**2. PrPSc accumulation**

**Figure 1A** shows the expression of total PrP and PrPSc in uninfected and prioninfected mouse brains. In this figure, we can easily recognize that the total amount of PrP in infected mouse brains is larger than in uninfected mouse brains. In cultured cells, such excessive expression of total PrP in infected cells was also confirmed (**Figure 1B**). These results indicate that the amount of PrPSc in infected cells is larger than PrPC in uninfected cells, and that PrPSc is protected against proteolytic degradation.

Why is PrPSc protected from proteolysis and over-accumulated? One possible reason is the protease resistance of PrPSc that is attributed to its β-rich structure at the C-terminal region. If such protease resistance mainly affected the inhibition of PrPSc degradation, most of the PrPSc could be found in the lysosome, which contains various kinds of hydrolytic enzymes and is a major compartment responsible for the digestion of macromolecules such as proteins. The majority of PrPSc is actually observed intracellularly, whereas PrPC mainly localizes to the cell surface (**Figure 2A**). However, detailed analyses of its intracellular distribution show that PrPSc is widely distributed in posttrans Golgi network (TGN) compartments [8–10] (**Figure 2B**). From these

**47**

**Figure 2.**

generative diseases.

*A Molecular Mechanism for Abnormal Prion Protein Accumulation*

observations, it seems that impairment of PrPSc trafficking into lysosomes as well as its protease-resistance causes inhibition of degradation and over-accumulation of PrPSc.

*PrPSc is widely distributed in post-Golgi compartments. (A) PrPC (green, uninfected cells) and PrPSc (green, infected cells) were visualized by immunofluorescence staining with mouse monoclonal anti-PrP antibody (SAF83) and anti-PrPSc antibody (132), respectively. (B) PrPSc indicated organelle markers in prion infected cells were doubly stained with anti-PrPSc antibody (132) and anti-transferrin receptor, Rab11, Rab5, Rab9 and* 

PrP would have to move by transport vesicles in post-TGN compartments, including TGN, endosomes, lysosomes, and the plasma membrane. Then, in this transport network, the PrP to be degraded could be sorted into transport carriers bound for late endosomal/lysosomal compartments. For this purpose, a sorting receptor might be useful and required because it can select and concentrate a target cargo protein into transport carriers and promote transport carrier formation. In our recent study, Sortilin has been identified as a sorting receptor that directs PrP into late endosomal/lysosomal compartments. Sortilin is a member of the VPS10P domain receptor family, which is comprised of five members (Sortilin, SorCS1, SorCS2, SorCS3, and SorLA). In this section, briefly, we describe Sortilin and other VPS10P receptors and their implications for neurode-

**3. Sortilin and other VPS10P domain receptors**

*LAMP1 antibody, respectively. DAPI was used for nuclear stain (blue).*

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

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

#### **Figure 2.**

*Prions - Some Physiological and Pathophysiological Aspects*

fractionated by detergent-based biochemical fractionation, most of the PrPSc was detected in detergent-resistant membrane (DRM) fractions [11], suggesting that PrPSc mainly exists in membrane bound form and PrPSc is degraded preferentially in lysosomes, but not by cytosolic proteasomes. PrPSc to be degraded in lysosomes might be preferentially selected and directed into the lysosomal degradation pathway by dedicated membrane trafficking machinery. Therefore, knowledge of the mechanism that sorts PrP into late endosomal/lysosomal compartments should

*PrP expression in mice brain and N2a cells. (A) Total PrP and PrPSc were compared between RML prion infected mouse brains at terminal stage and age matched uninfected mice brain by western blotting. (B) N2a cells were treated with uninfected or 22 L-prion infected mice brain homogenate. At 30 dpi, total PrP and PrPSc were detected by western blotting. Blots were probed with anti-PrP antibody (6D11) and anti-*β*-actin antibody.*

**Figure 1A** shows the expression of total PrP and PrPSc in uninfected and prioninfected mouse brains. In this figure, we can easily recognize that the total amount of PrP in infected mouse brains is larger than in uninfected mouse brains. In cultured cells, such excessive expression of total PrP in infected cells was also confirmed (**Figure 1B**). These results indicate that the amount of PrPSc in infected cells is larger than PrPC in uninfected cells, and that PrPSc is protected against proteolytic

Why is PrPSc protected from proteolysis and over-accumulated? One possible reason is the protease resistance of PrPSc that is attributed to its β-rich structure at the C-terminal region. If such protease resistance mainly affected the inhibition of PrPSc degradation, most of the PrPSc could be found in the lysosome, which contains various kinds of hydrolytic enzymes and is a major compartment responsible for the digestion of macromolecules such as proteins. The majority of PrPSc is actually observed

detailed analyses of its intracellular distribution show that PrPSc is widely distributed in posttrans Golgi network (TGN) compartments [8–10] (**Figure 2B**). From these

mainly localizes to the cell surface (**Figure 2A**). However,

be important for understanding the accumulation of PrPSc.

**2. PrPSc accumulation**

intracellularly, whereas PrPC

degradation.

**Figure 1.**

**46**

*PrPSc is widely distributed in post-Golgi compartments. (A) PrPC (green, uninfected cells) and PrPSc (green, infected cells) were visualized by immunofluorescence staining with mouse monoclonal anti-PrP antibody (SAF83) and anti-PrPSc antibody (132), respectively. (B) PrPSc indicated organelle markers in prion infected cells were doubly stained with anti-PrPSc antibody (132) and anti-transferrin receptor, Rab11, Rab5, Rab9 and LAMP1 antibody, respectively. DAPI was used for nuclear stain (blue).*

observations, it seems that impairment of PrPSc trafficking into lysosomes as well as its protease-resistance causes inhibition of degradation and over-accumulation of PrPSc.

### **3. Sortilin and other VPS10P domain receptors**

PrP would have to move by transport vesicles in post-TGN compartments, including TGN, endosomes, lysosomes, and the plasma membrane. Then, in this transport network, the PrP to be degraded could be sorted into transport carriers bound for late endosomal/lysosomal compartments. For this purpose, a sorting receptor might be useful and required because it can select and concentrate a target cargo protein into transport carriers and promote transport carrier formation. In our recent study, Sortilin has been identified as a sorting receptor that directs PrP into late endosomal/lysosomal compartments. Sortilin is a member of the VPS10P domain receptor family, which is comprised of five members (Sortilin, SorCS1, SorCS2, SorCS3, and SorLA). In this section, briefly, we describe Sortilin and other VPS10P receptors and their implications for neurodegenerative diseases.

VPS10P-domain receptors are multiligand type-I transmembrane proteins. They contain five members, Sortilin, SorLA, SorCS1, SorCS2, and SorCS3, and deliver a number of target cargo proteins to their destinations, interacting with them via VPS10P domains on the luminal/extracellular N-terminus (**Figure 3**). The whole luminal/extracellular region in Sortilin is composed of a simple VPS10P domain, but other receptors have additional modules (**Figure 3**).

VPS10P-domain receptors are expressed in the brain and are involved in neuronal function and viability [12, 13]. Sortilin binds to progranulin and mediates endocytosis and delivery of progranulin into lysosomes [14], and rare nonsynonymous variants in SORT1 increase the risk for frontotemporal lobar degeneration [15]. Sortilin also mediates trafficking of neuronal degeneration causative and related proteins. Sortilin has been identified as an amyloid precursor protein (APP) interaction partner and promotes α-cleavage of APP [16]. In addition, Sortilin interacts with BACE1, β-site APP cleavage enzyme 1, and mediates its retrograde trafficking from the plasma membrane to TGN via early endosomes [17]. It has been suggested that Sortilin is potentially associated with Parkinson's disease [18]. Moreover, recently, it has been reported that Sortilin is involved in tau prion replication [19].

As for other VPS10P receptors, it has been reported that SorLA is associated with sporadic and late-onset Alzheimer's disease (AD) [5, 20]. SorLA directs APP into the recycling pathway and protects APP from β-cleavage resulting in Aβ generation [5, 21, 22]. On the other hand, loss of SorLA shifts the traffic flow of APP to the late endosomal pathway and facilitates β-cleavage of APP and Aβ-generation [5, 21, 22]. In addition, a meta-analysis indicated that multiple SorLA variants are associated with the risk of Alzheimer's disease [23]. SorCS1 is also involved in APP transport and Aβ-generation and is identified as a risk factor for Alzheimer's disease [24, 25]. Variants of SorCS2 and SorCS3 are also associated with the risk of Alzheimer's disease [24, 25]. Although a number of studies have indicated that VPS10P-domain receptors are

#### **Figure 3.**

*VPS10P domain receptors. VPS10P-domain receptors are multiligand type-I transmembrane proteins. They contain five members, Sortilin, SorLA, SorCS1, SorCS2 and SorCS3. The extracellular/luminal region of VPS10P receptors contains VPS10P domain and additional domains. The intracellular domain of VPS10P receptors contains motifs for interaction with adaptor proteins. The propeptide at N-terminal region is cleaved by furin in the TGN.*

**49**

*A Molecular Mechanism for Abnormal Prion Protein Accumulation*

implicated in neurodegenerative diseases and their impairment could be a risk factor for diseases, the relation between VPS10P receptors and prion disease is not known.

Sortilin has been identified as a novel PrP-binding protein and is colocalized with PrPC both at the cell surface and intracellular compartments [11]. In Sortilinknockdown (Sortilin-KD) uninfected cells, most of the PrPC is localized at the cell surface, and PrPC expression is increased. In addition, a PrPC uptake experiment, in which cell surface PrPC was labeled with anti-PrP antibody and internalized labeled PrPC was measured after incubation, demonstrated that PrPC internalization was weakened by Sortilin-KD [11]. These results indicate that Sortilin acts as a cell

was also colocalized with Sortilin intracellularly [11]. This made us recollect

was costained for either Rab9 (a late endosomal

into late endosomes to degrade it.

distributed to both late and recycling endosomes in control cells,

is localized to recycling endosomes [11]. These observations indi-

that Sortilin could function intracellularly as a sorting receptor for PrP trafficking.

marker) or Rab11 (a recycling endosomal marker) by indirect immunofluorescence,

whereas, in Sortilin-depleted cells, it failed to localize to late endosomes, and most of

with NH4Cl, which increases lysosomal pH and inhibits proteolytic enzymes in lysosomes, PrPC was effectively accumulated in wt but not in ΔSort cells [11], and PrPC colocalization with LAMP1, a lysosomal marker, in NH4Cl-treated ΔSort cells was significantly lower than NH4Cl-treated wt cells [11]. These results suggest that

Moreover, when wild type (wt) and Sortilin-knockout (ΔSort) cells were treated

Altogether, it could be concluded that Sortilin functions as a cell surface receptor for PrPC internalization and a sorting receptor to direct PrPC to lysosomes via late endosomes (**Figure 4**). We would be able to extend such a role of Sortilin in PrPC trafficking to PrPSc because Sortilin directly interacted with PrPC through its highly flexible N-terminal domain and anti-Sortilin antibody coprecipitated both PrPC and

The inhibition of Sortilin inhibited PrPC internalization by ~20% in the PrPC uptake assay [11]. This result raises a question. Why is PrPC endocytosis inhibited partially even when Sortilin function is almost or completely abolished [11]? There are suggestive findings to answer this question. We examined the PrP distribution in uninfected wt cells and in uninfected ΔSort cells by detergent-based biochemical fractionation. Sixty three percent of PrPC in wt cells was detected in detergent resistant membrane (DRM) fractions, generally recognized as raft fractions, but thirtyseven percent of PrPC was also found in detergent soluble (nonraft) fractions [11]. Sortilin deficiency changed the PrPC distribution, and PrPC in nonraft fractions was reduced to ~15% in ΔSort cells [11]. At present, it is thought that both lipid raft- and clathrin-mediated endocytosis execute PrPC internalization [13, 26]. Sortilin was mostly isolated in nonraft fractions [11]. It has been reported that the cytoplasmic tail of Sortilin can interact with clathrin-associated adaptor protein complex, AP-2, at the plasma membrane and facilitate clathrin-mediated endocytosis [13, 27, 28]. We showed that the recombinant PrP devoid of its N-terminal domain (residues 23–88) (PrPΔ23–88) did not bind to Sortilin. Additionally, internalization and lysosomal degradation of PrPΔ23–88 were inhibited, and it accumulated at the cell surface [11]. These results are in good agreement with a previous report: the

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

**4. Role of Sortilin in PrP trafficking**

surface receptor for PrPC endocytosis.

cate that Sortilin is also required for sorting of PrPC

ΔSort cells failed to transport PrPC properly into lysosomes.

PrPSc. In practical terms, Sortilin is implicated in PrPSc degradation.

When the internalized labeled PrPC

PrPC

the internalized PrPC

the internalized PrPC

implicated in neurodegenerative diseases and their impairment could be a risk factor for diseases, the relation between VPS10P receptors and prion disease is not known.
