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

*Prions - Some Physiological and Pathophysiological Aspects*

but other receptors have additional modules (**Figure 3**).

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,

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

*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* 

**48**

**Figure 3.**

*by furin in the TGN.*

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 surface receptor for PrPC endocytosis.

PrPC was also colocalized with Sortilin intracellularly [11]. This made us recollect that Sortilin could function intracellularly as a sorting receptor for PrP trafficking. When the internalized labeled PrPC was costained for either Rab9 (a late endosomal marker) or Rab11 (a recycling endosomal marker) by indirect immunofluorescence, the internalized PrPC distributed to both late and recycling endosomes in control cells, whereas, in Sortilin-depleted cells, it failed to localize to late endosomes, and most of the internalized PrPC is localized to recycling endosomes [11]. These observations indicate that Sortilin is also required for sorting of PrPC into late endosomes to degrade it.

Moreover, when wild type (wt) and Sortilin-knockout (ΔSort) cells were treated 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 ΔSort cells failed to transport PrPC properly into lysosomes.

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

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

#### **Figure 4.**

*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: Plasma membrane.*

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