The cellular prion protein, a major player in the neuropathology of prion diseases, is believed to control both death and survival pathways in central neurons. However, the cellular and molecular mechanisms underlying these functions remain to be deciphered. This chapter presents cytopathological studies of the neurotoxic effects of infectious prions and cellular prion protein-deficiency on cerebellar neurons in wild-type and transgenic mice. The immunochemical and electron microscopy data collected in situ and ex vivo in cultured organotypic cerebellar slices indicate that an interplay between apoptotic and autophagic pathways is involved in neuronal death induced either by the infectious prions or by prion protein-deficiency.
- prion protein
1.1 Prion diseases
Transmissible spongiform encephalopathies (TSEs) or “prion diseases” are fatal neurodegenerative disorders in humans (Creutzfeldt-Jakob disease (CJD), Gerstmann-Straüssler-Scheinker syndrome (GSS), variant CJD (vCJD), fatal familial insomnia (FFI) and kuru) and in animals (bovine spongiform encephalopathy (BSE), transmissible mink encephalopathy (TME), chronic wasting disease of cervids (CWD), camel prion disease (CPD), and scrapie of sheep and goats) [1, 2, 3, 4]. Prevailing over a viral etiology, the conformational corruption of host-encoded cellular prion protein (PrPc) by a pathogenic isoform (PrPTSE) is now widely accepted as underlying prion transmission and pathogenesis in TSEs [5, 6, 7].
1.2 PrPc functions
PrPc has been implicated in neurotransmission, olfaction, proliferation and differentiation of neural precursor cells, neuritic growth, neuronal homeostasis, cell signaling, cell adhesion, myelin maintenance, copper and zinc transport, as well as neuroprotection against toxic insults, such as oxidative stress and excitotoxicity (see [14, 15] for reviews). Increasing evidence links prion protein misfolding and accumulation to neurodegeneration in prion diseases. Accordingly, several nonexclusive mechanisms of prion-mediated neurotoxicity are currently under investigation (see  for review). PrPc has been localized in three major sites: enriched in lipid rafts, anchored in the outer plasma membrane leaflet by its GPI tail , and intracellularly in the Golgi apparatus early and late endosomes [18, 19]. Since lipid rafts are pivotal microdomains for signal transduction, PrPc is likely triggering intracellular signaling pathways [20, 21]. The first evidence that PrPc might mediate extracellular signals was the caveolin-1-dependent coupling of PrPc to the tyrosine-protein kinase Fyn . From this pioneering work, accumulating data suggested that PrPc functions as a “dynamic cell surface platform for the assembly of signaling molecules,” partnering with other membrane proteins to transduce cellular signaling .
1.3 Synaptic PrPc
Whereas, PrPc is highly expressed in both neurons and glial cells of the CNS [19, 23, 24], it is preferentially localized in the pre- and postsynaptic terminals of neurons [19, 24, 25]. Immunocytochemical studies of primate and rodent brains [25, 26] including an EGFP-tagged PrPc in transgenic mice, showed that PrPc is enriched along axons and presynaptic terminals [27, 28, 29], and undergoes anterograde and retrograde transport [30, 31]. Such a synaptic targeting of PrPc suggests that it could be involved in preserving synaptic structure and function. Indeed, synaptic dysfunction and loss are early prominent events in prion diseases [32, 33]. However, a functional role of PrPc at synapses is not consistently supported by functional data and still remains contentious.
Insights into possible mechanisms by which PrPc modulates synaptic mechanisms and neuronal excitability at a molecular level have been provided by the documented interactions of PrPc with several ion channels including the voltage-gated calcium channels (VGCCs) , the N-methyl-D-aspartate glutamate receptors (NMDARs)  and the voltage-gated potassium channels Kv4.2 . PrPc has been shown to regulate NMDARs due to its affinity for copper that leads to inhibition of glutamate receptors and excitotoxicity [37, 38]. While interaction of PrPc with these channels may account for some of its functions, a toxic response can also be activated when PrPc misfolds. A structural change in cell surface PrPc has been proposed to simultaneously disrupt NMDAR function and plasma membrane permeability, leading to dysregulation of ion homeostasis and neuronal death [39, 40]. PrPc can also interact with kainate receptor subunits GluR6/7 , α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors subunits GluA1 and GluA2 [42, 43], and metabotropic glutamate receptors of group 1 mGluR1 and mGluR5 [44, 45]. PrPc can interact with the β-amyloid peptide (Aβ) and the later [45, 46] is believed to underlie the Aβ oligomer-induced disruption of LTP in Alzheimer’s disease . Thus, PrPc seems to behave as a cell surface receptor for synaptic oligomers of the Aβ peptide and, of other β-sheet-rich neurotoxic proteins .
1.4 PrPTSE-related neurotoxicity in prion diseases
The histopathological signature of TSEs notably relies on the aggregation of PrPTSE, vacuolation of the brain tissue, astrogliosis, and synaptic and neuronal loss. How neurons, the major targets of prions, die, remains a central question in prion diseases. The absence of neurodegenerative phenotypes after depletion of PrPc suggests that neurotoxicity is not due to a loss of PrPc function but rather results from a gain of toxicity upon its conversion to PrPTSE, which then acts on the central nervous system (CNS) . Although PrPc is required for propagation of infectious prions and PrPTSE-mediated toxicity , the mechanisms by which prions are lethal for neurons remain mostly unknown. Nevertheless, the endogenous PrPc conversion has been shown to cause neuronal dysfunction and death, rather than PrPTSE itself which does not seem to be directly neurotoxic. A precise understanding of the factors leading to neurotoxicity in prion infections is crucial to developing targeted therapies and investigating the role of PrPc in neurons should provide insight.
The conformational conversion of PrPc begins on the neuronal surface, where PrPc interacts with exogenous PrPTSE, and then proceeds within endogenous compartments suggesting that neurotoxicity may be triggered by PrPc misfolding both at the cell surface and inside the cell. In both acquired and genetic prion diseases, intracellular PrPc misfolding would ultimately alter synaptic proteostasis, either through an indirect unfolded protein response (UPR)-mediated mechanism , likely arising either from an impairment of the neuronal ubiquitin-proteasome system (UPS) , or a direct interference with secretory trafficking of PrPc-interacting cargoes . Common features associated with prion infections include Ca2+ dysregulation, release of reactive oxygen species, and induction of endoplasmic-reticulum (ER) stress, which has been recently suggested as an important player in pathogenesis . Prion-infected mice show brisk activation of the UPR and specifically of the PERK pathway, resulting in eIF2α phosphorylation and suppression of translational initiation. PERK inhibition protects mice from prion neurotoxicity, confirming an important pathogenic role of ER stress . Since UPR activation and/or increased eIF2α-P levels as well as UPS impairment are commonly seen in prion disorders and in Alzheimer’s and Parkinson’s diseases, translational control, and UPS stimulation strategies may offer a common therapeutic opportunity to prevent synaptic failure and neuronal loss in protein misfolding diseases [51, 54].
1.5 Loss of PrPc anti-inflammatory protective function in prion disease
A protective role of PrPc against a noxious insult mediated by the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) has recently been demonstrated . The α-secretase activity mediated by the TNFα-converting enzyme (TACE) was impaired at the surface of Fukuoka and 22L scrapie prion-infected neurons. Furthermore, the activity of 3-phosphoinositide-dependent kinase-1 (PDK1) which inactivates phosphorylation and caveolin-1-mediated internalization of TACE is increased in scrapie-infected neurons. PDK1 was shown to be controlled by RhoA-associated coiled-coil containing kinases (ROCK) which favored the PrPTSE production. In these neurons, exacerbated ROCK activity overstimulated PDK1 activity which canceled the neuroprotective α-cleavage of PrPc by TACE α-secretase, physiologically precluding PrPTSE production. Inhibition of ROCK lowered PrPTSE in prion-infected cells as well as in the brain of prion-diseased mice which had extended lifespans . Indeed, the dysregulation of TACE resulted in PrPTSE accumulation and reduced the shedding of TNFα receptor type 1 (TNFR1) from the neuronal plasma membrane. Inversely, inhibition of PDK1
Synaptolysis is believed to initiate the neurodegeneration arising after a decrease in depolarization-induced calcium transients that progressively impairs glutamate release . However, although cytoskeletal disruption in dendritic spines plays a major role in neuronal dysfunction, neither changes in postsynaptic densities and presynaptic compartment nor disruption of afferent innervation have been systematically observed, suggesting that even at terminal stages of the disease neuronal loss may not result from deafferentation as previously proposed in the hippocampus and cerebellum of scrapie-infected mice [33, 58, 59]. Thus, neuronal vulnerability to pathological protein misfolding appears to be more strongly dependent than previously thought, on the structure and function of target neurons.
Recent investigations of scrapie pathogenesis in the mouse cerebellum revealed an early upregulation of tumor necrosis factor-α receptor type 1 (TNFR1), a key mediator of neuroinflammation at the membrane of astrocytes enveloping Purkinje cell (PC) excitatory synapses already at the preclinical stage of the disease before PrP22L precipitation, GFAP astrogliosis, and PC death . The contribution of perisynaptic astrocytes to prion pathogenesis through TNFR1 upregulation remains to be clarified and, although the cell types responsible for PrP22L production in the cerebellum are still uncertain, these data suggest a critical role for astrocytes in prion pathogenesis.
2. Mechanisms of neuronal death in prion diseases
Despite the overall advances made in this field during the last decades, the sequence of cellular and molecular events leading to neuronal cell demise in TSEs remains obscure. At present, neuronal cell death can be envisioned as resulting from several parallel, interacting, or sequential pathways involving protein processing and proteasome dysfunction , oxidative stress , inflammation  apoptosis, and autophagy . The repertoire of pathways that lead to neuronal death is however limited . In TSEs, apoptosis is the most popular theory of cell death but is not convincingly documented. In all cases, the probable disruption of both neuronal metabolism and circuits generates a pro-apoptotic signal for neurons. In addition to disruption of cellular proteostasis, UPS dysfunction may lead to neurotoxicity by activating pro-apoptotic pathways. PrPTSE aggresomes can associate with pro-apoptotic factors such as vimentin and caspases . On the other hand, autophagy has been reported in TSEs, but its role in prion disease pathology is not well established . However, the extensive synaptic autophagy observed in prion diseases  has been proposed to contribute to overall synaptic degeneration, a major precocious pathological feature leading to neuronal death in TSEs. This chapter reports recent biochemical and cytopathological studies investigating the involvement of apoptosis and autophagy in neuronal loss induced by infectious prions as well as by PrPc-deficiency in the mouse cerebellum.
Among TSEs, scrapie is a natural ovine prion disease widely studied in mouse models using murine-adapted prion strains (22L, ME7) that, akin to natural prion strains, differ in their rate of disease progression (i.e., duration of the incubation period), as well as the extent and regional pattern of brain histopathology [66, 67]. For example, the characteristic of a prion strain mostly relies on specific biochemical properties related to PrPTSE misfolding. The variable susceptibility of neuronal types to prion infection also emerges as another critical parameter that underlies the complex mechanisms of prion pathogenesis [54, 68, 69] and affects PrPTSE progression along defined anatomical routes . The cellular and molecular mechanisms involved in targeting PrPTSE to specific neuronal populations [33, 71, 72] and neuron-to-neuron spreading of prions in the CNS remain elusive .
In several prion diseases, the cerebellum is a preferential prion target for scrapie [74, 75, 76, 77, 78], also observed in Creutzfeldt-Jakob disease (CJD) cases [79, 80, 81, 82, 83, 84, 85, 86, 87]. Cerebellar circuits are exquisitely patterned and the expression patterns of zebrins in PCs define a topographical map of genetically determined zones controlling sensory-motor behavior [88, 89]. Subsets of PCs expressing zebrins alternate with subsets of zebrin-free PCs, thus forming complementary stripes of biochemically distinct PCs . The most comprehensively studied zonal marker is zebrin II/aldolase C (ZII/AldC) . The expression of ZII/AldC by itself, however, is not sufficient to recapitulate the full complexity of the cerebellar cortex because of the many other PC subtypes [91, 92].
In a recent study , the parasagittal compartmentation of the cerebellar cortex restricted 22L scrapie pathogenesis, including PrP22L accumulation, PC neurodegeneration, and gliosis. Indeed, PCs displayed a differential, subtype-specific vulnerability to 22L prions with zebrin-expressing PCs being more resistant to prion toxicity, whereas in stripes where PrP22L accumulated most zebrin-deficient PCs were lost and spongiosis was accentuated ( Figure 1 ). Although this banding pattern of PrP22L accumulation is most likely delineated by structural constraints of compartmentation, different biochemical properties of PC subpopulations may well determine their differential resistance to scrapie prions.
2.1 Prion-induced apoptosis
2.1.1 Apoptotic pathways in prion-infected neurons
The mechanism of prion neurotoxicity requires neuronal expression of PrPc and is based on the subversion of its normal function triggered by an interaction with PrPTSE at the cell surface, thereby transducing a toxic signal into the cell. Nevertheless, this has been challenged by the discovery of a monomeric, highly α-helical form of PrPc with strong
Endoplasmic-reticulum stress has recently been implicated in an apoptotic regulatory pathway activated by changes in Ca2+ homeostasis or accumulation of aggregated proteins. In both these situations, Ca2+ is released and caspase-12 is activated . ER stress and caspase-12 activation have been identified in prion-infected N2a cells as well as in the brains of prion-diseased mice and CJD patients . The synaptic dysfunction and neuronal death caused by PrPTSE accumulation via dysregulation of the Ca2+-sensitive phosphatase calcineurin (CaN) provides further evidence of the role of ER stress and Ca2+ homeostasis in prion-induced neurodegeneration . The increase in Ca2+ cytosolic levels following hyperactivation of CaN dysregulates the pro-apoptotic Bcl-2-associated death promoter (Bad), and the transcription factor cAMP response element-binding (CREB). Dephosphorylated Bad interacts with Bax causing mitochondrial stress and apoptosis while dephosphorylated CREB cannot translocate into the nucleus to regulate the transcription of synaptic proteins, resulting in synaptic loss .
2.1.2 Mitochondrial apoptosis in prion-infected cerebellar neurons
PrPc has recently been suggested to participate in anti-apoptotic and anti-oxidative processes by interacting with the stress inducible protein 1 (STI-1) to regulate superoxide dismutase (SOD) activation . The PrPc octapeptide repeat region contains a B-cell-lymphoma 2 (Bcl-2) homology domain 2 (BH2) of the family of apoptosis regulating Bcl-2 proteins involved in the anti-apoptotic function of Bcl-2. A direct interaction between PrPc and the C-terminus of anti-apoptotic Bcl-2 has also been found [101, 102]. In addition, the third helix of PrPc impaired the BAX conformation changes required for apoptosis activation suggesting that PrPc may assure the neuroprotective function of Bcl-2 . Along this line,
Activation of the mitochondrial apoptotic pathway was observed when primary neurons were exposed to aggregated neurotoxic peptides like PrP106-126 or recombinant mutant PrP [107, 108, 109]. Apoptotic neuronal death demonstrated by activation of several caspases and DNA fragmentation is evident in natural prion diseases as well as in experimental models of TSEs [76, 110, 111]. In the cerebellum, apoptotic features have been observed in granule cells in CJD patients [112, 113] as well as in mice experimentally infected with CJD  and scrapie strains 301V, 87V, 22A , 79A , M1000/Fukuoka-1 , 127S , 22L, 139A, and RML [116, 117]. More recently, activation of caspase-3 was found in PCs of 22L-infected mice . However, cerebral upregulation of the pro-apoptotic factor BAX has been reported in some cases of scrapie-infected rodents [116, 118], whereas no changes in clinical illness and neuropathology could be detected in the brain of Bax-deficient mice infected with 6PB1 mouse-adapted BSE prions . This suggested that BAX-mediated cell death is not involved in the pathological mechanism induced by BSE. Nevertheless, BAX is known to be involved in neuronal death in Tg(PG14)  and Ngsk PrnP0/0  murine models of PrP-deficiency-linked diseases. In these cases, neuronal death is restricted to cerebellar neurons that are known to undergo BAX and BCL2-dependent apoptosis in other abnormal conditions [122, 123]. This led us to further investigate the involvement of intrinsic mitochondrial apoptotic pathways in a cerebellotropic prion disease such as the 22L scrapie. For this purpose, the pathogenesis of 22L scrapie in the brain of
2.1.3 Prion-induced neuronal death in cerebellar organotypic slice cultures (COCS)
In the recently developed prion cerebellar organotypic slice culture (COCS) assay, progressive spongiform neurodegeneration that closely reproduce features of prion disease can be induced
Furthermore, significant spine loss and altered dendritic morphology, analogous to that seen
Following infection of COCS from C57Bl6/J, ZH-I Prnp0/0, and Tga20 PrP-over-expressing mice with brain homogenate from C57Bl6/J infected intracerebrally (ic.) with either 22L or 139A scrapie prions, PrP22L and PrP139A accumulation could be detected on histoblots from wild-type and Tga20 COCS, respectively, 30 and 20 days post infection (dpi), but not on histoblots from ZH-I mice ( Figure 5 ). Furthermore, quantitative analysis of PCs in these COCs indicated that a severe loss of neurons was induced by 22L prions in wild-type slices at 30 dpi (22 ± 2 surviving PCs/slice) and in Tga20 slices at 20 dpi (293 ± 68 surviving PCs) as well as by 139A prions in wild-type slices at 30 dpi (145 ± 63 surviving PCs/slice) and in Tga20 slices at 20 dpi (191 ± 31 surviving PCs/slice) compared to noninfected control COCS (220 ± 27 surviving PCs/slice in wild-type slices and 357 ± 71 surviving PCs/slice in Tga20 slices) ( Figure 6 ). At 30 dpi, the trilaminar organization of the cerebellar cortex was evident in noninfected COCs, which did not exhibit any clear ultrastructural modifications ( Figure 7 ). Nevertheless, numerous vacuoles, autophagosomes, and lysosomes had formed in granule cells infected by 22L and 139A ( Figure 8 ). In diseased PCs, autophagosomes with double membranes and rough endoplasmic reticulum (Nissl bodies) formed compartmented organelles of various sizes (1–10 compartments) resembling different stages leading to multivesicular vacuoles ( Figure 9 ). Although further investigations are necessary, these ultrastructural alterations were not observed in noninfected slices suggesting that a specific effect of prions links prion-induced ER stress to this morphological ER modification.
2.2 Prion-induced autophagy
Autophagy and apoptosis are activated in many neurodegenerative diseases featured by ubiquitinated misfolded proteins. In neurons, the degradation of abnormal proteins such as α-synuclein in Parkinson’s disease, β-amyloid peptide in Alzheimer’s disease (AD), or PrP in TSEs occurs by autophagy [14, 132, 133, 134, 135]. These cardinal proteins contribute to synaptic dysregulation and altered organelles leading to apoptosis. The neurodegenerating neurons exhibit robust accumulation of cytosolic autophagosomes (see  for review, Figure 10 ) suggesting a dysregulation of the autophagic flux resulting from autophagic stress, due to an imbalance between protein synthesis and degradation . Autophagy reduces intraneuronal aggregates and slows down the progression of clinical disease in experimental models of AD [137, 138, 139] and prion diseases [140, 141]. Thus, dysregulation of the autophagic flux impairs the elimination of misfolded proteins and damaged organelles which then accumulate in the cytoplasm and contribute to cell dysfunction and death .
Together, spongiform vacuolation of the neuropil, synaptolysis, accompanied by neuronal cell loss and gliosis constitute the classical neuropathological quartet of TSEs. The typical “spongiform vacuoles” are believed to result from autophagy and develop within neuronal elements, myelinated axons, and myelin sheaths [143, 144]. Autophagic vacuoles are increased in prion-diseased neurons [64, 65, 145], and the scrapie responsive gene 1 (SRG1) protein is overexpressed and bound to neuronal autophagosomes in the brain of scrapie- and BSE-infected animals and CJD-diseased humans [146, 147]. In addition, LC3-II, a marker of autophagosomes is increased in the cytosol of neurons in scrapie-infected hamsters and CJD- and FFI-diseased patients.
Recent evidence indicated that PrPc, but not truncated PrP devoid of the N-terminal octapeptide repeat region, exerts a negative control on the induction of autophagy . Thus, the loss or subversion of PrPc function resulting from prion infection may upregulate autophagy in diseased neurons . While autophagy-inducing agents increased cellular clearance of PrPTSE [149, 150, 151], blocking the fusion of autophagosomes with lysosomes allowed visualization of PrPTSE in the autophagosomes suggesting that degradation of endosomal PrPTSE is by autophagy . However, saturation of the autolysosomal degradation process can release PrPTSE aggregates and degradation enzymes into the neuroplasm contributing to autophagy upregulation and neuronal death . Nevertheless, although autophagy-inducing agents delayed disease onset and PrPTSE accumulation in the CNS of mice , survival time was not modified . Along this line, neither autophagy-inducing nor -inhibiting treatments altered the time course or amplitude of prion-induced neuronal death, strongly suggesting that autophagy in protein misfolding diseases is a secondary mechanism in the neurodegenerative process [141, 154].
3. Neuronal death in prion protein-deficient mice
3.1 Impaired autophagy in Zrch-1 prion protein-deficient mice
With the exception of the
3.2 Neuronal loss in Dpl-expressing Ngsk prion protein-deficient mice
Nagasaki (Ngsk) PrP-deficient mice which have a deletion of the entire
Because Dpl neurotoxicity depends on PrPc-deficiency in PCs, investigating the underlying neurotoxic mechanism may provide important insight into the neuroprotective function of PrPc. The resistance of the PC population to neurotoxicity increased in the cerebellum of Ngsk mice, which were either deficient for the pro-apoptotic factor Bax  or over-express the anti-apoptotic factor Bcl-2 . Although this suggests that an intrinsic apoptotic process is involved in the death of the Ngsk
To further investigate the role of autophagy in the death of Ngsk
At 6.5–7 months of age, the amount of autophagic somato-dendritic compartments and axons of PC were significantly decreased in
This suggests that BAX-deficiency modulates autophagy in Ngsk
At 12 months of age, the amount of autophagic somato-dendritic compartments and axons of PCs in
The absence of BAX not only protected some PCs from neurotoxicity in the cerebellum of the Ngsk
The complex pattern of neuronal death observed in neurodegenerative diseases is believed to involve an extensive interplay between the major cell death pathways [177, 178]. This is likely the case in prion-infected, as well as PrP-deficient neurons such as PCs. We further investigated PC death in Ngsk
The neurotoxic effects of PrP-deficiency were quantitatively analyzed by counting PCs at 3, 5, 7, 12, and 21 days in COCS from wild-type, Ngsk
Furthermore, at the ultrastructural level, whereas autophagic organelles were rare in wild-type PCs after 7 and 12 DIV, Ngsk
This morphometric and quantitative analysis of COCS suggests that PrP-deficiency, rather than Dpl neurotoxicity, is responsible for the neuronal growth deficit and loss
Although the contribution of apoptosis to prion-induced death of central neurons including cerebellar ones is strongly supported, our studies of scrapie-infected PCs show that although caspase-3 is activated, the pro-apoptotic BAX/BCL-2-dependent mitochondrial pathway is not involved in the prion-induced death of these neurons. This is also the case for BSE-induced death of hippocampal and thalamic neurons , suggesting that prions exert neurotoxicity through BAX-independent activation of caspase-3. Ultrastructural evidence of ER stress and robust autophagy in the scrapie-infected cerebellar neurons both