Biochemistry and Cell Biology

Sortilin: a new player in dementia and Alzheimer-type neuropathology

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2018-0023.R1

Manuscript Type: Mini Review

Date Submitted by the Author: 09-Apr-2018

Complete List of Authors: Xu, Shu-Yin; Central South University Jiang, Juan; Central South University Pan, Aihua; Central South University Cai, Yan; CentralDraft South University Yan, Xiao-Xin; Central South Universuty School of Basic Medicine, Anatomy and Neurobiology

Is the invited manuscript for consideration in a Special N/A Issue? :

Keyword: aging, neurodegenerative diseases, Vps10p, amyloid plaques

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1 Sortilin: a new player in dementia and Alzheimer-type neuropathology 2 3 Shu-Yin Xu1, Juan Jiang1, Aihua Pan1, Cai Yan1,2#, Xiao-Xin Yan1# 4 5 1Department of Anatomy and Neurobiology, and 2Department of Histology and Embryology, 6 Xiangya School of Medicine, Central South University, Changsha, Hunan 410013, China 7 8 Abstract：：：Age-related dementias are now a major mortality factor among most human 9 populations in the world, with Alzheimer's disease (AD) being the leading 10 dementia-causing neurodegenerative disease. The pathogenic mechanism underlying 11 dementia disorders, and AD in specific, remained largely unclear. Efforts to develop drugs 12 targeting the major disease hallmark lesions, such as amyloid and tangle pathologies, have 13 been unsuccessful so far. The vacuolar sorting 10p (Vps10p) family plays a 14 critical role in membrane signal transduction and protein sorting and trafficking between 15 intracellular compartments. Data emergingDraft during the past few years point to an 16 involvement of this family in the development of AD. Specifically, the Vps10p member 17 sortilin has been shown to participate in amyloid plaque formation, tau phosphorylation, 18 abnormal protein sorting and . In this article, we update some latest findings 19 from animal experiments and human brain studies that suggest abnormal sortilin 20 expression in association with AD-type neuropathology, warranting further research that 21 might lead to novel concept for the development of AD therapeutics.

22 23 Key words：：：amyloid plaques, brain aging, neurodegenerative diseases, Vps10p

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25 #Corresponding author: Yan Cai or Xiao-Xin Yan, Department of Anatomy and Neurobiology, 26 Xiangya School of Medicine, Central South University, Changsha, Hunan 410013, China. Email: 27 [email protected]; [email protected].

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30 Introduction

31 As one of the most common neurodegenerative diseases, Alzheimer’s disease (AD) is 32 clinically manifested as memory loss, cognitive decline, personality change and various 33 neurological symptoms. The neuropathological hallmarks in the brain of AD subjects 34 include senile plaques containing extracellular β-amyloid peptide (Aβ) deposits, dystrophic 35 neurites filled with abnormal neuronal organelles and axonal , and intraneuronal 36 tangle formation rich of aggregated hyperphosphorylated tau (p-tau) proteins. The 37 mechanisms underlying AD pathogenesis remain largely elusive to date. As denoted 38 previously, several competing etiological and pathogenic hypotheses have been proposed, 39 including the amyloid cascade hypothesis, tau protein theory, prion theory, oxidative 40 stress theory, genetic susceptibility, signal transduction dysfunction, and 41 cholinergic hypothesis (Scheltens et al. 2016). Among the above, the amyloid hypothesis 42 has been mostly influential, which posits that Aβ products, either as extracellular deposits 43 or soluble forms, cause synaptic damage,Draft inflammatory glial activation, neuritic dystrophy 44 and tauopathy, leading to synaptic and neuronal loss and ultimately cognitive and 45 neurological deficits (Mullard, 2016). However, this hypothesis has been somewhat 46 questioned lately, because various Aβ-targeting therapies developed so far have 47 consistently failed in clinical trials (Franco and Cedazo-Minguez, 2014; Gold, 2017; Tse 48 and Herrup, 2017). Thus, in order to develop effective mechanism-based medicine, it is 49 important to broaden the understanding of AD pathology and pathogenesis. 50 In the past decade especially the last few years, increasing evidence suggests that 51 alterations in the vacuolar protein sorting 10p (Vps10p) family may relate to 52 the development of AD (Bagyinszky et al. 2014; Mufson et al. 2010; Nyborg et al. 2006). 53 The Vps10p members belong to the type I transmembrane proteins. Generally they 54 consist of a N-terminal extracellular sequence containing the Vps10p homology domain, 55 a transmembrane part and a short intracellular tail at the C-terminal (Quistgaard et al. 56 2014). There are five members in this protein family, including sortilin, sorting 57 protein-related receptor with A-type repeats (SorLA), and sortilin-related receptor CNS 58 (central ) expressed 1 (SorCS 1), SorCS2 and SorCS3 (Hermey 2009) (Fig. 59 1). Comparing to other members, sortilin has the simplest structure, though likely the 60 widest range of binding capability. As a membrane receptor or co-receptor, 61 sortilin plays important biological roles for signal transduction and protein sorting in

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62 cells. Relating to AD pathogenesis in specific, sortilin may participate in the development 63 of both the amyloid and the tau pathologies. In fact, sortilin itself appears to undergo 64 novel degradation process and could generate insoluble peptidic fragments that deposit 65 at neuritic plaques in the human brain (Hu et al. 2017). In this article, we will first 66 briefly review the structure and function of sortilin, and then update recent findings that 67 highlight a change in sortilin expression relative to AD-type amyloid and tau 68 pathologies. 69 70 Biochemical structure of sortilin protein

71 Sortilin was first identified from human brain tissue in 1997 (Petersen et al. 1997). 72 Full-length sortilin is a ~100 kDa transmembrane protein encoded by the SORT1 on 73 1p13.3. The sequence of sortilin consists of a N-terminal signal 74 peptide (1-33a.a), a pro-peptide (34-78a.a), the Vps10p domain (133-741a.a), a 75 transmembrane helix (759-780a.a) Draftand an intracellular C-terminal tail (781-831a.a). The 76 extracellular Vps10p domain and the intracellular tail are highly conserved in evolution. 77 The Vps10p domain is formed by folding of 10 cysteine-rich fragments（10CC） that 78 appear to arrange in a tunnel shape with a unique 10-bladed beta propeller, which may 79 serve as a channel for ligand binding (Quistgaard et al. 2009). The Vps10p domain 80 contains two lysosomal sorting motifs, MS1 (787-FLVHRY-792) and MS2 81 (823-HDDSDEDLL-831). The structure and function of those sorting motifs appear 82 similar to that of mannose 6-phosphate receptor (M6PR), which is involved in 83 transporting proteins from trans-Golgi network to endosomal- system (Petersen, 84 et al. 1997; Puertollano et al. 2001). The acidic-cluster-dileucine sequences within the 85 cytoplasmic tail can bind to the (Vps27p/Hrs/STAM) VHS domain of the Golgi-localizing, 86 γ -Adaptin Ear Homology Domain, ADP-ribosylation Factor-binding (GGA) sorting 87 proteins, which are key players in protein sorting at trans-Golgi network (Ghosh and 88 Kornfeld 2004; Nielsen et al. 2001). Overall, sortilin messenger and protein are expressed 89 widely in the CNS according to a rat study (Sarret et al. 2003). A recent study shows 90 expression of sortilin full-length protein in cortical and hippocampal in rodent 91 and human cerebrum (Hu, et al. 2017). Individual studies have suggested that sortilin is 92 also expressed in various types of peripheral cells, including hepatocytes, adipocytes, 93 skeletal myocytes, and macrophages (Kjolby et al. 2015).

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94 Synthesis and maturation of sortilin protein

95 The N-terminal of sortilin is synthesized in the early system 96 and contains a 44-residue-long protein (named as spadin). Spadin not only can promote 97 adequate folding, but also prevent premature binding of sortilin to ligands during its 98 synthesis. Spadin is further hydrolyzed in trans-Golgi network (TGN) by a protease called 99 . At this stage, sortilin is converted into a mature form, which can sort proteins and 100 transport them to their destined intracellular organelles or compartments. Sortilin can be 101 secreted by the TGN as clathrin-coated vesicles, which may mediate bi-directional 102 protein transportation between the TGN and plasma membrane. There are at least three 103 trafficking routes for activated sortilin (Fig.2). Firstly, in the constitutive secretory 104 pathway, secretory vesicles transport sortilin to the cell surface and fuse with it 105 immediately. Then about 5-10% of sortilin’s extracellular domain is cleaved and degraded, 106 releasing the soluble ligand-binding domain to the extracellular space. The remaining 107 sortilin keeps intact and serves as theDraft binding site for ligands. Sortilin binding with 108 ligands may exert different functions, such as signal transduction and receptor-mediated 109 . Following endocytosis, sortilin associated with vesicles is transported from 110 the plasma membrane to the early by adaptor protein 2 (AP2). Then the ligand 111 may be degraded in , while sortilin is transported reversely to the TGN with the 112 and AP2 complex. Secondly, anterograde transport of sortilin moves from the 113 TGN to early endosome by GGAs and AP1. While the ligand is degraded in the lysosome, 114 the majority of sortilin is palmitoylated and moves back to TGN for re-use (McCormick et 115 al. 2008). Upon successive rounds of transport, a portion of the receptor is ubiquitinated 116 and internalized into lysosomes for degradation (Dumaresq-Doiron et al. 2013). Thirdly, 117 in the regulated secretory pathway, sortilin assists ligands to be incorporated into 118 secretory granules after the cell is stimulated by extracellular signal. But this pathway 119 only exists in the cells that are capable of regulated (Carlo et al. 2014).

120 Major biological function of sortilin

121 As an important membrane signaling protein, it is expected that there could be 122 numerous sortilin ligands in central and peripheral cells (Eggert et al., 2017; Strong, 123 2018). Most sortilin ligands identified so far are associated with or 124 signal transduction. Ligands related to lipid metabolism include

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125 lipoproteinlipase, apolipoprotein A-V (ApoA-V) and ApoB100, which are generally 126 transported to the cell surface through constitutive secretion, and then degraded in 127 lysosomes. The neurotrophic signaling pathway appears fairly complex because sortilin 128 has been shown to participate in this system by three different ways (Nykjaer and 129 Willnow 2012). Thus, first, with the help of sortilin, mature neurotrophin (mNT) and 130 proneurotrophin (proNT) secreted by neurons and glial cells are released to the 131 extracellular space through regulatory secretory pathways. Second, sortilin can transport 132 the tyrosine kinase receptor (Trk) to axon terminals through anterograde trafficking. Trk 133 and p75 neurotrophin receptor (p75NTR) are involved in mNT signal transduction, which 134 help maintain the growth, development and differentiation of neurons (Bracci-Laudiero 135 and De Stefano, 2016). Third, on cell surface, sortilin and p75NTR work together as 136 partners in the transduction of proNT, which is responsible for mediating apoptosis 137 during differentiation, in aging and under certain pathological conditions (Lewin and 138 Nykjaer 2014; Liu et al. 2007). One of the first identified neuronal roles of sortilin is to 139 bind with and regulateDraft the signaling of this and other neuropeptides. In 140 addition, sortilin can bind to thyroglobulin and plays a role in the recycling of the latter. 141 Sortilin is also involved in the formation of the glucose transporter-4 (Glut4) vesicles, 142 which regulate glucose transport in response to insulin (Hermey 2009). To sum up, 143 sortilin travels between the cell surface and the endoplasmic reticulum, 144 and lysosomes to mediate diverse signal transduction, secretion and degradation of 145 various partner proteins, thereby fundamentally affecting their biological functions. 146 147 Genetic evidence for sortilin involvement in dementias

148 Emerging data from genome-wide association study (GWAS) suggest that the Vps10p 149 family proteins are genetically related to the risk of developing AD in human (Reitz et al. 150 2013). For instance, a number of studies have shown that alterations of SorLA single 151 nucleotide polymorphism (SNPs) are associated with AD-type brain imaging phenotypes, 152 such as leukoencephalopathy and hippocampal atrophy (Assareh et al. 2014). Specifically 153 in regard to sortilin, some SORT1 SNPs such as rs646776, rs599839 and rs12740374, 154 can affect the expression of SORT1 (Musunuru et al. 2010). For example, the rs646776 155 can increase the levels of transcriptional SORT1 mRNA, while the G allele of rs599839 is 156 important to promote SORT1 messenger expression. The rs12740374 with a secondary T

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157 allele can activate the CCAAT/enhance binding protein, which can elevate SORT1 mRNA 158 expression by more than 12 fold. So far, no SORT1 variations have been reported to 159 enhance the risk of AD. For instance, a study in Chinese Han population suggest that no 160 SORT1 SNPs variants are related to the risk of AD (Zeng et al. 2013). It should be noted 161 that certain SORT1 variants are associated with some cardiovascular conditions, such as 162 high plasma LDL-C level and (Kjolby, et al. 2015), which are known risk 163 factors for AD (Gottesman et al.2017). 164 Importantly, two late studies have extended evidence supporting a certain link 165 between genetic variants of sortilin and the risk of developing AD and frontotemporal 166 dementia. A study enrolled 620 AD patients and 1107 healthy controls shows that the 167 rs17646665 polymorphism in the non-coding region of the SORT1 gene is associated 168 with a reduced risk of AD (Andersson et al. 2016). During the revision of our manuscript, 169 a study on a Belgian cohort of 636 FTD patients and 1066 unaffected control individuals 170 reveals 5 patient-only nonsynonymous rare variants in SORT1 (Philtjens et al., 2018). The 171 rare coding variants in patients areDraft related to the β-propeller domain, including two 172 variants that are predicted to be the binding site for GRN. Analyzing a total of three 173 independent patient/control cohorts including 1155 FTD patients and 1161 controls from 174 Spain, Italy, and Portugal, the authors find 7 additional patient-only nonsynonymous 175 variants in European population. Thus, SORT1 appears to be a newly identified genetic 176 risk factor for FTD (Philtjens et al., 2018). 177 178 Pathological evidence for sortilin change relative to AD-type pathology

179 A limited number of studies have addressed sortilin expression in human brain tissue. 180 Levels of full-length sortilin in the cerebral cortex have been shown to be maintained in 181 subjects with mild cognitive impairment (MCI) and AD in an earlier report (Mufson, et al. 182 2010), while other studies report elevation of the protein in the cerebrum of AD patients 183 relative to aged controls (Coulson and Nykjaer, 2013; Finan et al. 2011; Saadipour et al. 184 2013) (Table.1). A recent study by our group shows that the levels of full-length sortilin 185 tend to be increased in neocortical lysates from aged and AD individuals relative to 186 mid-age subjects (Hu, et al. 2017). Immunohistochemical and immunoblotting 187 characterizations reveal for the first time that putative sortilin C-terminal fragments can 188 deposit extracellularly at senile plaques in aged and AD human brain. The morphological

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189 pattern of sortilin deposition at senile plaques appears to be comparable to that of 190 extracellular Aβ fibrils (Fig. 3). Notably, this extracellular sortilin neuropathology does not 191 occur in several commonly used transgenic mouse models of AD and even in aged Macaca 192 monkeys with overt cerebral amyloid deposition (Zhou et al., 2018). Thus, in reference to 193 their human brain counterparts, neuritic plaques seen in transgenic AD model mouse 194 represent an incomplete form of this disease pathological hallmark. The species 195 difference in neuritic plaque constituents is in line with the notion that during aging and 196 in AD there exist more complex secondary proteopathies in the brain of human relative to 197 rodents and non-human primates. The precise cellular/molecular mechanism underlying 198 extracellular sortilin deposition remains to be elucidated in future studies. Given that a 199 unifying explanation for Aβ deposition is still not established (Li et al. 2017; Yan et al. 200 2014), the finding of extracellular sortilin deposition at senile plaques in the human brain 201 extends a new reference system to explore how and why particular protein fragments 202 accumulate and deposit in the brain extracellular space. 203 Draft 204 Experimental study on sortilin modulation of Aβ generation

205 Amyloid precursor protein (APP) can be cleaved by two secretase-mediated 206 pathways. In the non-amyloidogenic pathway, APP is first cleaved byα-secretase, 207 releasing secreted amyloid precursor protein α (sAPPα), and α-site cleaved APP 208 C-terminal fragments (αCTFs) that can not form full-sequence Aβ peptides. The 209 amyloidogenic APP proteolytic pathway is initiated by β-secretase mediated cleavage to 210 produce β-site cleaved CTFs (βCTFs) that further produce monomeric Aβ species via 211 ɤ-secretase processing (Cai et al., 2010; Zhang et al., 2010; Liu et al., 2013). Overall, Aβ is 212 removed from the brain parenchyma via enzymatic degradation and other clearance 213 mechanisms. In theory, increased expression of APP, enhanced activity of β- andγ 214 -secretases or obstructed Aβ clearance can lead to abnormal elevation of Aβ in the brain, 215 which could potentially result in cerebral Aβ deposition (Yan et al., 2014; Li et al., 2017). 216 Using human embryonic kidney HEK293T cell line expressing SORT1 transgene, 217 Finan et al. (2011) show that sortilin and β-secretase interact with each other. sAPPβ and 218 Aβ can also interact with the expression of sortilin, suggestive of sortilin involvement in 219 β-secretase-mediated APP processing. Since sortilin and β-secretase both have 220 intracellular motifs with similar binding partners and intracellular transport pathways,

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221 the authors suggest that the C-terminal of sortilin binds to β-secretase, thereby guiding 222 its intracellular transport. In subsequent in vitro experiments using C-terminal truncated 223 sortilin constructs, these investigators report that sortilin could redistribute β-secretase 224 from the TGN to the endosome. Truncated sortilin loses the binding site of retromer, 225 resulting in a reduction of retrograde trafficking of β-secretase. Gustafsen et al. (2013) 226 demonstrate that levels of sAPPα are significantly increased, whereas levels of sAPPβ are 227 decreased in the cells expressing C-terminal truncated sortilin, relative to non-transfected 228 control cells. In addition, sAPP levels are increased after inhibition of lysosomal protease 229 activity relative to control. Together, these in vitro experimental results suggest that 230 sortilin affects APP processing by promoting the α-secretase pathway and the degradation 231 of sAPP in lysosomes. Thus, further studies are needed to consolidate that sortilin could 232 affect Aβ production by modulating APP trafficking and enzymatic processing in vivo. 233 234 Experimental study on sortilin regulationDraft of APP and Aβ degradation 235 Yang et al. (2013) has reported that the C-terminal of sortilin can regulate 236 non-specific degradation of APP. Specifically, the MS1 part of the C-terminal can bind to 237 APP and direct it to the lysosome. Knockout of this segment reduces the quantity of APP 238 targeting to lysosomes. In a subsequent study (Ruan et al. 2017), they show that aged 239 APP and presenilin 1 double transgenic mice (about 9 months old) with silenced SORT1 240 gene develop increased amyloid plaques in the forebrain, with astrocytic activation in the 241 and neuronal loss in the cortex. These pathological phenotypes could be 242 rescued by intra-hippocampal injection of a viral vector that mediate overexpression of 243 human sortilin. Therefore, it is suggested that sortilin plays a protective role in AD by 244 reducing amyloid pathogenesis. 245 Sortilin may also participate in Aβ degradation through receptor-mediated pathways. 246 Most of Aβ in brain may bind to ApoE, and then form ApoE/Aβ complex, which is 247 transported to the lysosome for degradation and then being released through the 248 extracellular fluid or the blood-brain barrier (BBB)(Fan et al. 2009). Low-density 249 receptor (LDLR) and LDL–related protein 1(LRP1) may serve as two major 250 receptors for transporting ApoE/Aβ through the BBB. Carlo et al. (2013) report that the 251 concentration of ApoE in the cortex and hippocampus of SORT1-/- mice increases 2 fold 252 relative to the wildtype controls. Thus, the capability for ApoE-assisted binding and

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253 degradation of Aβ appear to be both decreased without sortilin. However, it is also shown 254 that the levels of Aβ40 and the amount of senile plaques are higher in the SORT1-/- brain 255 as compared to control, possibly because the expression of LPR1 is not changed in the 256 knockouts (Carlo et al., 2013). 257 258 Experimental study on sortilin regulation of tau hyperphosphorylation

259 Neurofibrillary tangle is one of the pathological hallmarks of AD and is associated 260 with intraneuronal tau hyperphosphorylation. It is suggested that abnormally 261 phosphorylated tau proteins spread in the brain in a manner similar to prion propagation 262 (Fraser et al., 2014; Yin et al. 2014). Pathogenic prion PrPSc has virus-like infectivity 263 capable of inducing conformational transformation of the normal prion protein PrPC. 264 PrPSc can incessantly proliferate and damage brain tissue. Prion diseases, just like AD, 265 show neuronal tau hyperphosphorylation in the brain (Ballatore et al. 2007). Uchiyama et 266 al. ( 2017) demonstrate that sortilinDraft can bind to both PrPC and PrPSc, and guide them to 267 lysosome for degradation. However, because proliferating PrPSc increases sortilin 268 degradation in the lysosome, the positive effect of sortilin on prion propagation becomes 269 limited. Overall, the authors propose that sortilin can protect brain from injury during 270 PrPSc transmission (Sakaguchi and Uchiyama, 2017). 271 Recently, Johnson et al. (2017) demonstrate that abnormal tau phosphorylation in 272 Tg2541 transgenic mice is mainly located in the hindbrain, although there is no 273 significant difference in the levels of tau mRNA and protein between the forebrain and 274 hindbrain. The expression of sortilin in the forebrain is significantly higher than in the 275 hindbrain. Therefore, it is suggested that sortilin can inhibit abnormal tau spreading in 276 the forebrain of the transgenic mice with enhanced human mutant tau transgene. 277 278 Experimental evidence for sortilin involvement in proNT-mediated apoptosis

279 In AD pathogenesis, it is suggested that neurons and glial cells release proNT to 280 potentiate cellular apoptosis and this effect may relate to the massive loss of neurons in 281 the brains of AD patients (Fahnestock et al. 2001). As increased release of proNT in the 282 brain is also found in other conditions such as epilepsy, spinal nerve injury, retinal 283 ischemia and prion disease, it is proposed that proNT plays a significant role in neuronal 284 death by a mechanism involving receptor-mediated apoptosis (Hempstead, 2014; Glerup

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285 et al. 2014). Sortilin has been considered as a modulator in proNT-induced apoptosis in 286 neurons (Nykjaer et al. 2004). Thus, sortilin can bind to the proNT propeptides with high 287 affinity, while mature proNT binds to p75 NTR forming a receptor complex that initiates 288 apoptotic signaling (Nykjaer et al. 2004; Rogers et al. 2010). Structural analyses show 289 that proNT and p75NTR take the form of a 2:2 symmetrical configuration when forming a 290 trimmer. The binding affinity of this proNT/p75NTR complex to sortilin is 5 times higher 291 than proNT alone (Feng et al. 2010). NGF-deprived AD model mice exhibit typical 292 pathological characteristics of AD such as Aβ deposition and tau phosphorylation, along 293 with cholinergic deficit and working memory deficits. After crossbred with SORT1-/- 294 mice, their cholinergic function and working memory are improved, although Aβ and tau 295 phosphorylation are not changed in the descendants. These findings point to a specific 296 resistance of SORT1-/- mice to proNT-induced apoptosis (Capsoni et al. 2013). An in vitro 297 experiment shows that by silencing sortilin expression in neuroblastoma SH-SY5Y, Aβ 298 oligomers-induced is significantly mitigated (Takamura et al. 2012). 299 Draft 300 Conclusions and perspectives

301 Increasing in vitro and in vivo studies during the past few years report that sortilin 302 may be related genetically to the risk of development of AD and frontotemporal dementia, 303 and pathologically to AD-type lesions via participation in Aβ production and clearance, tau 304 phosphorylation and neuronal death. Sortilin may also influence the progression of AD 305 pathology by mediating signal transduction and intracellular transportation of other 306 molecules in neurons and glial cells (Wang et al. 2017). The finding that sortilin itself can 307 yield fragment products to deposit at senile plaques provides clear pathological evidence 308 for its involvement in this AD hallmark lesion. Therefore, future studies should be carried 309 out to elucidate the cellular and molecular mechanism by which sortilin affects AD 310 pathogenesis. It is worth noting that the levels of sortilin in circulation may serve as a 311 biomarker for coronary atherosclerosis and diabetes (Oh et al. 2017). Other reports 312 suggest that SorL1 is a target for the development of new drugs for the treatment of AD 313 (Na et al. 2017). The genetic and pathological evidence for sortilin involvement in 314 dementia and AD-type neuropathology warrants further investigation of the role of 315 sortilin in AD etiology and pathogenesis, which might extend new cutting edge for the 316 development of novel AD diagnostic and therapeutic options.

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317 Financial and competing interest’s disclosure 318 Ms. Shu-Yin Xu was awarded a postgraduate studentship from Central South University 319 (#2017zzts820). Dr. Yan Cai was awarded a grant from the National Natural Science 320 Foundation (NNSF) of China (#81200837). Prof. Xiao-Xin Yan is funded by a NNSF grant 321 (#91632116). The sponsors have no role in the design of this work; collection, analysis, 322 and interpretation of the data; writing of manuscript; or the decision to submit this 323 manuscript. The authors have no other relevant affiliations or financial involvement with 324 any organization or entity with a financial interest/conflict in the subject matter. No 325 writing assistance was utilized in the production of this manuscript. 326 327 Reference

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382 doi:10.1074/jbc.M110.170217. PMID:21245145. 383 Franco, R., Cedazo-Minguez, A. 2014. Successful therapies for Alzheimer's disease: why so 384 many in animal models and none in humans? Front Pharmacol. 5:146. 385 Fraser, P.E. 2014. Prions and prion-like proteins. J. Biol. Chem. 289(29):19839-19840. 386 doi:10.1074/jbc.R114.583492. PMID:24860092. 387 Ghosh, P., and Kornfeld, S.2004. The GGA proteins: key players in protein sorting at the 388 trans-Golgi network. Eur. J. Cell Biol. 83(6):257-262. doi:10.1078/0171-9335-00374. 389 PMID:15511083. 390 Glerup, S., Nykjaer, A., and Vaegter, C.B. 2014. Sortilins in neurotrophic factor signaling. 391 Handb. Exp. Pharmacol. 220:165-189. doi:10.1007/978-3-642-45106-5_7. 392 PMID:24668473. 393 Gold, M. 2017. Phase II clinical trials of anti-amyloid β antibodies: When is enough, 394 enough? Alzheimers Dement (N Y). 3(3):402-409. 395 Gottesman, R.F., Schneider, A.L., Zhou, Y., Coresh, J., Green, E., Gupta, N., et al.2017. 396 Association Between Midlife VascularDraft Risk Factors and Estimated Brain Amyloid 397 Deposition. Jama, 317(14):1443-1450.doi:10.1001/jama.2017.3090. 398 PMID:28399252. 399 Gustafsen, C., Glerup, S., Pallesen, L.T., Olsen, D., Andersen, O.M., Nykjaer, A., et al.2013. 400 Sortilin and SorLA display distinct roles in processing and trafficking of amyloid 401 precursor protein. J. Neurosci. 33(1):64-71. doi:10.1523/jneurosci.2371-12.2013. 402 PMID:23283322. 403 Hempstead, B.L. 2014. Deciphering proneurotrophin actions. Handb. Exp. Pharmacol. 404 220:17-32. doi:10.1007/978-3-642-45106-5_2. PMID:24668468. 405 Hermey, G. 2009. The Vps10p-domain receptor family. Cell Mol. Life Sci. 406 66(16):2677-2689. doi:10.1007/s00018-009-0043-1. PMID:19434368. 407 Hu, X., Hu, Z.L., Li, Z., Ruan, C.S., Qiu, W.Y., Pan, A., et al. 2017. Sortilin Fragments Deposit at 408 Senile Plaques in Human Cerebrum. Front Neuroanat. 11:45. 409 doi:10.3389/fnana.2017.00045. PMID:28638323. 410 Johnson, N.R., Condello, C., Guan, S., Oehler, A., Becker, J., Gavidia, M., et al. 2017. Evidence 411 for sortilin modulating regional accumulation of human tau prions in transgenic mice. 412 Proc. Natl. Acad. Sci. U S A. doi:10.1073/pnas.1717193114. PMID:29203673. 413 Kjolby, M., Nielsen, M.S., and Petersen, C.M. 2015. Sortilin, encoded by the cardiovascular 414 risk gene SORT1, and its suggested functions in cardiovascular disease. Curr.

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415 Atheroscler. Rep. 17(4):496. doi:10.1007/s11883-015-0496-7. PMID:25702058. 416 Lewin, G.R., and Nykjaer, A. 2014. Pro-, sortilin, and nociception. Eur. J. 417 Neurosci. 39(3):363-374. doi:10.1111/ejn.12466. PMID:24494677. 418 Li, J.M., Huang, L.L., Liu, F., Tang, B.S., and Yan, X.X. 2017. Can brain impermeable BACE1 419 inhibitors serve as anti-CAA medicine? BMC Neurol. 17(1):163. 420 doi:10.1186/s12883-017-0942-y. PMID:28841840. 421 Liu, F., Xue, Z.Q., Deng, S.H., Kun, X., Luo, X.G., Patrylo, P.R., et al. 2013. γ-secretase binding 422 sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a 423 putative origin of CSF Aβ. Eur. J. Neurosci. 37(10):1714-25. 424 Liu, Y., Deng, Y.B., and Zhou, L.H. 2007. Advance in research on the apoptosis of neuronal 425 cells mediated by p75NTR. Chinese Bulletin of Life Sciences(Chinese), 19(1):63-67. 426 McCormick, P.J., Dumaresq-Doiron, K., Pluviose, A.S., Pichette, V., Tosato, G., and Lefrancois, 427 S. 2008. Palmitoylation controls recycling in lysosomal sorting and trafficking. Traffic , 428 9(11):1984-1997. doi:10.1111/j.1600-0854.2008.00814.x. PMID:18817523. 429 Mufson, E.J., Wuu, J., Counts, S.E., andDraft Nykjaer, A. 2010. Preservation of cortical sortilin 430 protein levels in MCI and Alzheimer's disease. Neurosci. Lett. 471(3):129-133. 431 doi:10.1016/j.neulet.2010.01.023. PMID:20085800. 432 Mullard, A.2016.Alzheimer amyloid hypothesis lives on. Nat. Rev. Drug Discov. 16(1):3-5. 433 doi:10.1038/nrd.2016.281. PMID:28031570. 434 Musunuru, K., Strong, A., Frank-Kamenetsky, M., Lee, N.E., Ahfeldt, T., Sachs, K.V., et al. 435 2010. From noncoding variant to phenotype via SORT1 at the 1p13 locus. 436 Nature, 466(7307):714-719. doi:10.1038/nature09266. PMID:20686566. 437 Na, J.Y., Song, K., Lee, J.W., Kim, S., and Kwon, J. 2017. Sortilin-related receptor 1 interacts 438 with amyloid precursor protein and is activated by 6-shogaol, leading to inhibition of 439 the amyloidogenic pathway. Biochem. Biophys. Res. Commun. 484(4):890-895. 440 doi:10.1016/j.bbrc.2017.02.029. PMID:28188785. 441 Nielsen, M.S., Madsen, P., Christensen, E.I., Nykjaer, A., Gliemann, J., Kasper, D., et al. 2001. 442 The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS 443 domain of the GGA2 sorting protein. Embo. j. 20(9):2180-2190. 444 doi:10.1093/emboj/20.9.2180. PMID:11331584. 445 Nyborg, A.C., Ladd, T.B., Zwizinski, C.W., Lah, J.J., and Golde, T.E.2006.Sortilin, SorCS1b, and 446 SorLA Vps10p sorting receptors, are novel gamma-secretase substrates. Mol. 447 Neurodegener. 1:3. doi:10.1186/1750-1326-1-3. PMID:16930450.

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448 Nykjaer, A., Lee, R., Teng, K.K., Jansen, P., Madsen, P., Nielsen, M.S., et al. 2004. Sortilin is 449 essential for proNGF-induced neuronal cell death. Nature, 427(6977):843-848. 450 doi:10.1038/nature02319. PMID:14985763. 451 Nykjaer, A., and Willnow, T.E. 2012. Sortilin: a receptor to regulate neuronal viability and 452 function. Trends Neurosci. 35(4):261-270.doi:10.1016/j.tins.2012.01.003. 453 PMID:22341525. 454 Oh, T.J., Ahn, C.H., Kim, B.R., Kim, K.M., Moon, J.H., Lim, S., et al. 2017. Circulating sortilin 455 level as a potential biomarker for coronary atherosclerosis and diabetes mellitus. 456 Cardiovasc. Diabetol. 16(1):92. doi:10.1186/s12933-017-0568-9. PMID:28728579. 457 Petersen, C.M., Nielsen, M.S., Nykjaer, A., Jacobsen, L., Tommerup, N., Rasmussen, H.H., et 458 al. 1997. Molecular identification of a novel candidate sorting receptor purified from 459 human brain by receptor-associated protein affinity chromatography. J. Biol. Chem. 460 272(6):3599-3605. PMID:9013611. 461 Philtjens, S., Van Mossevelde, S., van der Zee, J., Wauters, E., Dillen, L., Vandenbulcke, M., et 462 al. 2018. Rare nonsynonymous variantsDraft in SORT1 are associated with increased risk 463 for frontotemporal dementia. Neurobiol. Aging, pii: S0197-4580(18)30054-X. 464 Puertollano, R., Aguilar, R.C., Gorshkova, I., Crouch, R.J., and Bonifacino, J.S.2001.Sorting of 465 mannose 6-phosphate receptors mediated by the GGAs. Science, 466 292(5522):1712-1716. doi:10.1126/science.1060750. PMID:11387475. 467 Quistgaard, E.M., Groftehauge, M.K., Madsen, P., Pallesen, L.T., Christensen, B., Sorensen, 468 E.S., et al. 2014.Revisiting the structure of the Vps10 domain of human sortilin and its 469 interaction with neurotensin. Protein Sci. 23(9):1291-1300. doi:10.1002/pro.2512. 470 PMID:24985322. 471 Quistgaard, E.M., Madsen, P., Groftehauge, M.K., Nissen, P., Petersen, C.M., and Thirup, S.S. 472 2009. Ligands bind to Sortilin in the tunnel of a ten-bladed beta-propeller domain. Nat. 473 Struct. Mol. Biol. 16(1):96-98 .doi:10.1038/nsmb.1543. PMID:19122660. 474 Reitz, C., Tosto, G., Vardarajan, B., Rogaeva, E., Ghani, M., Rogers, R.S., et al. 2013. 475 Independent and epistatic effects of variants in VPS10-d receptors on Alzheimer 476 disease risk and processing of the amyloid precursor protein (APP). Transl. Psychiatry, 477 3:e256. doi:10.1038/tp.2013.13. PMID:23673467. 478 Rogers, M.L., Bailey, S., Matusica, D., Nicholson, I., Muyderman, H., Pagadala, P.C., et al. 2010. 479 ProNGF mediates death of Natural Killer cells through activation of the 480 p75NTR-sortilin complex. J. Neuroimmunol. 226(1-2):93-103.

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481 doi:10.1016/j.jneuroim.2010.05.040. PMID:20547427. 482 Ruan, C.S., Liu, J., Yang, M., Saadipour, K., Zeng, Y.Q., Liao, H., et al. 2017. Sortilin inhibits 483 amyloid pathology by regulating non-specific degradation of APP. Exp. Neurol. 299(Pt 484 A):75-85. doi:10.1016/j.expneurol.2017.10.018. PMID:29056359. 485 Saadipour, K., Yang, M., Lim, Y., Georgiou, K., Sun, Y., Keating, D., et al. 2013. Amyloid 486 beta1-42 (Abeta42) up-regulates the expression of sortilin via the p75(NTR)/RhoA 487 signaling pathway. J. Neurochem. 127(2):152-162. doi:10.1111/jnc.12383. 488 PMID:23895422. 489 Sakaguchi, S., and Uchiyama, K. 2017. Novel amplification mechanism of prions through 490 disrupting sortilin-mediated trafficking. Prion:1-7. 491 doi:10.1080/19336896.2017.1391435. PMID:29099278. 492 Sarret, P., Krzywkowski, P., Segal, L., Nielsen, M.S., Petersen, C.M., Mazella, J., et al. 2003. 493 Distribution of NTS3 receptor/sortilin mRNA and protein in the rat central nervous 494 system. J. Comp Neurol 461(4):483-505.doi:10.1002/cne.10708. PMID:12746864. 495 Scheltens, P., Blennow, K., Breteler, M.M.,Draft de Strooper, B., Frisoni, G.B., Salloway, S., et 496 al.2016.Alzheimer's disease. Lancet, 388(10043):505-517. 497 doi:10.1016/s0140-6736(15)01124-1. PMID:26921134. 498 Strong, A. 2018. Revisiting Old Friends: Sortilin-1, low-density lipoprotein receptor, and 499 prorenin receptor as modulators of lipoprotein and energy metabolism. Circ Res., 500 122(5):652-654. 501 Takamura, A., Sato, Y., Watabe, D., Okamoto, Y., Nakata, T., Kawarabayashi, T., et al. 2012. 502 Sortilin is required for toxic action of Abeta oligomers (AbetaOs): extracellular AbetaOs 503 trigger apoptosis, and intraneuronal AbetaOs impair degradation pathways. Life Sci. 504 91(23-24):1177-1186. doi:10.1016/j.lfs.2012.04.038. PMID:22579764. 505 Tse, K.H., Herrup, K. 2017. Re-imagining Alzheimer's disease - the diminishing 506 importance of amyloid and a glimpse of what lies ahead. J. Neurochem. 507 143(4):432-444. 508 Uchiyama, K., Tomita, M., Yano, M., Chida, J., Hara, H., Das, N.R., et al. 2017. Prions amplify 509 through degradation of the VPS10P sorting receptor sortilin. PLoS Pathog. 510 13(6):e1006470. doi:10.1371/journal.ppat.1006470. PMID:28665987. 511 Wang, Y., Qin, X., and Paudel, H.K. 2017. Amyloid beta peptide promotes lysosomal 512 degradation of clusterin via sortilin in hippocampal primary neurons. Neurobiol. Dis. 513 103:78-88.doi:10.1016/j.nbd.2017.04.003. PMID:28396259.

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514 Yan, X.X., Ma, C., Gai, W.P., Cai, H., and Luo, X.G. 2014. Can BACE1 inhibition mitigate early 515 axonal pathology in neurological diseases? J. Alzheimers Dis. 38(4):705-718. 516 doi:10.3233/jad-131400. PMID:24081378. 517 Yang, M., Virassamy, B., Vijayaraj, S.L., Lim, Y., Saadipour, K., Wang, Y.J., et al. 2013. The 518 intracellular domain of sortilin interacts with amyloid precursor protein and regulates 519 its lysosomal and lipid raft trafficking. PLoS One, 8(5):e63049. 520 doi:10.1371/journal.pone.0063049. PMID:23704887. 521 Yin, R.H., Tan, L., Jiang, T., and Yu, J.T. 2014. Prion-like Mechanisms in Alzheimer's Disease. 522 Curr. Alzheimer Res. 11(8):755-764. PMID:25212914. 523 Zeng, F., Deng, Y.P., Yi, X., Cao, H.Y., Zou, H.Q., Wang, X., et al. 2013. No association of 524 SORT1 gene polymorphism with sporadic Alzheimer's disease in the Chinese Han 525 population. Neuroreport, 24(9):464-468. doi:10.1097/WNR.0b013e3283619f43. 526 PMID:23660633. 527 Zhang, X.M., Xiong, K., Cai, Y., Cai, H., Luo, X.G., Feng, J.C., et al. 2010. Functional deprivation 528 promotes amyloid plaque pathogenesisDraft in Tg2576 mouse olfactory bulb and piriform 529 cortex. Eur. J. Neurosci. 31(4):710-21. 530 Zhou, F.Q., Jiang, J., Griffith, C.M., Patrylo, P.R., Cai, H., Chu, Y., et al. 2018. Lack of 531 human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease 532 model mice and macaques . Alzheimers Res. Ther., DOI: 10.1186/s13195-018-0370-2. 533 534 535 536 537 538 Figure legends 539 Figure 1. Molecular architecture of the vacuolar protein sorting 10 (Vps10p) receptor 540 family proteins. The extracellular domains of all the receptors contain one Vps10p domain 541 (Vps10p-D). SorLA has the largest extracellular part. Its Vps10p-D is followed by an 542 epidermal growth factor-type repeat, a cluster of 11 complement-type repeats and 6 543 fibronectin-type III repeats. SorCS1, SorCS2 and SorCS3 are distributed in different areas 544 of cell, all of them contain a leucine-rich segment between the Vps10p-D and the 545 transmembrane. SorLA, sorting protein-related receptor with A-type repeats; SorCS, 546 sortilin-related receptor CNS expressed.

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547 548 Figure 2. Routes of sortilin trafficking. (a) via constitutive secretory pathway that is 549 responsible for translating ligands to the cell surface directly; (b) from cell surface, with 550 ligands being targeted for lysosomal degradation; (c) via GGA anterograde transport and 551 retromer recycling path, after transporting ligands to , most of sortilin return 552 to the TGN for re-use; (d) via regulatory secretory pathway that exists in cells to regulate 553 secretion. TGN; trans Golgi network; E: endosome; L: Lysosome. 554 555 Figure 3. Extracellular deposition of putative C-terminal sortilin fragments (Sort-CFTs) as 556 senile plaque-like lesions in the temporal lobe neocortex and dentate gyrus (DG) of 557 Alzheimer’s disease human brain. Sortilin immunolabeling is visualized with a rabbit 558 antibody against the C-terminal domain (Hu et al., 2017). Cortical layers are indicated by 559 Arabic numbers. WM: white matter; ML: molecular layer; GCL: granule cell layer. Scale bar 560 = 200 µm in A applying to B, equivalentDraft to 20 µm in C-F.

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Figure 1: Molecular architecture of the vacuolar protein sorting 10 (Vps10p) receptor family proteins. The extracellular domains of all the receptors contain one Vps10p domain (Vps10p-D). SorLA has the largest extracellular part. Its Vps10p-D is followed by an epidermal growth factor-type repeat, a cluster of 11 complement-type repeats and 6 fibronectin-type III repeats. SorCS1, SorCS2 and SorCS3 are distributed in different areas of cell, all of them contain a leucine-rich segment between the Vps10p-D and the transmembrane. SorLA, sorting protein-related receptor with A-type repeats; SorCS, sortilin-related receptor CNS expressed.

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Figure 2. Routes of sortilin trafficking. (a) via constitutive secretory pathway that is responsible for translating ligands to the cell surface directly; (b) from cell surface, with ligands being targeted for lysosomal degradation; (c) via GGA anterograde transport and retromer recycling path, after transporting ligands to endosomes, most of sortilin return to the TGN for re-use; (d) via regulatory secretory pathway that exists in cells to regulate secretion. TGN; trans Golgi network; E: endosome; L: Lysosome.

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Figure 3. Extracellular deposition of putative Cterminal sortilin fragments (SortCFTs) as senile plaquelike lesions in the temporal lobe neocortex and dentate gyrus (DG) of Alzheimer’s disease human brain. Sortilin immunolabeling is visualized with a rabbit antibody against the Cterminal domain (Hu et al., 2017). Cortical layers are indicated by Arabic numbers. WM: white matter; ML: molecular layer; GCL: granule cell layer. Scale bar = 200 µm in A applying to B, equivalent to 20 µm in CF.

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Table 1. Sortilin protein levels reported in Alzheimer's disease and control human brains Sample size and Areas examined Mean Sortilin change relative Tissue resource Reference mean age at death PMD(hours) to control (years) AD(n=8,76.0) basic forebrain Not indicated no difference London Al-Shawi et NC(n=7,75.0) nuclei hippocampus Neurodegenerative al.(2008) Diseases Brain Bank, UK

MCI(n=20,83.6) frontal cortex MCI(6.3) no difference Rush University Medical Mufson et AD(n=21,86.3) temporal cortex AD(6.5) Center, USA al.(2010) NC(n=17, 82.8) NC(5.0) AD(n=12,76.6) temporal cortex AD(4.9) elevated New York Brain Bank at Finan et NC(n=12,79.9) NC(4.3) Columbia University, al.(2011) USA AD(n=4,81.0) cortex Not indicated elevated South Australia Brain at Saadipour et NC(n=4,85.6) Flinders University, al.(2013) Australia AD(n=9,87.1) middle temporal AD(6.9) aged elevated in AD and aged Central South University, Hu et al.(2017) aged NC(n=9,80.0) gyrus NC(8.8) mid- relative to mid-age China mid-age age NC(11.1) groups NC(n=9,56.4) MCI: mild cognitive impairment; AD: Alzheimer's disease; NC: normal control; PMD: postmortem delay.

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