulting in the rapid release of calcium to the cytoplasm and the activation of UPR, an adaptive signaling reaction that augments the cell's capacity to produce properly folded proteins and decreases the unfolded protein load [89] (Fig. 2). There are three main ER resident transmembrane signaling proteins that operate as stress sensors activating UPR signaling responses. These proteins include double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), activating transcription factor (ATF) 6, and inositol requiring kinase 1 (IRE1). In cells undergoing ER stress, IRE1α dimerizes and autophosphorylates, leading to its activation in the cytosolic domain. Active IRE1α processes the mRNA encoding XBP1, which is a transcription factor and up-regulates many essential UPR genes involved in folding, ERAD, organelle biogenesis, and protein quality control. In addition, active IRE1α activates alarm responses mediated by the JNK. Activation of PERK decreases the general protein synthesis rate through phosphorylation of the initiation factor eIF2α. eIF2α phosphorylation increases the translation of the ATF4 mRNA, which encodes a transcription factor that induces the expression of genes involved in amino acid metabolism, antioxidant responses, apoptosis, and autophagy. ATF6 is a type II ER transmembrane protein encoding a bZIP transcriptional factor on its cytosolic domain and is localized at the ER in unstressed cells. Upon ER stress induction, ATF6 is processed at the golgi apparatus (GA) releasing its cytosolic domain, which then translocates to the nucleus where it increases the expression of some ER chaperones, ERAD-related genes, and proteins involved in ER and GA biogenesis [124].

Figure 2

A schematic diagram of mammalian unfolded protein response signaling and connection to neuronal cell death machinery in prion disease The major unfolded protein response (UPR) signaling pathways induced by endoplasmic reticulum (ER) stress in prion diseases are depicted [90]. Under normal conditions, the protein chaperone glucose-regulated protein (GRP78) binds the termini of inositol-requiring kinase 1 (IRE1α) [91–94], PRKR (double-stranded RNA-activated protein kinase)-like endoplasmic reticulum kinase (PERK) [95] and activating transcription factor 6 (ATF6) [96–101] in the lumen of the ER, serving as a negative regulator of their activation. Unfolded proteins such as PrP^Sc in the ER cause GRP78 to release IRE1α, PERK and ATF6 [102]. Oligomerization of PERK activates its intrinsic kinase activity, and results in the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) [103,104] and suppression of global mRNA translation [105]. Activation of PERK leads to the inhibition of cap-dependent translation but paradoxically increases translation of the potent transcription factor, AFT4 [106]. Upon ER stress, PERK-mediated phosphorylation of NRF2 promotes its dissociation from KEAP1 (Kelch-like Ech-associated protein 1), leading to the nuclear accumulation of NRF2 [106]. Inactivated ATF6α translocate to the Golgi apparatus where it is cleaved by site 1 and site 2 proteases for proteolytic processing to release active ATF6α, which controls expression of UPR and ER-assisted degradation (ERAD) genes [101,107,108]. In the nucleus, ATF6 activates transcription of XBP1 and molecular chaperones such as GRP78 and GRP94 [109]. Upon release, IRE1α oligomerize in ER membranes. Oligomerized IRE1α binds to TNF receptor-associated factor 2 (TRAF2), activating apoptosis signal-regulating kinase 1 (ASK1) and downstream kinases that activate p38 MAPK (mitogen-activated protein kinase) and Jun N-terminal kinase (JNK) [110–112]. The intrinsic ribonuclease activity of IRE1α also results in X-box-binding protein 1 (XBP1) production [91]. ATF4 [105,113], XBP1, and ATF6 all converge on the promoter of the gene encoding C/EBP homologous protein (CHOP) , which transcriptionally controls expression of the genes encoding BIM (also known as BCL2L11) and B-cell leukemia/lymphoma 2 (Bcl-2) [114–116]. The p38 MAPK stimulates CHOP activity. JNK activates BIM, but inhibits Bcl-2 [117]. Bcl-2-associated X protein (Bax) and Bcl-2 antagonist/killer (BAK) are also reported to interact with and activate IRE1α [118]. Caspase-12 activation is linked to the ER stress pathway through the ER transmembrane kinase Ire1α and the adapter protein TRAF2 [110,119,120]. Following ER stress, caspase-12, as a member of the initiator caspases group, directly cleaves caspase-9, leading to caspase-9-dependent activation of caspase-3 [121,122]. GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein [123]; NRF2, nuclear respiratory factor 2; HO-1, heme oxygenase-1; EDEM [118], α-mannosidase–like protein; GADD34, growth arrest and DNA damage-inducible gene 34; MKK, mitogen activated protein kinase kinase; S1P, site-1 protease; S2P, site-2 protease.

Prolonged ER stress leads to apoptosis mediated by different regulators [125,126], including members of the Bcl-2 family [110,111,126,127]. Activation of ASK1 and JNK also regulates apoptosis under ER stress conditions [128,129]. Sustained PERK signaling was proposed as a pro-apoptotic effector [123], possibly through the induction of CHOP/GADD153 and the Bcl-2 family members BIM and PUMA [130–133]. Many additional components of the ER stress apoptosis pathway have been identified [110,111]. Furthermore, ER stress can trigger PrP^C misfolding and aggregation [134–136], and facilitates the conversion of PrP^C into PrP^Sc in a cell-free system [135]. Similarly, proteasome inhibition leads to the accumulation of a protease resistant form of PrP^C derived from the ERAD [137,138]. These observations may be relevant for understanding the occurrence of sporadic forms of CJD, the most common prion disease in humans, where alteration in the folding/quality control process or the ER environment may be a key event in initiating PrP misfolding. To evaluate the possible involvement of the UPR in prion disease, researchers tested the susceptibility of a brain-specific XBP1 conditional knock-out mouse to scrapie prion pathogenesis [69]. Surprisingly, no effect was observed on the activation of ER stress responses, PrP^Sc levels, neuronal loss or animal survival. Since the UPR in mammals is not limited to the IRE1/XBP1 pathway, activation of these alternative UPR pathways may well compensate for XBP1 deficiency in the prion model employed.

It is worth noting that upregulation of caspase-12 (caspase-4 in humans) in vitro and in the brain of prion-infected mice and patients with sporadic and variant CJD has been observed [139]. Caspase-12 is ubiquitously expressed and synthesized as an inactive pro-enzyme. Upon proteolytical processing, the active form of caspase-12 is generated consisting of a regulatory pro-domain and two catalytic (p20 and p10) subunits. The mechanism of caspases-12 activation is unclear but unlike other caspases, caspase-12 is remarkably specific to insults that elicit ER stress [140]. Caspase-12 normally resides on the ER membrane; however, upon activation, it translocates to the cytoplasm where it cleaves downstream caspases [89]. Although caspase-12 and caspase-4 are involved in ER stress-mediated apoptosis, the upstream events leading to their activation are unclear. However, the role of caspase-12-mediated apoptosis in prion disease has been questioned by a study showing that caspase-12 deletion did not change the progression of the disease in vivo [141]. Caspase-3 is also activated in prion-infected cultured cells and brains of diverse animals and humans affected by TSE [139,142].

Disruption of calcium (Ca²⁺) homeostasis in the cell is probably the most adverse and immediate effect caused by ER stress [143,144]. Calcium is a second messenger in cellular signaling pathways; thus, maintaining a specific concentration of calcium in the cytoplasm is critical for normal cellular biology. The ER is the main site for intracellular storage of calcium. In neurons, the effect of calcium is particularly deleterious, because calcium waves are important for neuronal activity [143]. The release of calcium from the ER to the cytoplasm occurs when cells are exposed to misfolded prion protein [139,141,145]. Disturbances in ER calcium regulation can induce UPR and also independently perturb cellular events that are critically linked to cell life and death.

Alteration of ER homeostasis and subsequent ER stress has also been implicated in the pathogenesis of prion diseases. PrP^Sc induces an increase of cystolic calcium released mainly from the ER, which leads to loss of Δψm, increased ROS and cell death. This release of calcium is dependent on the apoptosis triggering domain (residues 106–126) of prion protein, meanwhile, functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide [70,118]. These effects could be inhibited by blocking the release of calcium from ER or by the addition of antioxidants [144]. Reticulon 3, an ER-localized protein that can cause rapid depletion of ER calcium stores, is upregulated in the ME7/CV mouse scrapie model [146]. The resulting loss of calcium from the ER would inhibit the activity of several ER chaperones and enzymes triggering ER stress [147]. On the other hand, calcium release appears to be one of the first changes after prion infection in cells. Increased calcium in the cytoplasm deregulates downstream targets including calcineurin (CaN), a type 2B phosphatase [148]. The activity of this enzyme is regulated by the calcium–calmodulin complex. Optimal activity of CaN is required to maintain the proper phosphorylation state of protein targets, such as the apoptosis inducer Bad, or the transcription factor CREB (cAMP response element-binding). Hyperactivation of CaN reduces the phosphorylation of Bad, which then disassociates from the scaffolding protein 14-3-3 and interacts with Bax to form channels in the mitochondrial membrane [149]. As a result, cytochrome c is released into the cytoplasm, leading to caspase activation and finally apoptosis. CREB, which is dephosphorylated by hyperactivated CaN, cannot translocate to the nucleus where it regulates the expression of genes required for synaptic plasticity [150]. CaN activation is implicated in neuronal death induced both by PrP^Sc and PrP synthetic peptides [151,152]. Moreover, CaN activity increases in the brain at the beginning of the symptomatic phase of prion disease [151]. Strikingly, blocking CaN activity in sick prion-infected mice increases animal survival, decreases the progression of deterioration and reduces neurodegeneration [151]. These data support the concept that PrP^Sc induces synaptic damage and neuronal death in TSEs through ER stress, at the same time, calcium homeostasis and CaN activity are changed.

In summary, alternative apoptotic pathways operate simultaneously in the brains of animals with prion disease. It is strongly implicate that the ER and specifically signals associated with prion protein infections [144]. This could explain why elimination of one route to apoptosis by gene knockout does not alter the progression of the disease substantially. It is also possible that neuronal death is not contributing much to prion disease pathology. The exact mechanism of neuronal death contributing to prion disease awaits further investigation.

Based on the available data, researchers propose a model of TSE disease progression [153] (Fig. 3) in which the primary abnormality is the formation and accumulation of PrP^Sc, first in peripheral tissues and later in the brain. The initial formation of PrP^Sc, either by template conversion upon infection or spontaneous misfolding in sporadic and inherited cases of the disease, begins a long and silent presymptomatic period in which PrP^Sc accumulates progressively. PrP^Sc accumulation leads to ER stress and activation of the UPR as a defense mechanism to combat protein misfolding through reduction of overall protein expression and upregulation of various chaperones. Chronic ER stress can directly cause neuronal death and possibly spongiform degeneration. Brain inflammation in the form of reactive astrocytes and activated microglia also begins during the presymptomatic phase and probably acts as a protective mechanism to remove misfolded aggregates. Both inflammation and autophagy become chronic, and at this stage they could also contribute to neuronal death and perhaps brain vacuolation. If the body cannot reduce and control PrP^Sc formation and the misfolded aggregates accumulate to high levels, several degenerative processes manifest. From the point of view of the clinical disease, the most important consequence of PrP^Sc might be the induction of synaptic alterations, which are probably responsible for the initial disease symptoms. At this early stage, the disease process might be reversible if the neurological abnormalities can be halted. Progressive synaptic dysfunction leads to dendritic loss, which might be sufficient to induce neuronal death. Extensive neuronal death produced by diverse pathways as well as profuse vacuolization of the brain are probably responsible for the severe clinical deterioration typical of the end stage of the disease, which often results in the death of the individual (Fig. 3).

Figure 3

Schematic model of neurodegeneration in prion diseases The disease process starts with the conformational changes of PrP^C induced by prion infections, mutations or unknown factors, leading to the production and accumulation of misfolded PrP^Sc proteins. PrP^Sc slowly but gradually accumulates in the brain during a long and clinically silent pre-symptomatic phase. PrP^Sc accumulation triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) as well as MT dysfunction and oxidative stress, which represent the first line of defense against protein misfolding and the crucial role to initiate the signaling cascades involved in apoptosis, respectively. The pathological protein may also interact with different neuronal cell-surface receptors, triggering signal transduction cascades which result in cellular stress and neuronal dysfunction. Other early consequences of PrP^Sc accumulation are brain inflammation (in the form of astrocytosis and microglial activation) and autophagy. Both inflammation and autophagy might initially be defensive mechanisms, but later could also contribute to neuronal death and perhaps brain vacuolation. The first neuronal damage leading to noticeable clinical consequences is probably synaptic disruption, marking the end of the pre-symptomatic phase and beginning of the early clinical phase of the disease. Synaptic dysfunction produces loss of dendrites and finally neuronal death. The end and irreversible stages of the disease are characterized by massive spongiform degeneration and neuronal death, which are probably triggered by a variety of interconnecting cellular pathways. This figure is reviewed in [151].

Concluding Remarks

Whereas apoptosis in TSEs is relatively well understood, autophagy is not. There are several uncertainties in our thinking on neuronal autophagy in TSEs. First, autophagy is regarded as a short-term response to nutrient deficiency [29], which is not the case in slow transmissible diseases like TSEs. However, it seems that autophagy is activated to prevent apoptosis; when autophagy is blocked, apoptosis ensues. On the other hand, when apoptosis is blocked as in Bax/Bak-deficient mice, autophagy is activated as a cell-survival mechanism [154]. Nevertheless, when all subcellular organelles are ‘self-eaten’, the cell eventually dies. An analogous situation was observed when inhibition of autophagosomes and lysosomes was accomplished via targeting of LAMP2 by RNA interference [155]. A dual role for autophagy was envisaged, i.e. protective role against apoptosis and detrimental role in cell death, and both are mediated by the same set of ATG genes [29]. Thus, the abundant presence of autophagy in TSEs suggests that neurons either try to escape apoptosis or die by autophagy via one of the PCD pathways. Another uncertainty is how autophagy contributes to overall pathology underlying TSEs. The hallmark of TSEs is vacuolation, intracellular ‘empty’ spaces surrounded by a single or double membrane. The histogenesis of vacuoles is not well understood and most ultrastructural studies suffer from the inability to ascertain the subcellular organelles from which vacuoles originate, although dilated ER or mitochondria have been suggested [156]. As dystrophic neurites are abundant in TSEs [157], it is plausible that (macro)autophagy plays a role in neuronal degeneration. However, considering the evidence that autophagy may prevent apoptosis, it is also possible that abundant presence of autophagic vacuoles within dystrophic neuritis actually reflects neurons struggling to survive in the noxious environment of misfolded PrP.

As mitochondrial dysfunction and oxidative stress may determine neuronal death/survival after neurodegeneration, recent research indicates that oxidative stress may regulate p53-dependent transcription, p53 translocation, and pro-survival Akt signaling through phosphorylation. Decreasing oxidative stress by SOD1 overexpression affords neuroprotection [158]. On the other hand, another review highlighted the importance of further investigations in the diverse functions of caspase-3, which is the downstream factor of mitochondria-mediated apoptosis, in the nervous system [139]. The intricate pathways contributing simultaneously to brain dysfunction pose an insurmountable challenge for developing a treatment for the disease. The molecular mechanisms of the mitochondrial imbalance-mediated neuronal death might contribute to multiple targets treatment for TSEs [159] and other neurodegenerative diseases.

In the meantime, ER stress responses and in particular UPR in neurodegeneration is a fast emerging research field. As reviewed here, there is evidence of misfolded proteins accumulation and the involvement of the UPR in prion diseases and several other human neurodegenerative conditions. Although strong correlations between them exist, direct evidence to causally link the UPR and ER stress to neurological disorders in vivo is lacking. Predicting whether and how ER stress affects neuronal survival is difficult because activation of the UPR may decrease neurodegeneration by increasing protein folding, protein quality control, and autophagy, and extensive or chronic ER stress may result in irreversible neuronal damage and apoptosis. At the same time, the importance of ER stress and the UPR in the pathophysiology of neurodegeneration is unclear. Is ER stress the cause or simply an effect of disease pathology? The elucidation of the exact role of ER stress in neurodegenerative disorders requires studies the individual arms of the UPR, namely PERK, IRE1, and ATF6. If ER stress is a critical factor of neurodegeneration in these disorders, it raises the hope for the development of a common neuroprotective therapy for the treatment of neurodegenerative conditions.

Furthermore, we know little about the cell types in the brain that are primarily affected by mitochondria and ER stress; nor have we conclusively identified the endogenous stimuli that evoke the oxidative stress and UPR. The high secretory ability of neuronal populations might display increased sensitivity to genetic and/or environmental factors that disrupt mitochondria, ER and other organelles function. In this context, understanding the possible role of organelles in neuronal cells such as oligodendrocytes, Schwann cells, and neuropeptide-secretory neurons is of particular relevance for future therapeutic intervention. From our current knowledge of the molecular basis of prion-induced neurodegeneration, strategies boosting the natural defensive pathways (UPR, brain inflammation and autophagy) or inhibition of the most damaging processes (synaptic loss, spongiform degeneration and neuronal death) might have therapeutic benefits.