**Abstract**

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.

**Keywords:** prion protein, Doppel, apoptosis, autophagy, cerebellum, mouse

### **1. Introduction**

### **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) [1–4]. 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 [5–7].

#### **1.2 PrPc functions**

*Prnp*-knockout mice were generated in order to investigate the physiological functions of PrPc . In either mixed C57BL/6 j x129/Sv(ev) (Zurich I, ZrchI, *Prnp*ZH1/ZH1, [8]) or pure 129/Ola (Npu, Edinburgh, Edbg, [9]) or C57BL/6J (Zurich III, ZrchIII, *Prnp*ZH3/ZH3, [10]) genetic backgrounds, the first *Prnp* null mouse strains produced were viable with no clear abnormality except for their resistance to prion infection [11] and absence of obvious neurodegeneration. Similar absence of neurodegeneration or histopathology resulted from depletion of neuronal PrPc in adult conditional *Prnp*-knockout NFH-Cre/tg37 mice [12]. Thus, a physiological function of PrPc that is essential for life seemed to be ruled out unless it is highly redundant or is compensated. Nevertheless, looking at different neuronal and other cell functions in PrPc -ablated mice has revealed a number of differences that can be attributed to the physiological functions of PrPc (see [13] for review).

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 [16] 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 [17], 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 [21]. 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 [22].

## **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 [27–29], 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 voltagegated calcium channels (VGCCs) [34], the N-methyl-D-aspartate glutamate receptors (NMDARs) [35] and the voltage-gated potassium channels Kv4.2 [36]. 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 [41], 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

**9**

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

of other β-sheet-rich neurotoxic proteins [40].

**1.4 PrPTSE-related neurotoxicity in prion diseases**

suggests that neurotoxicity is not due to a loss of PrPc

nervous system (CNS) [48]. Although PrPc

The conformational conversion of PrPc

and investigating the role of PrPc

diseases, intracellular PrPc

misfolding diseases [51, 54].

A protective role of PrPc

PrPc

of PrPc

oligomer-induced disruption of LTP in Alzheimer's disease [47]. Thus, PrPc

to behave as a cell surface receptor for synaptic oligomers of the Aβ peptide and,

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

from a gain of toxicity upon its conversion to PrPTSE, which then acts on the central

prions and PrPTSE-mediated toxicity [49], the mechanisms by which prions are lethal

in neurons should provide insight.

misfolding would ultimately alter synaptic proteostasis,

against a noxious insult mediated by the pro-inflamma-

by TACE α-secretase,

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

interacts with exogenous PrPTSE, and then proceeds within endogenous

ing both at the cell surface and inside the cell. In both acquired and genetic prion


either through an indirect unfolded protein response (UPR)-mediated mechanism [50], likely arising either from an impairment of the neuronal ubiquitinproteasome system (UPS) [51], or a direct interference with secretory trafficking

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 [53]. 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 [50]. 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

**1.5 Loss of PrPc anti-inflammatory protective function in prion disease**

which canceled the neuroprotective α-cleavage of PrPc

tory cytokine tumor necrosis factor-α (TNFα) has recently been demonstrated [55]. 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 RhoAassociated coiled-coil containing kinases (ROCK) which favored the PrPTSE production. In these neurons, exacerbated ROCK activity overstimulated PDK1 activity

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

for neurons remain mostly unknown. Nevertheless, the endogenous PrPc

compartments suggesting that neurotoxicity may be triggered by PrPc

seems

conversion

misfold-

function but rather results

is required for propagation of infectious

begins on the neuronal surface, where

*Prions - Some Physiological and Pathophysiological Aspects*

[12]. Thus, a physiological function of PrPc

microdomains for signal transduction, PrPc

[25, 26] including an EGFP-tagged PrPc

pathways [20, 21]. The first evidence that PrPc

was the caveolin-1-dependent coupling of PrPc

at different neuronal and other cell functions in PrPc

tion of neuronal PrPc

(see [13] for review).

[16] for review). PrPc

**1.3 Synaptic PrPc**

Whereas, PrPc

a functional role of PrPc

and still remains contentious.

documented interactions of PrPc

neuronal death [39, 40]. PrPc

response can also be activated when PrPc

PrPc

mouse strains produced were viable with no clear abnormality except for their resistance to prion infection [11] and absence of obvious neurodegeneration. Similar absence of neurodegeneration or histopathology resulted from deple-

ruled out unless it is highly redundant or is compensated. Nevertheless, looking

number of differences that can be attributed to the physiological functions of 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

anchored in the outer plasma membrane leaflet by its GPI tail [17], and intracellularly in the Golgi apparatus early and late endosomes [18, 19]. Since lipid rafts are pivotal

a "dynamic cell surface platform for the assembly of signaling molecules," partnering

[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

enriched along axons and presynaptic terminals [27–29], and undergoes anterograde

could be involved in preserving synaptic structure and function. Indeed, synaptic dysfunction and loss are early prominent events in prion diseases [32, 33]. However,

nisms and neuronal excitability at a molecular level have been provided by the

has been shown to regulate NMDARs due to its affinity for copper that leads

with these channels may account for some of its functions, a toxic

has been proposed to simultaneously disrupt NMDAR function and

gated calcium channels (VGCCs) [34], the N-methyl-D-aspartate glutamate receptors (NMDARs) [35] and the voltage-gated potassium channels Kv4.2 [36].

to inhibition of glutamate receptors and excitotoxicity [37, 38]. While interac-

plasma membrane permeability, leading to dysregulation of ion homeostasis and

GluR6/7 [41], α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors subunits GluA1 and GluA2 [42, 43], and metabotropic glutamate

β-amyloid peptide (Aβ) and the later [45, 46] is believed to underlie the Aβ

[21]. From this pioneering work, accumulating data suggested that PrPc

with other membrane proteins to transduce cellular signaling [22].

and retrograde transport [30, 31]. Such a synaptic targeting of PrPc

Insights into possible mechanisms by which PrPc

receptors of group 1 mGluR1 and mGluR5 [44, 45]. PrPc

in adult conditional *Prnp*-knockout NFH-Cre/tg37 mice

has been localized in three major sites: enriched in lipid rafts,

is highly expressed in both neurons and glial cells of the CNS

that is essential for life seemed to be

is likely triggering intracellular signaling

might mediate extracellular signals

to the tyrosine-protein kinase Fyn

in transgenic mice, showed that PrPc

with several ion channels including the voltage-

misfolds. A structural change in cell

can also interact with kainate receptor subunits

at synapses is not consistently supported by functional data

functions as

is

suggests that it

modulates synaptic mecha-

can interact with the


**8**

PrPc

tion of PrPc

surface PrPc

oligomer-induced disruption of LTP in Alzheimer's disease [47]. 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 [40].

## **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) [48]. Although PrPc is required for propagation of infectious prions and PrPTSE-mediated toxicity [49], 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 [50], likely arising either from an impairment of the neuronal ubiquitinproteasome system (UPS) [51], or a direct interference with secretory trafficking of PrPc -interacting cargoes [52]. 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 [53]. 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 [50]. 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 [55]. 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 RhoAassociated 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 [56]. 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 *in vitro* promoted TACE localization at the plasma membrane, restoring TACE-dependent α-secretase activity and shedding of PrPc and TNFR1, thereby attenuating PrPTSE-induced neurotoxicity. Similarly, inhibition or siRNA-mediated silencing of PDK1 extended survival and reduced motor impairment of scrapie-diseased mice [55]. Mechanistically, PrPc coupling to the NADPH oxidase-TACE α-secretase signaling pathway limits the sensitivity of recipient cells to TNFα by promoting TACE-mediated cleavage of TNFα receptors (TNFRs) and the release of soluble TNFRs. PrPc expression was further shown to be necessary for maintaining TACE α-secretase at the plasma membrane and its TNFR shedding activity. The loss of PrPc provoked TACE internalization, canceling TACE-mediated cleavage of TNFR. This rendered PrPc -depleted cells and *Prnp*-knockout mice highly vulnerable to pro-inflammatory TNFα insult. Thus, abnormal trafficking and activity of TACE in prion diseases likely originates from a loss of PrPc cytoprotective function [57].

Synaptolysis is believed to initiate the neurodegeneration arising after a decrease in depolarization-induced calcium transients that progressively impairs glutamate release [34]. 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 [59]. 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 [60], oxidative stress [61], inflammation [55] apoptosis, and autophagy [62]. The repertoire of pathways that lead to neuronal death is however limited [63]. 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 [60]. On the other hand, autophagy has been reported in TSEs, but its role in prion disease pathology is not well established [64]. However, the extensive synaptic autophagy observed

**11**

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

spreading of prions in the CNS remain elusive [73].

well determine their differential resistance to scrapie prions.

neurons independently of endogenous membrane-bound PrPc

noncellular-autonomous mechanisms propagating prion spread [95].

*2.1.1 Apoptotic pathways in prion-infected neurons*

**2.1 Prion-induced apoptosis**

α-helical form of PrPc

ablation of PrPc

prions as well as by PrPc

in prion diseases [65] 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


In several prion diseases, the cerebellum is a preferential prion target for scrapie [74–78], also observed in Creutzfeldt-Jakob disease (CJD) cases [79–87]. 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 zebrinfree PCs, thus forming complementary stripes of biochemically distinct PCs [88]. The most comprehensively studied zonal marker is zebrin II/aldolase C (ZII/AldC) [90]. 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 [59], the parasagittal compartmentation of the cerebellar cortex restricted 22L scrapie pathogenesis, including PrP22L accumulation, PC neurodegeneration, and gliosis. Indeed, PCs displayed a differential, subtypespecific 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

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

autophagy and apoptosis with a molecular signature similar to that observed in prion-infected animal brains [93]. This toxic PrP (TPrP) killed PrP-deficient neurons *in vitro* suggesting that a PrP-derived toxic signal can be generated within

disease progression in mice even though glial replication was maintained and PrPTSE accumulated [94]. Thus, prion pathogenesis is governed by both cell-autonomous mechanisms responsible for cellular dysfunction and neurodegeneration and

with strong *in vitro* and *in vivo* neurotoxicity that elicits

expression in neurons reversed neurodegeneration and affected

. Indeed, postnatal

#### *Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

*Prions - Some Physiological and Pathophysiological Aspects*

receptors (TNFRs) and the release of soluble TNFRs. PrPc

canceling TACE-mediated cleavage of TNFR. This rendered PrPc

previously thought, on the structure and function of target neurons.

**2. Mechanisms of neuronal death in prion diseases**

and its TNFR shedding activity. The loss of PrPc

cytoprotective function [57].

ity and shedding of PrPc

loss of PrPc

prion pathogenesis.

extended lifespans [56]. 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 *in vitro* promoted TACE localization at the plasma membrane, restoring TACE-dependent α-secretase activ-

icity. Similarly, inhibition or siRNA-mediated silencing of PDK1 extended survival and reduced motor impairment of scrapie-diseased mice [55]. Mechanistically, PrPc coupling to the NADPH oxidase-TACE α-secretase signaling pathway limits the sensitivity of recipient cells to TNFα by promoting TACE-mediated cleavage of TNFα

shown to be necessary for maintaining TACE α-secretase at the plasma membrane

*Prnp*-knockout mice highly vulnerable to pro-inflammatory TNFα insult. Thus, abnormal trafficking and activity of TACE in prion diseases likely originates from a

Synaptolysis is believed to initiate the neurodegeneration arising after a decrease in depolarization-induced calcium transients that progressively impairs glutamate release [34]. 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

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 [59]. 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

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 [60], oxidative stress [61], inflammation [55] apoptosis, and autophagy [62]. The repertoire of pathways that lead to neuronal death is however limited [63]. 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 [60]. On the other hand, autophagy has been reported in TSEs, but its role in prion disease pathology is not well established [64]. However, the extensive synaptic autophagy observed

and TNFR1, thereby attenuating PrPTSE-induced neurotox-

expression was further


provoked TACE internalization,

**10**

in prion diseases [65] 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 [70]. 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 [73].

In several prion diseases, the cerebellum is a preferential prion target for scrapie [74–78], also observed in Creutzfeldt-Jakob disease (CJD) cases [79–87]. 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 zebrinfree PCs, thus forming complementary stripes of biochemically distinct PCs [88]. The most comprehensively studied zonal marker is zebrin II/aldolase C (ZII/AldC) [90]. 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 [59], the parasagittal compartmentation of the cerebellar cortex restricted 22L scrapie pathogenesis, including PrP22L accumulation, PC neurodegeneration, and gliosis. Indeed, PCs displayed a differential, subtypespecific 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 *in vitro* and *in vivo* neurotoxicity that elicits autophagy and apoptosis with a molecular signature similar to that observed in prion-infected animal brains [93]. This toxic PrP (TPrP) killed PrP-deficient neurons *in vitro* suggesting that a PrP-derived toxic signal can be generated within neurons independently of endogenous membrane-bound PrPc . Indeed, postnatal ablation of PrPc expression in neurons reversed neurodegeneration and affected disease progression in mice even though glial replication was maintained and PrPTSE accumulated [94]. Thus, prion pathogenesis is governed by both cell-autonomous mechanisms responsible for cellular dysfunction and neurodegeneration and noncellular-autonomous mechanisms propagating prion spread [95].

#### **Figure 1.**

*Banding pattern of PrP22L, EAAT4 zebrin and PC loss in the EAAT4-eGFP mouse cerebellum* **A***–***H***. The pattern of PrP22L deposits (immunoperoxidase (immunoHRP) in A and E is artificially visualized in red in C and G) correlated with the banding pattern of the zebrin excitatory amino acid transporter 4 (EAAT4-eGFP, green in B, F) in merged PrP22L-EAAT4 images C and G in the cerebellar vermis (A–C) and hemispheres (E–G) infected with 22L ic. (clinical stage 145 dpi). D, H. EAAT4-eGFP PCs in the same regions of the vermis (D) and hemisphere (H) of a noninfected EAAT4-eGFP mouse as shown in the cerebellum of the 22L-infected EAAT4-eGFP mouse (A–G). The zebrin bands are numbered according to the current nomenclature in A–K and indicated by arrowheads in F.* **I***–***K***. In the cerebellum infected icb. (preclinical stage), two bands of PrP22L deposits (6 and 7) are visualized by immunoHRP in I and artificially visualized in green in K. These cross crus2 and paramedian lobule (PM) and display a marked loss of CaBP-immunofluorescent PCs (red). Scale bars = 50 μm.* **L***. Quantitative analysis of EAAT4-expressing and -nonexpressing PCs in the cerebellum of EAAT4-eGFP mice infected i.c. (clinical stage). The EAAT4-nonexpressing PCs are more sensitive to 22L toxicity. \*p < 0.05. The number of mice analyzed is indicated on the bars in the graph.*

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 [96]. ER stress and caspase-12 activation have been identified in prioninfected N2a cells as well as in the brains of prion-diseased mice and CJD patients [97]. 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 [98]. 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 [99].

#### *2.1.2 Mitochondrial apoptosis in prion-infected cerebellar neurons*

PrPc has recently been suggested to participate in anti-apoptotic and antioxidative processes by interacting with the stress inducible protein 1 (STI-1) to regulate superoxide dismutase (SOD) activation [100]. The PrPc octapeptide repeat

**13**

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

impairs *Bax*-dependent neuronal death [106].

PrPc

[104, 105]. PrPc

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

the BAX conformation changes required for apoptosis activation suggesting that

Furthermore, transgenic expression of *Bax* or *Bax* and *Prnp* indicated that *Prnp*

Activation of the mitochondrial apoptotic pathway was observed when primary neurons were exposed to aggregated neurotoxic peptides like PrP106-126 or recombinant mutant PrP [107–109]. 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 [111] and scrapie strains 301V, 87V, 22A [76], 79A [110], M1000/Fukuoka-1 [114], 127S [115], 22L, 139A, and RML [116, 117]. More recently, activation of caspase-3 was found in PCs of 22L-infected mice [59]. 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 [119]. 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) [120] and Ngsk PrnP0/0 [121] 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 *Bax*-KO (*Bax*<sup>−</sup>/<sup>−</sup>) mice [124] and in mice expressing a human Bcl-2 transgene [125] was analyzed. Clinical signs of 22L scrapie (mainly ataxia) were similar to those previously described for C57Bl/6 mice [126]. *Bax*<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice infected by either intraperitoneal (ip.) or intracerebellar (icb.) route displayed ataxia 10–15 days sooner than wild-type mice. Survival times however, were similar in all genotypes (i.e., 223 dpi ip. and 129 dpi icb.). whereas 22L induced more severe cerebellar spongiosis via the icb. route than the ip. route, similar lesion profiles [71] were induced by 22L ip. in the brain of *Bax*<sup>−</sup>/<sup>−</sup> and wild-type mice and lesion profiles were not different in the brain of *Bax*<sup>−</sup>/<sup>−</sup>, HuBcl-2 and wild-type mice infected with 22L icb. (**Figure 2**). Anatomopathological analysis of the cerebral and cerebellar cortices of the 22L-diseased *Bax*<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice did not reveal any modified patterns of vacuolation, astrogliosis, and PrP22L deposits irrespective of the inoculation route. Synaptophysin and calcium-binding protein (CaBP) immunohistochemistry also revealed severe synapse and PC loss in all cases (**Figure 3**). Finally, quantitative analysis of the cerebellar granule cells immunolabeled for the nuclear marker NeuN revealed a significant loss of neurons in all genotypes infected by the icb. route (**Figure 4**). Surprisingly, no significant difference could be detected between *Bax*<sup>−</sup>/<sup>−</sup> and wild-type mice infected by the icb. route, whereas HuBcl-2 mice whose granule cells are rescued from developmental cell death [127] lost more granule cells than wild-type and Bax<sup>−</sup>/<sup>−</sup> mice (**Figure 4**). These data indicate that neither suppression of *Bax* nor overexpression of Bcl-2 protected cerebellar neurons from 22L scrapie-induced neurotoxicity. Thus, the granule cell

 may assure the neuroprotective function of Bcl-2 [103]. Along this line, *Prnp*0/0 neurons were more susceptible to apoptotic stimuli such as serum deprivation than

also protected primary neurons against BAX-dependent apoptosis.

impaired

or Bcl-2 expression

Bcl-2 has also been found [101, 102]. In addition, the third helix of PrPc

their wild-type counterparts, whereas they were rescued by PrPc

*Prions - Some Physiological and Pathophysiological Aspects*

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 [96]. ER stress and caspase-12 activation have been identified in prioninfected N2a cells as well as in the brains of prion-diseased mice and CJD patients [97]. 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 [98]. 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

*Banding pattern of PrP22L, EAAT4 zebrin and PC loss in the EAAT4-eGFP mouse cerebellum* **A***–***H***. The pattern of PrP22L deposits (immunoperoxidase (immunoHRP) in A and E is artificially visualized in red in C and G) correlated with the banding pattern of the zebrin excitatory amino acid transporter 4 (EAAT4-eGFP, green in B, F) in merged PrP22L-EAAT4 images C and G in the cerebellar vermis (A–C) and hemispheres (E–G) infected with 22L ic. (clinical stage 145 dpi). D, H. EAAT4-eGFP PCs in the same regions of the vermis (D) and hemisphere (H) of a noninfected EAAT4-eGFP mouse as shown in the cerebellum of the 22L-infected EAAT4-eGFP mouse (A–G). The zebrin bands are numbered according to the current nomenclature in A–K and indicated by arrowheads in F.* **I***–***K***. In the cerebellum infected icb. (preclinical stage), two bands of PrP22L deposits (6 and 7) are visualized by immunoHRP in I and artificially visualized in green in K. These cross crus2 and paramedian lobule (PM) and display a marked loss of CaBP-immunofluorescent PCs (red). Scale bars = 50 μm.* **L***. Quantitative analysis of EAAT4-expressing and -nonexpressing PCs in the cerebellum of EAAT4-eGFP mice infected i.c. (clinical stage). The EAAT4-nonexpressing PCs are more sensitive to 22L* 

 has recently been suggested to participate in anti-apoptotic and antioxidative processes by interacting with the stress inducible protein 1 (STI-1) to

octapeptide repeat

transcription of synaptic proteins, resulting in synaptic loss [99].

*toxicity. \*p < 0.05. The number of mice analyzed is indicated on the bars in the graph.*

*2.1.2 Mitochondrial apoptosis in prion-infected cerebellar neurons*

regulate superoxide dismutase (SOD) activation [100]. The PrPc

**12**

PrPc

**Figure 1.**

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 [103]. Along this line, *Prnp*0/0 neurons were more susceptible to apoptotic stimuli such as serum deprivation than their wild-type counterparts, whereas they were rescued by PrPc or Bcl-2 expression [104, 105]. PrPc also protected primary neurons against BAX-dependent apoptosis. Furthermore, transgenic expression of *Bax* or *Bax* and *Prnp* indicated that *Prnp* impairs *Bax*-dependent neuronal death [106].

Activation of the mitochondrial apoptotic pathway was observed when primary neurons were exposed to aggregated neurotoxic peptides like PrP106-126 or recombinant mutant PrP [107–109]. 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 [111] and scrapie strains 301V, 87V, 22A [76], 79A [110], M1000/Fukuoka-1 [114], 127S [115], 22L, 139A, and RML [116, 117]. More recently, activation of caspase-3 was found in PCs of 22L-infected mice [59]. 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 [119]. 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) [120] and Ngsk PrnP0/0 [121] 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 *Bax*-KO (*Bax*<sup>−</sup>/<sup>−</sup>) mice [124] and in mice expressing a human Bcl-2 transgene [125] was analyzed. Clinical signs of 22L scrapie (mainly ataxia) were similar to those previously described for C57Bl/6 mice [126]. *Bax*<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice infected by either intraperitoneal (ip.) or intracerebellar (icb.) route displayed ataxia 10–15 days sooner than wild-type mice. Survival times however, were similar in all genotypes (i.e., 223 dpi ip. and 129 dpi icb.). whereas 22L induced more severe cerebellar spongiosis via the icb. route than the ip. route, similar lesion profiles [71] were induced by 22L ip. in the brain of *Bax*<sup>−</sup>/<sup>−</sup> and wild-type mice and lesion profiles were not different in the brain of *Bax*<sup>−</sup>/<sup>−</sup>, HuBcl-2 and wild-type mice infected with 22L icb. (**Figure 2**). Anatomopathological analysis of the cerebral and cerebellar cortices of the 22L-diseased *Bax*<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice did not reveal any modified patterns of vacuolation, astrogliosis, and PrP22L deposits irrespective of the inoculation route. Synaptophysin and calcium-binding protein (CaBP) immunohistochemistry also revealed severe synapse and PC loss in all cases (**Figure 3**). Finally, quantitative analysis of the cerebellar granule cells immunolabeled for the nuclear marker NeuN revealed a significant loss of neurons in all genotypes infected by the icb. route (**Figure 4**). Surprisingly, no significant difference could be detected between *Bax*<sup>−</sup>/<sup>−</sup> and wild-type mice infected by the icb. route, whereas HuBcl-2 mice whose granule cells are rescued from developmental cell death [127] lost more granule cells than wild-type and Bax<sup>−</sup>/<sup>−</sup> mice (**Figure 4**). These data indicate that neither suppression of *Bax* nor overexpression of Bcl-2 protected cerebellar neurons from 22L scrapie-induced neurotoxicity. Thus, the granule cell

**Figure 2.**

*Spongiosis lesion profiles in the brain of wild-type (WT), Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice infected ip. and icb. with the 22L scrapie prion strain.* **A***. Very similar lesion profiles were induced by 22L scrapie ip. in Bax<sup>−</sup>/<sup>−</sup> and WT mice. 22L induced more severe cerebellar spongiosis via the icb. route than via the ip. route. 1: cingulate and 2nd motor cortices, 2: lateral and medial septum, 3: caudate putamen, 4: retrosplenial cortex, 5: hippocampus, 6: thalamus, 7: hypothalamus, 8: superior colliculus, 9A: cerebellar molecular layer, 9B: cerebellar granular layer, 9C: cerebellar white matter, 10: medulla.* **B***. Very similar lesion profiles were induced by 22L scrapie icb. in Bax<sup>−</sup>/<sup>−</sup>, HuBcl-2 and WT mice.*

and PC death induced by 22L scrapie does not seem to involve BAX and cannot be counteracted by overexpression of the anti-apoptotic factor BCL-2. However, cleaved caspase-3 and -9 were observed in the brains of Bax<sup>−</sup>/<sup>−</sup> mice, suggesting that apoptosis may occur through (an) alternative mechanism(s) in TSEs of infectious origin. Indeed, apoptotic features have been reported in the brain of wild-type mice infected with RML, in the absence of Bax upregulation [116], while other proteins involved in cell death including those associated with the mitochondrial inner membrane, the UPS and the endoplasmic-reticulum-associated protein degradation (ERAD) pathway [128] were upregulated.

#### *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 *ex vivo* [117, 129]. Infecting COCS with three different scrapie strains (RML, 22L, 139A) produced three distinct patterns of prion protein

**15**

**Figure 4.**

*brain homogenate.*

**Figure 3.**

deposition accompanied by salient features of prion disease pathogenesis such as severe neuronal loss, a pro-inflammatory response, and typical neuropathological changes (spongiform vacuolation, tubulovesicular structures, neuronal dystrophy,

*Quantitative analysis of cerebellar GCs immunostained for the nuclear marker NeuN revealed a significant loss of neurons in all genotypes infected icb., but not ip. \*p < 0.05; \*\*p < 0.01. Whereas Bax<sup>−</sup>/<sup>−</sup> and WT mice lost a similar amount of GCs, the HuBcl-2 mice lost more GCs than the WT and Bax<sup>−</sup>/<sup>−</sup> mice. NIB, noninfected* 

*Anatomopathology of 22L scrapie ip. and icb. in the cerebellum of WT, Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice. Neither Bax knockout nor HuBcl-2 overexpression modified vacuolation (Mason's trichrome), astrogliosis (GFAP immunoHRP) and PrP22L accumulation (PrP immunoHRP) patterns in the cerebellar cortex of the 22L ip. and icb. infected Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice compared to the WT mice. Synaptophysin and CaBP reveal respectively synapse and PC loss in the cerebellum of all mice. Loss of Neun-immunostained GCs is also prominent in the cerebellum of the WT, Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 infected icb., yet seemed less pronounced in the mice infected ip.*

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

#### *Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

#### **Figure 3.**

*Prions - Some Physiological and Pathophysiological Aspects*

and PC death induced by 22L scrapie does not seem to involve BAX and cannot be counteracted by overexpression of the anti-apoptotic factor BCL-2. However, cleaved caspase-3 and -9 were observed in the brains of Bax<sup>−</sup>/<sup>−</sup> mice, suggesting that apoptosis may occur through (an) alternative mechanism(s) in TSEs of infectious origin. Indeed, apoptotic features have been reported in the brain of wild-type mice infected with RML, in the absence of Bax upregulation [116], while other proteins involved in cell death including those associated with the mitochondrial inner membrane, the UPS and the endoplasmic-reticulum-associated protein degradation (ERAD)

*Spongiosis lesion profiles in the brain of wild-type (WT), Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice infected ip. and icb. with the 22L scrapie prion strain.* **A***. Very similar lesion profiles were induced by 22L scrapie ip. in Bax<sup>−</sup>/<sup>−</sup> and WT mice. 22L induced more severe cerebellar spongiosis via the icb. route than via the ip. route. 1: cingulate and 2nd motor cortices, 2: lateral and medial septum, 3: caudate putamen, 4: retrosplenial cortex, 5: hippocampus, 6: thalamus, 7: hypothalamus, 8: superior colliculus, 9A: cerebellar molecular layer, 9B: cerebellar granular layer, 9C: cerebellar white matter, 10: medulla.* **B***. Very similar lesion profiles were induced by 22L scrapie icb. in* 

*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 *ex vivo* [117, 129]. Infecting COCS with three different scrapie strains (RML, 22L, 139A) produced three distinct patterns of prion protein

**14**

**Figure 2.**

pathway [128] were upregulated.

*Bax<sup>−</sup>/<sup>−</sup>, HuBcl-2 and WT mice.*

*Anatomopathology of 22L scrapie ip. and icb. in the cerebellum of WT, Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice. Neither Bax knockout nor HuBcl-2 overexpression modified vacuolation (Mason's trichrome), astrogliosis (GFAP immunoHRP) and PrP22L accumulation (PrP immunoHRP) patterns in the cerebellar cortex of the 22L ip. and icb. infected Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 mice compared to the WT mice. Synaptophysin and CaBP reveal respectively synapse and PC loss in the cerebellum of all mice. Loss of Neun-immunostained GCs is also prominent in the cerebellum of the WT, Bax<sup>−</sup>/<sup>−</sup> and HuBcl-2 infected icb., yet seemed less pronounced in the mice infected ip.*

#### **Figure 4.**

*Quantitative analysis of cerebellar GCs immunostained for the nuclear marker NeuN revealed a significant loss of neurons in all genotypes infected icb., but not ip. \*p < 0.05; \*\*p < 0.01. Whereas Bax<sup>−</sup>/<sup>−</sup> and WT mice lost a similar amount of GCs, the HuBcl-2 mice lost more GCs than the WT and Bax<sup>−</sup>/<sup>−</sup> mice. NIB, noninfected brain homogenate.*

deposition accompanied by salient features of prion disease pathogenesis such as severe neuronal loss, a pro-inflammatory response, and typical neuropathological changes (spongiform vacuolation, tubulovesicular structures, neuronal dystrophy, and gliosis). Neurodegeneration did not occur when PrP was genetically removed from neurons and was abrogated by compounds known to antagonize prion replication. Also, calpain inhibitors, but not caspase inhibitors, prevented neurotoxicity and fodrin cleavage; whereas, prion replication was unimpeded indicating that inhibiting calpain uncouples prion replication and neurotoxicity. These data validate the COCS as a powerful model system that faithfully reproduces many morphological hallmarks of prion infections and shows that prion neurotoxicity in cerebellar granule cells is calpain-dependent but caspase-independent.

Furthermore, significant spine loss and altered dendritic morphology, analogous to that seen *in vivo* were induced by RML scrapie in COCS [130], while the deposition pattern and subcellular distribution of PrP22L (i.e., granular deposits associated with neurons, astrocytes, and microglia but not PCs in the neuropil of the PC and molecular layers [131]), closely resembled that observed *in vivo* [59].

Following infection of COCS from C57Bl6/J, ZH-I Prnp0/0, and Tga20 PrP-overexpressing 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

#### **Figure 5.**

*Histoblots of cultured organotypic cerebellar slices (COCS) infected with the 22L and 139A scrapie strains. PrPc was detected in histoblots of noninfected (sham) COCS from WT C57Bl6/J (A) and Tga20 PrP-overexpressing (C), but not PrP-deficient ZH-I PrnP0/0 (B) mice. PrPc was completely digested by proteinase K (PK) in these COCS. After 30 and 20 days postinfection (dpi), PK revealed undigested PrP22L and PrP139A respectively in the WT and Tga20, but not ZH-I Prnp0/0 infected COCS.*

**17**

**Figure 7.**

**Figure 6.**

*with 22L and 139A scrapie prions at 30 dpi.*

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

*Mean numbers of CaBP-immunofluorescent PCs in WT and Tga20 COCS noninfected (sham) and infected* 

*Ultrastructural features of the C57Bl6/J mouse cerebellar cortex in noninfected COCS after 30 DIV.* **A***. Laminar organization of the cerebellar cortex with PC at the interface between internal granular layer (IGL) and molecular layer (ML).* **B***. Granule cells in the IGL.* **C***. IGL neuropil.* **D***. PC dendrite (d) in the ML neuropil.*  **E***,* **F***. Asymmetrical synapses (arrows) on interneurons dendrites (E) and PC spines (F).* **G***. PC. N, PC nucleus.* **H***. A smooth saccule (arrow) typically separates a mitochondrion from the plasma membrane in the PC neuroplasm.* **I***. Nissl body in the PC neuroplasm.* **J***. Degenerated cell with electron-dense vacuolated cytoplasm.* **K***. Autophagic digestion of a mitochondrion (\*).* **L***. Autophagic profiles in a PC axon (\*). Scale* 

*bars = 10 μm in A, 2 μm in B–D, G, J, 500 nm in E, F, H, I, K, L.*

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

#### **Figure 6.**

*Prions - Some Physiological and Pathophysiological Aspects*

and gliosis). Neurodegeneration did not occur when PrP was genetically removed from neurons and was abrogated by compounds known to antagonize prion replication. Also, calpain inhibitors, but not caspase inhibitors, prevented neurotoxicity and fodrin cleavage; whereas, prion replication was unimpeded indicating that inhibiting calpain uncouples prion replication and neurotoxicity. These data validate the COCS as a powerful model system that faithfully reproduces many morphological hallmarks of prion infections and shows that prion neurotoxicity in

Furthermore, significant spine loss and altered dendritic morphology, analogous to that seen *in vivo* were induced by RML scrapie in COCS [130], while the deposition pattern and subcellular distribution of PrP22L (i.e., granular deposits associated with neurons, astrocytes, and microglia but not PCs in the neuropil of the PC and

Following infection of COCS from C57Bl6/J, ZH-I Prnp0/0, and Tga20 PrP-overexpressing 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

*Histoblots of cultured organotypic cerebellar slices (COCS) infected with the 22L and 139A scrapie strains. PrPc was detected in histoblots of noninfected (sham) COCS from WT C57Bl6/J (A) and Tga20 PrP-overexpressing* 

*COCS. After 30 and 20 days postinfection (dpi), PK revealed undigested PrP22L and PrP139A respectively in the* 

 *was completely digested by proteinase K (PK) in these* 

cerebellar granule cells is calpain-dependent but caspase-independent.

molecular layers [131]), closely resembled that observed *in vivo* [59].

**16**

**Figure 5.**

*(C), but not PrP-deficient ZH-I PrnP0/0 (B) mice. PrPc*

*WT and Tga20, but not ZH-I Prnp0/0 infected COCS.*

*Mean numbers of CaBP-immunofluorescent PCs in WT and Tga20 COCS noninfected (sham) and infected with 22L and 139A scrapie prions at 30 dpi.*

#### **Figure 7.**

*Ultrastructural features of the C57Bl6/J mouse cerebellar cortex in noninfected COCS after 30 DIV.* **A***. Laminar organization of the cerebellar cortex with PC at the interface between internal granular layer (IGL) and molecular layer (ML).* **B***. Granule cells in the IGL.* **C***. IGL neuropil.* **D***. PC dendrite (d) in the ML neuropil.*  **E***,* **F***. Asymmetrical synapses (arrows) on interneurons dendrites (E) and PC spines (F).* **G***. PC. N, PC nucleus.* **H***. A smooth saccule (arrow) typically separates a mitochondrion from the plasma membrane in the PC neuroplasm.* **I***. Nissl body in the PC neuroplasm.* **J***. Degenerated cell with electron-dense vacuolated cytoplasm.* **K***. Autophagic digestion of a mitochondrion (\*).* **L***. Autophagic profiles in a PC axon (\*). Scale bars = 10 μm in A, 2 μm in B–D, G, J, 500 nm in E, F, H, I, K, L.*

#### **Figure 8.**

*Cytopathology of the C57Bl6/J mouse cerebellar cortex in COCS infected with 22L (A–F) and 139A (G–L) scrapie prions at 30 dpi.* **A***. IGL.* **B***–***D***. Autophagic profiles in the IGL neuropil.* **B***. Magnification of the inset in A.* **D***. Electron-dense lysosomes.* **E***,* **F***. Various stages of ER-derived reticulated organelles (arrows) in the neuroplasm of a PC.* **G***. Neurodegenerating profiles (\*) in the IGL neuropil.* **H***,* **I***. PC neuroplasm containing different stages of ER-derived reticulated organelles (arrows) and vacuoles. Scale bars = 2 μm in A–D, G–I, 500 nm in E, F.*

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, 132–135]. These cardinal proteins contribute to synaptic dysregulation and altered organelles leading to apoptosis. The neurodegenerating neurons exhibit robust accumulation of cytosolic autophagosomes (see [14] for review, **Figure 10**) suggesting a dysregulation of the autophagic flux resulting from autophagic stress, due to an imbalance between protein synthesis and degradation [136]. Autophagy reduces intraneuronal aggregates and slows down the progression of clinical disease in experimental models of AD [137–139] and prion diseases [140, 141]. Thus, dysregulation of the autophagic

**19**

**Figure 9.**

flux impairs the elimination of misfolded proteins and damaged organelles which then accumulate in the cytoplasm and contribute to cell dysfunction and death [142]. 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

*Cytopathological formation and evolution of ER-derived profiles in PCs of COCS infected with 139A scrapie prions at 30 dpi.* **A***,* **B***. Reticulation and sequestration of neuroplasm by ER saccules forming small double-membrane vesicles (arrows) containing ribosome-like particles (arrowheads in B) on both external and internal faces. ER, endoplasmic reticulum.* **C***,* **D***. Large compartmented ER-derived organelles which still display membrane-bound ribosomes (arrowheads). C. High magnification of* **Figure 8H***. D. See the enlargement between membranes (\*).* **E***. Fusion (arrows) of small ER-derived double-membraned vacuoles with lysosomes (L).* **F***. Large ER-derived double-membraned vacuoles with enlarged intermembrane space (\*)* 

*transforming into multivesicular vacuoles (arrows). Scale bars = 500 nm.*

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

*Prions - Some Physiological and Pathophysiological Aspects*

*Cytopathology of the C57Bl6/J mouse cerebellar cortex in COCS infected with 22L (A–F) and 139A (G–L) scrapie prions at 30 dpi.* **A***. IGL.* **B***–***D***. Autophagic profiles in the IGL neuropil.* **B***. Magnification of the inset in A.* **D***. Electron-dense lysosomes.* **E***,* **F***. Various stages of ER-derived reticulated organelles (arrows) in the neuroplasm of a PC.* **G***. Neurodegenerating profiles (\*) in the IGL neuropil.* **H***,* **I***. PC neuroplasm containing different stages of ER-derived reticulated organelles (arrows) and vacuoles. Scale bars = 2 μm in A–D, G–I,* 

alterations were not observed in noninfected slices suggesting that a specific effect of prions links prion-induced ER stress to this morphological ER modification.

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, 132–135]. These cardinal proteins contribute to synaptic dysregulation and altered organelles leading to apoptosis. The neurodegenerating neurons exhibit robust accumulation of cytosolic autophagosomes (see [14] for review, **Figure 10**) suggesting a dysregulation of the autophagic flux resulting from autophagic stress, due to an imbalance between protein synthesis and degradation [136]. Autophagy reduces intraneuronal aggregates and slows down the progression of clinical disease in experimental models of AD [137–139] and prion diseases [140, 141]. Thus, dysregulation of the autophagic

**18**

**Figure 8.**

*500 nm in E, F.*

**2.2 Prion-induced autophagy**

#### **Figure 9.**

*Cytopathological formation and evolution of ER-derived profiles in PCs of COCS infected with 139A scrapie prions at 30 dpi.* **A***,* **B***. Reticulation and sequestration of neuroplasm by ER saccules forming small double-membrane vesicles (arrows) containing ribosome-like particles (arrowheads in B) on both external and internal faces. ER, endoplasmic reticulum.* **C***,* **D***. Large compartmented ER-derived organelles which still display membrane-bound ribosomes (arrowheads). C. High magnification of* **Figure 8H***. D. See the enlargement between membranes (\*).* **E***. Fusion (arrows) of small ER-derived double-membraned vacuoles with lysosomes (L).* **F***. Large ER-derived double-membraned vacuoles with enlarged intermembrane space (\*) transforming into multivesicular vacuoles (arrows). Scale bars = 500 nm.*

flux impairs the elimination of misfolded proteins and damaged organelles which then accumulate in the cytoplasm and contribute to cell dysfunction and death [142].

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

#### **Figure 10.**

*Autophagy in PCs of 4.5 (A–E) and 12 (F) month-old control Bax+/+; Prnp+/+ (E) and Bax<sup>−</sup>/<sup>−</sup>;Ngsk Prnp0/0 (A–D, F) mice. Ultrastructural autophagic stages from phagophores to autolysosomes.* **A***. Phagophore (\*) and double-membraned autophagosome (arrowhead).* **B***. Sequestration of two mitochondria in an autophagosome (arrowhead). Go, Golgi dichtyosome.* **C***. Fusion of an autophagosome (arrowhead) with a lysosome (\*). Ly, lysosomes.* **D***. Autolysosomes (\*). A–D. Scale bars = 500 nm.* **E***. The somato-dendritic cytoplasm of this control Bax+/+; Prnp+/+ PC contains a few lysosomes and lipofuchsin bodies (arrowheads). N, nucleus; n, nucleolus.*  **F***. Autophagic PC with numerous autophagic organelles (arrowheads) accumulating in the neuroplasm. ML, molecular layer. IGL, internal granular layer. E, F. Arrows show PC axon. Scale bars = 2 μm.*

[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 [148]. Thus, the loss or subversion of PrPc function resulting from prion infection may upregulate autophagy in diseased neurons [16]. While

**21**

proposed to interfere with PrPc

hypothesis, PrPc

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

**3. Neuronal death in prion protein-deficient mice**

**3.1 Impaired autophagy in Zrch-1 prion protein-deficient mice**

[148, 157] suggesting that suppression of the protective effects of PrPc

**3.2 Neuronal loss in Dpl-expressing Ngsk prion protein-deficient mice**

to the aberrant overexpression of the *Prnd* gene encoding the PrPc

Nagasaki (Ngsk) PrP-deficient mice which have a deletion of the entire *Prnp* gene [161–163] develop progressive cerebellar ataxia, which was later discovered to result from the absence of a splice acceptor site in exon 3 of *Prnp* [164]. This leads

[165, 166] that causes selective degeneration of cerebellar PCs. Notably, the reintroduction of *Prnp* in mice overexpressing *Prnd* in the brain rescued the phenotype, suggesting a functional link between the two proteins [167]. Dpl has been shown to have intrinsic neurotoxic properties in cerebellar neurons [168] and has been

and Dpl bind a common ligand LPrP, where PrPc

cell survival signal while Dpl binding activates a death signaling cascade. In PrPc

deficient *Prnp*-knockout mice that do not express Dpl, the existence of a protein

and affect cell survival [100]. According to this

models do not show gross abnormalities indicating that PrPc

With the exception of the *Prnp*-knockout models in which ectopic expression of Doppel (Dpl) in the CNS leads to PC death, most other *Prnp*-knockout mouse

embryonic development and adulthood. Nevertheless, PrP-deficient mice exhibit an increased predilection for seizures, motor and cognitive disabilities, reduced synaptic inhibition, and long term potentiation in the hippocampus. Also, altered development of the granule cell layer, dysregulation of the cerebellar network and age-dependent spongiform changes with reactive astrogliosis have been observed [155, 156]. In cultures of PrP-deficient hippocampal neurons, autophagy is upregulated in the absence of serum or by hydrogen peroxide-induced oxidative stress

the autophagic flux in PrP-deficient neurons *in vivo*. Indeed, ultrastructural examination of hippocampus and cerebral cortex of ZH-I *Prnp*0/0 mice revealed an accumulation of autophagosomes containing incompletely digested material increasing from 3 to 12 months of age [158]. In addition, an ultrastructural examination of PCs in the cerebellum of ZH-I *Prnp*0/0 mice revealed significant autophagic accumulation in the somato-dendritic compartment of these neurons from 6 to 14.5 months of age (**Figure 11**). Since autophagic cell death is known to induce neurodegeneration [136, 159, 160], these signs of autophagy blockade could reflect a sustained, progressive autophagic neuronal loss in the CNS of the ZH-I *Prnp*0/0 mice.

may be dispensable for

could impair

paralogue Dpl

binding induces a


process [141, 154].

autophagy-inducing agents increased cellular clearance of PrPTSE [149–151], blocking the fusion of autophagosomes with lysosomes allowed visualization of PrPTSE in the autophagosomes suggesting that degradation of endosomal PrPTSE is by autophagy [134]. However, saturation of the autolysosomal degradation process can release PrPTSE aggregates and degradation enzymes into the neuroplasm contributing to autophagy upregulation and neuronal death [134]. Nevertheless, although autophagy-inducing agents delayed disease onset and PrPTSE accumulation in the CNS of mice [152], survival time was not modified [153]. 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

*Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

*Prions - Some Physiological and Pathophysiological Aspects*

[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

*molecular layer. IGL, internal granular layer. E, F. Arrows show PC axon. Scale bars = 2 μm.*

*Autophagy in PCs of 4.5 (A–E) and 12 (F) month-old control Bax+/+; Prnp+/+ (E) and Bax<sup>−</sup>/<sup>−</sup>;Ngsk Prnp0/0 (A–D, F) mice. Ultrastructural autophagic stages from phagophores to autolysosomes.* **A***. Phagophore (\*) and double-membraned autophagosome (arrowhead).* **B***. Sequestration of two mitochondria in an autophagosome (arrowhead). Go, Golgi dichtyosome.* **C***. Fusion of an autophagosome (arrowhead) with a lysosome (\*). Ly, lysosomes.* **D***. Autolysosomes (\*). A–D. Scale bars = 500 nm.* **E***. The somato-dendritic cytoplasm of this control Bax+/+; Prnp+/+ PC contains a few lysosomes and lipofuchsin bodies (arrowheads). N, nucleus; n, nucleolus.*  **F***. Autophagic PC with numerous autophagic organelles (arrowheads) accumulating in the neuroplasm. ML,* 

N-terminal octapeptide repeat region, exerts a negative control on the induc-

from prion infection may upregulate autophagy in diseased neurons [16]. While

tion of autophagy [148]. Thus, the loss or subversion of PrPc

, but not truncated PrP devoid of the

function resulting

**20**

**Figure 10.**

FFI-diseased patients.

Recent evidence indicated that PrPc

autophagy-inducing agents increased cellular clearance of PrPTSE [149–151], blocking the fusion of autophagosomes with lysosomes allowed visualization of PrPTSE in the autophagosomes suggesting that degradation of endosomal PrPTSE is by autophagy [134]. However, saturation of the autolysosomal degradation process can release PrPTSE aggregates and degradation enzymes into the neuroplasm contributing to autophagy upregulation and neuronal death [134]. Nevertheless, although autophagy-inducing agents delayed disease onset and PrPTSE accumulation in the CNS of mice [152], survival time was not modified [153]. 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].
