**5. Dysfunction of Sortilin by prion infection**

Sortilin expression also affects PrPSc levels. Sortilin-KD increased PrPSc in prion infected cells, similarly to PrPC in uninfected cells [11]. On the contrary, overexpression of Sortilin in infected cells reduced PrPSc [11]. Furthermore, when we investigated PrPSc accumulation in Sort1+/+ and Sort1−/− mouse brains after intracerebral prion inoculation, PrPSc levels in Sort1−/− mouse brains were significantly higher than in Sort1+/+ mouse brains at the early stages of disease (at 45, 60, 90 dpi) [11], suggesting an inhibition of PrPSc degradation. Namely, dysfunction of Sortilin causes excessive accumulation. If so, does prion infection inhibit Sortilin function? Notably, Sortilin in infected cells was ~50% lower than in uninfected cells [11]. Moreover, in infected mouse brains at terminal stage, Sortilin also fell to ~45% as compared with age-matched uninfected mice [11]. These observations suggested that prion infection downregulated Sortilin expression. To confirm this, uninfected cells were treated with RML prion-infected mouse brain homogenate, and Sortilin and PrPSc in individual cells were visualized by double immunofluorescence staining at 9 dpi (**Figure 5**). In cells displaying bright green signals derived from PrPSc, little Sortilin (red) was detected, whereas the bright red fluorescence of Sortilin was observed in the others; that is, Sortilin expression was reduced by prion infection.

**51**

**Figure 6.**

*membrane.*

*A Molecular Mechanism for Abnormal Prion Protein Accumulation*

To clarify why Sortilin is reduced by prion infection, we examined mRNA transcript levels by RT-PCR. There was little difference in Sortilin mRNA abundance between uninfected and infected cells. This suggested that the degradation of Sortilin was facilitated in prion infected cells. Hence, we treated cells with inhibitors of proteolytic degradation. The expression of Sortilin was almost the same in both untreated and MG132-treated cells but increased in NH4Cl-treated cells [11]. In particular, Sortilin expression was dramatically improved in NH4Cl-treated prioninfected cells, and another lysosomal inhibitor, concanamycin A, also improved Sortilin expression in infected cells [11], suggesting that Sortilin is over-degraded in

*Prion infection reduces Sortilin expression. Immunofluorescence staining of Sortilin (red) and PrPSc (green) 9 days after infection of uninfected cells with RML prions. Four horizontal serial images at 1 μm interval were collected, and orthogonally projected image was created. DAPI was used for nuclear stain (blue). Yellow arrow* 

Sortilin has been identified as a novel PrP-binding protein and functions as a sorting receptor to direct PrP into late endosomal/lysosomal compartments.

*Possible mechanism for PrPSc over-accumulation by prion infection. (I) the entry of Sortilin into the lysosomal degradation pathway is facilitated (green arrow) by prion infection (yellow arrow), (II) Sortilin is overdegraded in lysosomes, (III) trafficking of PrPSc to late endosomal/lysosomal compartments is restricted (red broken arrow), and (IV) PrPSc is protected against its degradation in lysosomes and is excessively accumulated. Red arrows indicate Sortilin-mediated PrP-trafficking pathway and blue arrows indicate other PrP-trafficking pathways. EE: Early endosomes, LE: Late endosomes, RE: Recycling endosomes, Lys: Lysosomes, PM: Plasma* 

*DOI: http://dx.doi.org/10.5772/intechopen.78951*

prion-infected cells in lysosomes.

**6. Conclusions**

**Figure 5.**

*indicates PrPSc-positive cell.*

*A Molecular Mechanism for Abnormal Prion Protein Accumulation DOI: http://dx.doi.org/10.5772/intechopen.78951*

#### **Figure 5.**

*Prions - Some Physiological and Pathophysiological Aspects*

largely enters the recycling pathway (**Figure 4**).

**5. Dysfunction of Sortilin by prion infection**

N-terminal domain (residues 23–107) of PrPC is sufficient for its endocytosis mediated by clathrin [29]. It is therefore inferred that Sortilin internalizes PrPC from nonraft domains at the cell surface by clathrin-coated vesicles. Moreover, it has been shown that the expression of total PrPC was not changed even when the flotillin-1– mediated lipid raft-dependent endocytosis of PrPC was inhibited by the knockdown of flotillin-1 [30]. Their and our results suggest that Sortilin-mediated endocytosis directs PrPC into the late endosomal/lysosomal degradation pathway, whereas PrPC that is internalized from the lipid raft domain in a Sortilin-independent manner

*Role of Sortilin in PrP-trafficking. Sortilin internalizes PrP from nonraft domain and direct into late endosomal/lysosomal degradation pathway. PrP internalized from lipid raft domain in Sortilin-independent manner would be largely recycled into cell surface. PrP might be also internalized from nonraft domain in Sortilin-independent manner. Red arrows indicate Sortilin mediated PrP-trafficking pathway. Blue line is lipid raft domain. EE: Early endosome, LE: Late endosomes, RE: Recycling endosomes, Lys: Lysosomes, PM:* 

Sortilin expression also affects PrPSc levels. Sortilin-KD increased PrPSc in prion infected cells, similarly to PrPC in uninfected cells [11]. On the contrary, overexpression of Sortilin in infected cells reduced PrPSc [11]. Furthermore, when we investigated PrPSc accumulation in Sort1+/+ and Sort1−/− mouse brains after intracerebral prion inoculation, PrPSc levels in Sort1−/− mouse brains were significantly higher than in Sort1+/+ mouse brains at the early stages of disease (at 45, 60, 90 dpi) [11], suggesting an inhibition of PrPSc degradation. Namely, dysfunction of Sortilin causes excessive accumulation. If so, does prion infection inhibit Sortilin function? Notably, Sortilin in infected cells was ~50% lower than in uninfected cells [11]. Moreover, in infected mouse brains at terminal stage, Sortilin also fell to ~45% as compared with age-matched uninfected mice [11]. These observations suggested that prion infection downregulated Sortilin expression. To confirm this, uninfected cells were treated with RML prion-infected mouse brain homogenate, and Sortilin and PrPSc in individual cells were visualized by double immunofluorescence staining at 9 dpi (**Figure 5**). In cells displaying bright green signals derived from PrPSc, little Sortilin (red) was detected, whereas the bright red fluorescence of Sortilin was observed in the others; that is, Sortilin expression was reduced by prion infection.

**50**

**Figure 4.**

*Plasma membrane.*

*Prion infection reduces Sortilin expression. Immunofluorescence staining of Sortilin (red) and PrPSc (green) 9 days after infection of uninfected cells with RML prions. Four horizontal serial images at 1 μm interval were collected, and orthogonally projected image was created. DAPI was used for nuclear stain (blue). Yellow arrow indicates PrPSc-positive cell.*

To clarify why Sortilin is reduced by prion infection, we examined mRNA transcript levels by RT-PCR. There was little difference in Sortilin mRNA abundance between uninfected and infected cells. This suggested that the degradation of Sortilin was facilitated in prion infected cells. Hence, we treated cells with inhibitors of proteolytic degradation. The expression of Sortilin was almost the same in both untreated and MG132-treated cells but increased in NH4Cl-treated cells [11]. In particular, Sortilin expression was dramatically improved in NH4Cl-treated prioninfected cells, and another lysosomal inhibitor, concanamycin A, also improved Sortilin expression in infected cells [11], suggesting that Sortilin is over-degraded in prion-infected cells in lysosomes.

#### **6. Conclusions**

Sortilin has been identified as a novel PrP-binding protein and functions as a sorting receptor to direct PrP into late endosomal/lysosomal compartments.

#### **Figure 6.**

*Possible mechanism for PrPSc over-accumulation by prion infection. (I) the entry of Sortilin into the lysosomal degradation pathway is facilitated (green arrow) by prion infection (yellow arrow), (II) Sortilin is overdegraded in lysosomes, (III) trafficking of PrPSc to late endosomal/lysosomal compartments is restricted (red broken arrow), and (IV) PrPSc is protected against its degradation in lysosomes and is excessively accumulated. Red arrows indicate Sortilin-mediated PrP-trafficking pathway and blue arrows indicate other PrP-trafficking pathways. EE: Early endosomes, LE: Late endosomes, RE: Recycling endosomes, Lys: Lysosomes, PM: Plasma membrane.*

Dysfunction of Sortilin induces delayed degradation and excessive accumulation of PrP. Notably, prion infection downregulated Sortilin expression by facilitating Sortilin degradation in lysosomes. Finally, we summarize a possible mechanism of excessive accumulation of PrPSc during prion infection (**Figure 6**): (I) the entry of Sortilin into the lysosomal degradation pathway is facilitated by prion infection, (II) Sortilin is over-degraded in lysosomes, (III) trafficking of PrPSc to late endosomal/lysosomal compartments is restricted, and (IV) PrPSc is protected against its degradation in lysosomes and is excessively accumulated. However, it still remains unclear how prion infection facilitates Sortilin degradation in lysosomes.
