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

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

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 models do not show gross abnormalities indicating that PrPc may be dispensable for 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 [148, 157] suggesting that suppression of the protective effects of PrPc could impair 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.

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

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 to the aberrant overexpression of the *Prnd* gene encoding the PrPc paralogue Dpl [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 proposed to interfere with PrPc and affect cell survival [100]. According to this hypothesis, PrPc and Dpl bind a common ligand LPrP, where PrPc binding induces a 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

#### **Figure 11.**

*Autophagy in ZH-I Prnp0/0 PCs.* **A***. Mitophagy in the PC neuroplasm of a 4.5 months-old ZH-I Prnp0/0 mouse. Arrowhead shows the double membrane of an autophagic vacuole sequestrating a mitochondrion (m). Scale bar = 500 nm.* **B***,* **C***. 12 months-old ZH-I Prnp0/0 mice. PC layer. B. Autophagic PC containing numerous autophagosomes and autolysosomes (arrowheads). Scale bar = 2 μm. C. PC layer. Degenerating PC axons containing autophagosomes and lysosomes (\*). Scale bar =500 nm.*

π has been proposed to induce a cell survival signal when bound to LPrP [169]. For the moment, LPrP and π remain to be identified, as well as the neuronal death pathways involved in Dpl-induced PC loss.

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 [121] or over-express the anti-apoptotic factor Bcl-2 [170]. Although this suggests that an intrinsic apoptotic process is involved in the death of the Ngsk *Prnp*0/0 PCs, a significant PC loss still occurred in both

**23**

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

to be determined.

(**Figure 13**).

PCs, nor in the absence of PrPc

the same PCs that are rescued by *Bax* deletion.

(Bax<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0) and (HuBcl-2; Ngsk *Prnp*0/0) double mutants. Thus, the Ngsk condition, i.e., Dpl neurotoxicity and PrP-deficiency, could activate BAXindependent mechanisms in the Ngsk *Prnp*0/0 PCs. These neurons exhibited robust autophagy well before significant neuronal death in the cerebellar cortex of the Ngsk *Prnp*0/0 mice [135, 171] suggesting that either "reactive" autophagy is initially induced as a neuroprotective response to Dpl neurotoxicity or impaired autophagy results from PrP-deficiency as in ZH-I *Prnp*0/0 mice (see above and [158]). Indeed, the increased expression of the autophagic markers SCRG1, LC3-II, and P62 proteins without any changes in mRNA levels, indicates that the ultimate steps of autophagic degradation are impaired in Ngsk *Prnp*0/0 PCs [135]. Probably due to this impairment of autophagic proteolysis, LC3-II-, and Lamp-1 labeled autophagosomes and autolysosomes [172] accumulate in the Ngsk *Prnp*0/0 PCs. How apoptosis and autophagy are involved in Ngsk *Prnp*0/0 PC death remains

To further investigate the role of autophagy in the death of Ngsk *Prnp*0/0 PCs, a quantitative analysis of autophagic PCs was performed at the ultrastructural level in the cerebellum of Ngsk *Prnp*0/0, *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0, ZCH-I *Prnp*0/0 and control *Bax*+/+;*Prnp*+/+ mice (**Figure 12**). At 4.5 months of age, equivalent amounts of autophagic somato-dendritic compartments and axons of PCs were found in the cerebella of Ngsk *Prnp*0/0 and *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0 mutants and were significantly more than those in ZCH-I *Prnp*0/0 and control *Bax*+/+;Prnp+/+ cerebella. Interestingly, the amounts of autophagic axons and somato-dendritic compartments of PCs in the ZCH-I *Prnp*0/0 and control *Bax*+/+;*Prnp*+/+ cerebella were not different. These data suggest that while autophagy induction is already visible in PCs with the Ngsk condition, it is not induced in control *Bax*+/+;*Prnp*+/+

to be induced by Dpl neurotoxicity in the Ngsk condition whether BAX is present or not; whereas PrP-deficiency alone has no autophagy-inducing effect at this age

At 6.5–7 months of age, the amount of autophagic somato-dendritic compart-

At 12 months of age, the amount of autophagic somato-dendritic compartments and axons of PCs in *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebella was equivalent to that found in 4.5 month-old cerebella suggesting that autophagy remains stable in this PC population, at a level similar to that maintained in the ZH-I *Prnp*0/0, and this likely results from PrP-deficiency. Indeed, many more autophagic PC somatodendritic compartments and axons were observed in ZH-I *Prnp*0/0 cerebella than in the cerebella of 12 month-old control *Bax*+/+;*Prnp*+/+ mice which did not contain autophagic PCs. However, the autophagic PC somato-dendritic compartments were

ments and axons of PC were significantly decreased in *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0 cerebella compared with Ngsk *Prnp*0/0 cerebella. Consequently, the amount of autophagic PC profiles in the *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0 and ZH-I *Prnp*0/0 cerebella was equivalent, yet more than in the control *Bax*+/+;*Prnp*+/+ cerebella. Furthermore, autophagic PC somato-dendritic compartments and axons did not change from 4.5 to 6.5–7 months of age in the *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0, whereas many more PCs were autophagic in the 6.5–7 month-old compared to the 4.5 month-old Ngsk *Prnp*0/0 cerebella. This increase was also observed in ZH-I *Prnp*0/0 cerebella, while no autophagic PCs were found in 6.5–7 month-old control *Bax*+/+;*Prnp*+/+ cerebella (**Figure 13**). This suggests that BAX-deficiency modulates autophagy in Ngsk *Prnp*0/0 PCs after 4.5 months of age. Autophagy in the ZH-I *Prnp*0/0 PCs had increased to the same level as observed in the *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebella. Thus, the persistent autophagy in the PCs of the *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 double mutants is likely related to PrP-deficiency. Also, autophagy- and *Bax*-dependent apoptosis are likely to occur in

in the ZCH-I *Prnp*0/0 PCs. Thus, autophagy seems

*Prions - Some Physiological and Pathophysiological Aspects*

**22**

**Figure 11.**

*Autophagy in ZH-I Prnp0/0 PCs.* **A***. Mitophagy in the PC neuroplasm of a 4.5 months-old ZH-I Prnp0/0 mouse. Arrowhead shows the double membrane of an autophagic vacuole sequestrating a mitochondrion (m). Scale bar = 500 nm.* **B***,* **C***. 12 months-old ZH-I Prnp0/0 mice. PC layer. B. Autophagic PC containing numerous autophagosomes and autolysosomes (arrowheads). Scale bar = 2 μm. C. PC layer. Degenerating PC axons* 

π has been proposed to induce a cell survival signal when bound to LPrP [169]. For the moment, LPrP and π remain to be identified, as well as the neuronal death

the underlying neurotoxic mechanism may provide important insight into the

toxicity increased in the cerebellum of Ngsk mice, which were either deficient for the pro-apoptotic factor Bax [121] or over-express the anti-apoptotic factor Bcl-2 [170]. Although this suggests that an intrinsic apoptotic process is involved in the death of the Ngsk *Prnp*0/0 PCs, a significant PC loss still occurred in both


. The resistance of the PC population to neuro-

*containing autophagosomes and lysosomes (\*). Scale bar =500 nm.*

Because Dpl neurotoxicity depends on PrPc

pathways involved in Dpl-induced PC loss.

neuroprotective function of PrPc

(Bax<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0) and (HuBcl-2; Ngsk *Prnp*0/0) double mutants. Thus, the Ngsk condition, i.e., Dpl neurotoxicity and PrP-deficiency, could activate BAXindependent mechanisms in the Ngsk *Prnp*0/0 PCs. These neurons exhibited robust autophagy well before significant neuronal death in the cerebellar cortex of the Ngsk *Prnp*0/0 mice [135, 171] suggesting that either "reactive" autophagy is initially induced as a neuroprotective response to Dpl neurotoxicity or impaired autophagy results from PrP-deficiency as in ZH-I *Prnp*0/0 mice (see above and [158]). Indeed, the increased expression of the autophagic markers SCRG1, LC3-II, and P62 proteins without any changes in mRNA levels, indicates that the ultimate steps of autophagic degradation are impaired in Ngsk *Prnp*0/0 PCs [135]. Probably due to this impairment of autophagic proteolysis, LC3-II-, and Lamp-1 labeled autophagosomes and autolysosomes [172] accumulate in the Ngsk *Prnp*0/0 PCs. How apoptosis and autophagy are involved in Ngsk *Prnp*0/0 PC death remains to be determined.

To further investigate the role of autophagy in the death of Ngsk *Prnp*0/0 PCs, a quantitative analysis of autophagic PCs was performed at the ultrastructural level in the cerebellum of Ngsk *Prnp*0/0, *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0, ZCH-I *Prnp*0/0 and control *Bax*+/+;*Prnp*+/+ mice (**Figure 12**). At 4.5 months of age, equivalent amounts of autophagic somato-dendritic compartments and axons of PCs were found in the cerebella of Ngsk *Prnp*0/0 and *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0 mutants and were significantly more than those in ZCH-I *Prnp*0/0 and control *Bax*+/+;Prnp+/+ cerebella. Interestingly, the amounts of autophagic axons and somato-dendritic compartments of PCs in the ZCH-I *Prnp*0/0 and control *Bax*+/+;*Prnp*+/+ cerebella were not different. These data suggest that while autophagy induction is already visible in PCs with the Ngsk condition, it is not induced in control *Bax*+/+;*Prnp*+/+ PCs, nor in the absence of PrPc in the ZCH-I *Prnp*0/0 PCs. Thus, autophagy seems to be induced by Dpl neurotoxicity in the Ngsk condition whether BAX is present or not; whereas PrP-deficiency alone has no autophagy-inducing effect at this age (**Figure 13**).

At 6.5–7 months of age, the amount of autophagic somato-dendritic compartments and axons of PC were significantly decreased in *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0 cerebella compared with Ngsk *Prnp*0/0 cerebella. Consequently, the amount of autophagic PC profiles in the *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0 and ZH-I *Prnp*0/0 cerebella was equivalent, yet more than in the control *Bax*+/+;*Prnp*+/+ cerebella. Furthermore, autophagic PC somato-dendritic compartments and axons did not change from 4.5 to 6.5–7 months of age in the *Bax*<sup>−</sup>/<sup>−</sup>; Ngsk *Prnp*0/0, whereas many more PCs were autophagic in the 6.5–7 month-old compared to the 4.5 month-old Ngsk *Prnp*0/0 cerebella. This increase was also observed in ZH-I *Prnp*0/0 cerebella, while no autophagic PCs were found in 6.5–7 month-old control *Bax*+/+;*Prnp*+/+ cerebella (**Figure 13**).

This suggests that BAX-deficiency modulates autophagy in Ngsk *Prnp*0/0 PCs after 4.5 months of age. Autophagy in the ZH-I *Prnp*0/0 PCs had increased to the same level as observed in the *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebella. Thus, the persistent autophagy in the PCs of the *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 double mutants is likely related to PrP-deficiency. Also, autophagy- and *Bax*-dependent apoptosis are likely to occur in the same PCs that are rescued by *Bax* deletion.

At 12 months of age, the amount of autophagic somato-dendritic compartments and axons of PCs in *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebella was equivalent to that found in 4.5 month-old cerebella suggesting that autophagy remains stable in this PC population, at a level similar to that maintained in the ZH-I *Prnp*0/0, and this likely results from PrP-deficiency. Indeed, many more autophagic PC somatodendritic compartments and axons were observed in ZH-I *Prnp*0/0 cerebella than in the cerebella of 12 month-old control *Bax*+/+;*Prnp*+/+ mice which did not contain autophagic PCs. However, the autophagic PC somato-dendritic compartments were

#### **Figure 12.**

*Quantitative analysis of autophagy in PCs of control Bax+/+;Prnp+/+ and PrP-deficient Bax+/+;Ngsk Prnp0/0, Bax<sup>−</sup>/<sup>−</sup>;Ngsk Prnp0/0 and ZH-I Prnp0/0 mutant mice. Autophagic somato-dendritic and axonal profiles were counted in 200 PCs in transverse cerebellar sections (50 PCs per hemisphere and hemivermis) from each mouse at 4.5, 6.5–7 and 12 months of age (n = 3 mice/age/genotype). PC soma, primary dendrite and axons were autophagic when containing three or more autophagic profiles (phagophore, autophagosome, autophagolysosome). Data are given as mean values ± standard deviation (SD). Statistical comparisons between ages and genotypes were performed using a two-tailed Student's t test (Statistica).* **A***. Mean percentages of autophagic PC somato-dendritic and axonal compartments. \*, #, @, \$, §: p < 0.01.* **B***. Mean percentages of autophagic PC presynaptic boutons making symmetrical synapses on somato-dendritic profiles of deep cerebellar neurons. The PC presynaptic boutons were autophagic when containing at least one autophagic organelle. Autophagic PC presynaptic boutons were counted in 300 presynaptic boutons selected randomly in either left or right fastigial, interposed and dentate nuclei (100 boutons/nucleus) in three 12 month-old mice of each genotype. Statistical comparisons between genotypes were performed using a two-tailed Student's t test (Statistica) and given as mean values ± standard deviation (SD). \*, p < 0.01.*

**25**

**Figure 13.**

*myelinated axon (\*). Scale bars = 2 μm.*

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

still more in Ngsk *Prnp*0/0 cerebella (16.36 ± 7.9) compared with *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 (5.08 ± 5) cerebella, and there was a significant increase from 6.5–7 (14.38 ± 7.8) to 12 months of age. The increased amount of autophagic PC axons in *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebella was stable during this same period (3.9 ± 5.6 at 6.5–7 months; 5.08 ± 5.1 at 12 months), suggesting that the initiation of axonal autophagy peaks at 6.5–7 months of age (**Figure 12**) [135, 171, 173–176]. In agreement, an examination of autophagy in the presynaptic terminals of PCs impinging on the somato-dendritic compartments of the deep nuclear neurons in the fastigial, interposed and dentate

*Autophagy in PCs of 7 (A, B) and 12 (C, D) month-old Bax<sup>−</sup>/<sup>−</sup>;Ngsk Prnp0/0 mice.* **A***. PC-like somatodendritic profile containing numerous autophagic vacuoles and autolysosomes (arrowheads) in the PC layer.*  **B***. Autolysosomes (arrowheads) in a dystrophic PC-like, myelinated axonal profile in the internal granular layer.* **C***. Autophagic vacuoles and autolysosomes in a PC-like somato-dendritic profile.* **D***. Autophagic PC-like*  *Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

*Prions - Some Physiological and Pathophysiological Aspects*

**24**

**Figure 12.**

*Quantitative analysis of autophagy in PCs of control Bax+/+;Prnp+/+ and PrP-deficient Bax+/+;Ngsk Prnp0/0, Bax<sup>−</sup>/<sup>−</sup>;Ngsk Prnp0/0 and ZH-I Prnp0/0 mutant mice. Autophagic somato-dendritic and axonal profiles were counted in 200 PCs in transverse cerebellar sections (50 PCs per hemisphere and hemivermis) from each mouse at 4.5, 6.5–7 and 12 months of age (n = 3 mice/age/genotype). PC soma, primary dendrite and axons were autophagic when containing three or more autophagic profiles (phagophore, autophagosome, autophagolysosome). Data are given as mean values ± standard deviation (SD). Statistical comparisons between ages and genotypes were performed using a two-tailed Student's t test (Statistica).* **A***. Mean percentages of autophagic PC somato-dendritic and axonal compartments. \*, #, @, \$, §: p < 0.01.* **B***. Mean percentages of autophagic PC presynaptic boutons making symmetrical synapses on somato-dendritic profiles of deep cerebellar neurons. The PC presynaptic boutons were autophagic when containing at least one autophagic organelle. Autophagic PC presynaptic boutons were counted in 300 presynaptic boutons selected randomly in either left or right fastigial, interposed and dentate nuclei (100 boutons/nucleus) in three 12 month-old mice of each genotype. Statistical comparisons between genotypes were performed using a two-tailed Student's t test* 

*(Statistica) and given as mean values ± standard deviation (SD). \*, p < 0.01.*

#### **Figure 13.**

*Autophagy in PCs of 7 (A, B) and 12 (C, D) month-old Bax<sup>−</sup>/<sup>−</sup>;Ngsk Prnp0/0 mice.* **A***. PC-like somatodendritic profile containing numerous autophagic vacuoles and autolysosomes (arrowheads) in the PC layer.*  **B***. Autolysosomes (arrowheads) in a dystrophic PC-like, myelinated axonal profile in the internal granular layer.* **C***. Autophagic vacuoles and autolysosomes in a PC-like somato-dendritic profile.* **D***. Autophagic PC-like myelinated axon (\*). Scale bars = 2 μm.*

still more in Ngsk *Prnp*0/0 cerebella (16.36 ± 7.9) compared with *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 (5.08 ± 5) cerebella, and there was a significant increase from 6.5–7 (14.38 ± 7.8) to 12 months of age. The increased amount of autophagic PC axons in *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebella was stable during this same period (3.9 ± 5.6 at 6.5–7 months; 5.08 ± 5.1 at 12 months), suggesting that the initiation of axonal autophagy peaks at 6.5–7 months of age (**Figure 12**) [135, 171, 173–176]. In agreement, an examination of autophagy in the presynaptic terminals of PCs impinging on the somato-dendritic compartments of the deep nuclear neurons in the fastigial, interposed and dentate

#### **Figure 14.**

*Autophagy in the deep cerebellar nuclei of 13 month-old Ngsk (A, C–F) and 10 month-old ZH-I (B) Prnp0/0 mice.* **A***–***E***. PC presynaptic boutons establishing symmetrical synapses (arrowheads) with somato-dendritic profiles of deep cerebellar neurons (DCN) and containing different stages of double-membrane wraps sequestrating neuroplasm (\* in A, B, D, E) and mitochondria (m in C, F).* **F***. Myelinated PC-like axon with mitophagic profiles. Scale bars = 500 nm.*

**27**

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

independent of Dpl expression in COCS.

death in *ex vivo* cultures.

early as 3DIV [14].

*Prnp*0/0 PCs.

The absence of BAX not only protected some PCs from neurotoxicity in the cerebellum of the Ngsk *Prnp*0/0 mice [121], but also decreased the number of autophagic neurons suggesting that the PCs rescued by *Bax* deficiency do not display activated autophagy, whereas the autophagic PCs in the *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebellum are likely to result from PrP-deficiency as in the ZH-I *Prnp*0/0 cerebellum. Nevertheless, the persistent loss of Bax<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 PCs could result from an increased sensitivity of these PCs to the Ngsk condition compared to ZH-I

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 *Prnp*0/0 and ZH-I *Prnp*0/0 COCS by measuring PC survival and development using morphometric methods [179] in COCS from these PrP-deficient mice. Similar timing and amplitude of PC growth impairment and death were observed in all PrP-deficient genotypes. Indeed, PC surface, perimeter, and dendritic extension increased between 7 and 21 DIV in the wild-type COCS, while no significant variation of surface and perimeter could be measured in the PrP-deficient mutant COCS during this period (**Figure 15**). Similarly, wild-type and PrP-deficient PCs displayed equivalent maximal dendritic extension after 7 days *ex vivo*, but wildtype PCs continued to increase their maximal dendritic length until 21 DIV, while the dendrites of PrP-deficient PCs did not grow during this period [14, 180]. Thus, PrP-deficient PCs exhibit a similar developmental deficit which seems to be

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 *Prnp*+/0, Ngsk *Prnp*0/0, and ZH-I *Prnp*0/0. Whereas, wild-type PCs' numbers remained stable during the whole period, severe PC loss (68–69%) had occurred at 7 DIV and slightly increased up to 21 DIV in all PrP-deficient mutant COCS. PC loss displayed similar kinetics and amplitude in Ngsk *Prnp*+/0, Ngsk *Prnp*0/0, and ZH-I *Prnp*0/0 COCS suggesting that despite detectable levels of 15–20 kDa glycosylated form of Dpl in the Ngsk *Prnp*0/0 COCS (**Figure 16**), it may be not implicated in PC

Furthermore, at the ultrastructural level, whereas autophagic organelles were rare in wild-type PCs after 7 and 12 DIV, Ngsk *Prnp*0/0 PCs contained numerous autophagosomes and autophagolysosomes at different maturation stages (**Figure 17**). During the period of PC death in the *Prnp*0/0 COCS (i.e., 3, 5, and 7 DIV) Western blotting of apoptotic and autophagic markers revealed a 4- to 5-fold increase in markers of autophagosomal formation such as LC3B-II (at 5 DIV), p62, and beclin-1 (at 3 and 5 DIV) in the ZH-I and Ngsk *Prnp*0/0 COCS and the lysosomal receptor LAMP-1 in the Ngsk *Prnp*0/0 COCS at 7 DIV (**Figure 18**). Increased amounts of activated caspase-3 indicated the apoptosis in protein extracts of COCS from both *Prnp*0/0 genotypes as

This morphometric and quantitative analysis of COCS suggests that PrPdeficiency, rather than Dpl neurotoxicity, is responsible for the neuronal growth deficit and loss *ex vivo*. Indeed, the neurotoxic properties of Dpl did not seem to contribute to Ngsk PC loss in the COCS, whereas Dpl-induced PC loss is detectable in 6-month-old Ngsk *Prnp*0/0 mice. A possible explanation for this difference is that COCS are not mature enough to model 6-month-old cerebellar tissue. Nevertheless, in Ngsk *Prnp*0/0 and ZH-I *Prnp*0/0 COCs, activation of autophagy and apoptosis is contemporaneous with the atrophy and death of PCs during the first week of culture suggesting that PrP-deficiency is solely responsible for neuronal death in this

deep cerebellar nuclei, revealed a significantly greater amount of autophagic PC presynaptic boutons in the deep nuclei of all mutants compared to control *Bax*+/+;*Prnp*+/+ mice (**Figure 14**).

*Prions - Some Physiological and Pathophysiological Aspects*

**26**

**Figure 14.**

*mitophagic profiles. Scale bars = 500 nm.*

*Bax*+/+;*Prnp*+/+ mice (**Figure 14**).

*Autophagy in the deep cerebellar nuclei of 13 month-old Ngsk (A, C–F) and 10 month-old ZH-I (B) Prnp0/0 mice.* **A***–***E***. PC presynaptic boutons establishing symmetrical synapses (arrowheads) with somato-dendritic profiles of deep cerebellar neurons (DCN) and containing different stages of double-membrane wraps sequestrating neuroplasm (\* in A, B, D, E) and mitochondria (m in C, F).* **F***. Myelinated PC-like axon with* 

deep cerebellar nuclei, revealed a significantly greater amount of autophagic PC presynaptic boutons in the deep nuclei of all mutants compared to control

The absence of BAX not only protected some PCs from neurotoxicity in the cerebellum of the Ngsk *Prnp*0/0 mice [121], but also decreased the number of autophagic neurons suggesting that the PCs rescued by *Bax* deficiency do not display activated autophagy, whereas the autophagic PCs in the *Bax*<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 cerebellum are likely to result from PrP-deficiency as in the ZH-I *Prnp*0/0 cerebellum. Nevertheless, the persistent loss of Bax<sup>−</sup>/<sup>−</sup>;Ngsk *Prnp*0/0 PCs could result from an increased sensitivity of these PCs to the Ngsk condition compared to ZH-I *Prnp*0/0 PCs.

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 *Prnp*0/0 and ZH-I *Prnp*0/0 COCS by measuring PC survival and development using morphometric methods [179] in COCS from these PrP-deficient mice. Similar timing and amplitude of PC growth impairment and death were observed in all PrP-deficient genotypes. Indeed, PC surface, perimeter, and dendritic extension increased between 7 and 21 DIV in the wild-type COCS, while no significant variation of surface and perimeter could be measured in the PrP-deficient mutant COCS during this period (**Figure 15**). Similarly, wild-type and PrP-deficient PCs displayed equivalent maximal dendritic extension after 7 days *ex vivo*, but wildtype PCs continued to increase their maximal dendritic length until 21 DIV, while the dendrites of PrP-deficient PCs did not grow during this period [14, 180]. Thus, PrP-deficient PCs exhibit a similar developmental deficit which seems to be independent of Dpl expression in COCS.

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 *Prnp*+/0, Ngsk *Prnp*0/0, and ZH-I *Prnp*0/0. Whereas, wild-type PCs' numbers remained stable during the whole period, severe PC loss (68–69%) had occurred at 7 DIV and slightly increased up to 21 DIV in all PrP-deficient mutant COCS. PC loss displayed similar kinetics and amplitude in Ngsk *Prnp*+/0, Ngsk *Prnp*0/0, and ZH-I *Prnp*0/0 COCS suggesting that despite detectable levels of 15–20 kDa glycosylated form of Dpl in the Ngsk *Prnp*0/0 COCS (**Figure 16**), it may be not implicated in PC death in *ex vivo* cultures.

Furthermore, at the ultrastructural level, whereas autophagic organelles were rare in wild-type PCs after 7 and 12 DIV, Ngsk *Prnp*0/0 PCs contained numerous autophagosomes and autophagolysosomes at different maturation stages (**Figure 17**). During the period of PC death in the *Prnp*0/0 COCS (i.e., 3, 5, and 7 DIV) Western blotting of apoptotic and autophagic markers revealed a 4- to 5-fold increase in markers of autophagosomal formation such as LC3B-II (at 5 DIV), p62, and beclin-1 (at 3 and 5 DIV) in the ZH-I and Ngsk *Prnp*0/0 COCS and the lysosomal receptor LAMP-1 in the Ngsk *Prnp*0/0 COCS at 7 DIV (**Figure 18**). Increased amounts of activated caspase-3 indicated the apoptosis in protein extracts of COCS from both *Prnp*0/0 genotypes as early as 3DIV [14].

This morphometric and quantitative analysis of COCS suggests that PrPdeficiency, rather than Dpl neurotoxicity, is responsible for the neuronal growth deficit and loss *ex vivo*. Indeed, the neurotoxic properties of Dpl did not seem to contribute to Ngsk PC loss in the COCS, whereas Dpl-induced PC loss is detectable in 6-month-old Ngsk *Prnp*0/0 mice. A possible explanation for this difference is that COCS are not mature enough to model 6-month-old cerebellar tissue. Nevertheless, in Ngsk *Prnp*0/0 and ZH-I *Prnp*0/0 COCs, activation of autophagy and apoptosis is contemporaneous with the atrophy and death of PCs during the first week of culture suggesting that PrP-deficiency is solely responsible for neuronal death in this

#### **Figure 15.**

*PC growth deficits and loss in PrP-deficient COCS.* **A***,* **B***. PC area (A) and perimeter (B) of WT PCs increased from DIV7 to DIV21, whereas both dimensions in Ngsk Prnp+/0, Ngsk Prnp0/0 and ZH-I Prnp0/0 PCs did not change during the same period. A. At DIV7, WT PC area was larger than area of PrP-deficient PCs.*  **C***. While the longest dendrite of WT PCs had significantly grown from DIV7 to DIV21, the longest dendrite of PrP-deficient PCs displayed similar growth impairment suggesting that in both Ngsk and ZH-I conditions, PrP-deficiency is responsible for PC growth deficits.* **D***–***F***. PC loss occurred progressively during the DIV7-DIV21 period in WT COCS (40% at DIV21) while similar loss of PrP-deficient PCs had occurred in the Ngsk Prnp+/0, Ngsk Prnp0/0 (40%) and ZH-I Prnp0/0 (55%) COCS as early as DIV7. E. The Ngsk Prnp0/0 COCS had lost many more PCs than the WT COCS over the DIV3-DIV7 period indicating a neurotoxic effect during this period that is attributable to PrP-deficiency since the Ngsk and the ZH-I conditions induced similar neuronal loss at DIV7.*

*ex vivo* system and that PrPc is neuroprotective for cerebellar PCs. As ZH-I *Prnp*0/0 PCs survive *in vivo*, PC death in ZH-I *Prnp*0/0 and Ngsk *Prnp*0/0 COCS could result from a noxious exacerbation of PrP-deficiency by *ex vivo* conditions.

**29**

**Figure 17.**

*n, nucleus. Scale bar = 2 μm.*

**Figure 16.**

*N-glucosidase (PNGase).*

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

*Western blot of Dpl in Ngsk Prnp0/0 DIV7 COCS and 12 month-old mouse cerebellum. Dpl was detected in a Ngsk Prnp0/0 COCS at DIV7 and in situ in the cerebellar extract from a 12 month-old Ngsk Prnp0/0 mouse but not in the cerebellum of a wild-type (WT) mouse. Dpl migrates at 15–20 kDa after deglycosylation by peptide* 

*Autophagy in Ngsk Prnp0/0 PCs ex vivo.* **A***. PC cytoplasm in a 12 DIV WT COCS. m, mitochondrion; l, lysosome. Scale bar = 500 nm.* **B–D***. Autophagic PC cytoplasm in 7 DIV Ngsk Prnp0/0 COCSs. Asterisks indicate nascent autophagic vacuoles in B and different maturation stages of autophagolysosomes in C and D.*  *Prion Proteins and Neuronal Death in the Cerebellum DOI: http://dx.doi.org/10.5772/intechopen.80701*

#### **Figure 16.**

*Prions - Some Physiological and Pathophysiological Aspects*

**28**

**Figure 15.**

*ex vivo* system and that PrPc

is neuroprotective for cerebellar PCs. As ZH-I *Prnp*0/0

PCs survive *in vivo*, PC death in ZH-I *Prnp*0/0 and Ngsk *Prnp*0/0 COCS could result

*PC growth deficits and loss in PrP-deficient COCS.* **A***,* **B***. PC area (A) and perimeter (B) of WT PCs increased from DIV7 to DIV21, whereas both dimensions in Ngsk Prnp+/0, Ngsk Prnp0/0 and ZH-I Prnp0/0 PCs did not change during the same period. A. At DIV7, WT PC area was larger than area of PrP-deficient PCs.*  **C***. While the longest dendrite of WT PCs had significantly grown from DIV7 to DIV21, the longest dendrite of PrP-deficient PCs displayed similar growth impairment suggesting that in both Ngsk and ZH-I conditions, PrP-deficiency is responsible for PC growth deficits.* **D***–***F***. PC loss occurred progressively during the DIV7-DIV21 period in WT COCS (40% at DIV21) while similar loss of PrP-deficient PCs had occurred in the Ngsk Prnp+/0, Ngsk Prnp0/0 (40%) and ZH-I Prnp0/0 (55%) COCS as early as DIV7. E. The Ngsk Prnp0/0 COCS had lost many more PCs than the WT COCS over the DIV3-DIV7 period indicating a neurotoxic effect during this period that is attributable to PrP-deficiency since the Ngsk and the ZH-I conditions induced similar neuronal loss at DIV7.*

from a noxious exacerbation of PrP-deficiency by *ex vivo* conditions.

*Western blot of Dpl in Ngsk Prnp0/0 DIV7 COCS and 12 month-old mouse cerebellum. Dpl was detected in a Ngsk Prnp0/0 COCS at DIV7 and in situ in the cerebellar extract from a 12 month-old Ngsk Prnp0/0 mouse but not in the cerebellum of a wild-type (WT) mouse. Dpl migrates at 15–20 kDa after deglycosylation by peptide N-glucosidase (PNGase).*

#### **Figure 17.**

*Autophagy in Ngsk Prnp0/0 PCs ex vivo.* **A***. PC cytoplasm in a 12 DIV WT COCS. m, mitochondrion; l, lysosome. Scale bar = 500 nm.* **B–D***. Autophagic PC cytoplasm in 7 DIV Ngsk Prnp0/0 COCSs. Asterisks indicate nascent autophagic vacuoles in B and different maturation stages of autophagolysosomes in C and D. n, nucleus. Scale bar = 2 μm.*

**Figure 18.**

*Western blot of autophagic markers p62, beclin-1 and LAMP-1.* **A***. p62 and* **B***. Beclin-1. The markers were weakly expressed in WT COCS, but increased in DIV3 and DIV5 COCS from PrP-deficient mice.* **C***,* **D***. LAMP-1 did not vary in WT and ZH-1 COCS from DIV3 to DIV7, but increased in DIV7 Ngsk Prnp0/0 COCS indicating increased lysosomal activity (p < 0.05; n = 3 mice/genotype and DIV).*
