18 Jul 2023

With its 2,214 amino acids and its dynamic lifestyle as an endosomal recycling receptor, SORL1 is rife with opportunities for mutations that could thwart its function. The size and splendor of this endosomal receptor is on display in Alzforum’s new entry to the Mutations database, where the protein’s many domains, and their vulnerabilities, are laid bare residue by residue (Part 1 of this series).

While protein-truncating variants in the gene invariably lead to Alzheimer's disease, hundreds of rare missense variants have been identified, too, most of them with unknown pathogenicity. This spurred Olav Andersen and colleagues at Aarhus University in Denmark to go through the entire protein, drawing on multiple sources of data to estimate the potential damage wrought by mutations at every residue.

The resulting compendium prioritizes SORL1 missense variants as high or moderate priority based on their likelihood of causing AD, but how well does this scheme reflect levels of disease observed among carriers of these variants in the real world? Importantly: Do the cumulative data gathered on these missense mutations help clinch the case for counting SORL1 among the causal AD genes? Taken together, a trio of new studies in carriers of these mutations answer “yes.”

Making Sense of Missense
Having collaborated with Andersen to create the compendium of prioritized variants, Henne Holstege at Amsterdam University Medical Center set out to put their predictions to the test in an exome and whole genome-sequenced cohort of 18,959 AD cases and 21,893 controls. Samples came from participants in the Alzheimer Disease European Sequencing (ADES) consortium, the Alzheimer’s Disease Sequencing Project (ADSP), the StEP-AD consortium, the Knight-ADRC, and the UCSF/NYGC/UAB cohort. After quality control, the researchers identified 646 unique SORL1 coding variants; of those, 52 were non-rare variants predicted to be benign, while 74 were protein-truncating. The remaining 520 were missense variants, which Holstege grouped into 111 high, 67 moderate, 73 low, and 269 no-priority variants. How would AD risk compare across these genetic groups?

In short, the findings confirmed the prioritization system. As expected, protein-truncating variants occurred almost exclusively in AD cases, cranking up risk for early onset AD 36-fold, and for late-onset AD nearly 10-fold. Rare missense variants deemed “high priority” increased risk of early onset AD 10-fold, and quadrupled risk for late-onset disease. Holstege emphasized that these risk numbers are conservative, given the relatively young average age of controls, many of whom are still in their 30s and 40s and therefore may yet develop AD.

Among AD cases in the cohort, the researchers estimated that carrying a protein-truncating or high-priority missense variant hastens AD onset by 10 and 8.2 years, respectively. Missense variants of moderate priority trended toward increasing risk of late, but not early, onset AD, while lower-priority variants did not affect AD risk or age at onset.

Priorities Aligned. Among people with AD, those carrying protein-truncating variants (black) or high-priority missense variants (blue) got their disease about a decade before those with wild-type SORL1 (red, left). Right: Missense variants in the YWTD (purple) or the calcium cage (light blue) domains triggered AD even earlier than protein truncating variants. [Courtesy of Holstege et al., medRxiv, 2023.]

The researchers came to unexpected conclusions when they broke down the lot of high-priority missense variants based on the protein domains in which they occurred. For example, people who carry variants that mangled the structure of the YWTD domain, which is key to the receptor’s initial folding within the ER, had an earlier disease onset than carriers of protein-truncating variants. Missense variants in any of four residues that form the “calcium cage,” a structure that nestles a critical calcium ion within each of SORL1’s 11 CR domains, also developed AD at younger ages than did carriers of protein-truncating variants. This suggested that these particularly severe missense variants work in a dominant-negative fashion, ensnaring wild-type SORL1 and preventing its proper trafficking, as well.

Conversely, the scientists found that a variant in the VPS10p loop domain, Y391C, which is predicted to interfere with SORL1 binding to Aβ, caused AD in all its carriers, but had a milder impact on age at onset than that seen in carriers of protein-truncating variants. To Holstege, this suggests that while messing with SORL1’s binding to Aβ ultimately leads to AD, variants that derail proper trafficking of SORL1 do so more swiftly.

Holstege also investigated how ApoE genotype influences age at onset in SORL1 mutation carriers. First, she found that among people with AD who did not carry a damaging SORL1 variant, ApoE4 noncarriers developed symptoms at an average of 75 years old, while carriers of one or two copies of ApoE4 developed AD at 70 and 65, respectively. In carriers of protein-truncating or high-priority missense variants of SORL1, ApoE4 hastened age at onset on top of the effect of SORL1.

Holstege told Alzforum that whether ApoE4 and some SORL1 variants may synergize to hasten AD remains an open question, but their effects are at least additive. ApoE genotype could explain the large variation in age at onset observed among unrelated carriers of the same SORL1 variant, Holstege said. For example, the age at onset of 12 Y391C carriers with AD ranged from 60 to 86 years, while five carriers of the R953H mutation developed AD between 46 and 78. Indeed, the researchers found that for these mutations and others, older cases were more likely to carry a protective ApoE2 allele, while earlier-onset cases were more likely to carry at least one ApoE4.These findings posted to medRxiv on July 16, and the scientists presented them in a poster at the Alzheimer’s Association International Conference being held in Amsterdam.

Missense Variants Snag SORL1, Cause Alzheimer's
Two other manuscripts posted on medRxiv support the idea that high-priority missense variants in SORL1 cause AD. One, led by Andersen and posted July 13, identified the Y1816C variant in three families. It lies within the third 3Fn domain of SORL1 that the researchers recently found was necessary for SORL1 to dimerize within the endosome.

Y1816C tracked with AD in three unrelated families from the Netherlands, Spain, and Italy. None of the families had available DNA samples from multiple generations, only from the second or third generations, but they had clinical history of AD in prior generations. Therefore, the findings hint at autosomal-dominant inheritance, but stop short of proving it. Five additional carriers of this missense variant—all with early onset AD—were found in Holstege’s exome-sequencing study (Holstege et al., 2022).

Piecing Together Pedigrees. The Y1816C variant lands within the third 3Fn domain of SORL1 (left). In a Spanish family, one parent had early onset AD. The proband (black arrow) carried Y1816C, as did two affected siblings and another who was not (yet) affected at age 55. ApoE genotype is listed for third generation. One unaffected sibling is a noncarrier. [Courtesy of Jensen et al., 2023.]

First author Anne Mette Jensen and colleagues performed cell biology studies to learn how the mutant might cause AD. First, a bit about the typical travels of normal SORL1. Recent studies suggest that after its translation in the ER, normal SORL1 journeys to the endosome at least partially by way of the cell surface, where it is recycled into the endosomal compartment to perform its sorting function. Then, according to recently published work from Andersen’s group, SORL1 dimerizes via its 3Fn and VPS10p domains, facilitating its interaction with the retromer, which subsequently whisks SORL1 and its associated cargoes up to the cell surface (Jensen et al., 2023). Once on the surface, SORL1 gets snipped by a protease there, releasing a soluble fragment, sSORL1 (Christensen et al., 2020). 

In the case of Y1816C-SORL1, the scientists found that while most of the mutant protein manages to traffic from its translational birthplace in the ER to the endosome, it gets stuck there, likely due to its inability to dimerize via the mutated 3Fn domain. In keeping with this idea, they also found that Y1816C-SORL1 never made it to the cell surface. Furthermore, soluble SORL1 was severely reduced in cell culture media and in the CSF of a Y1816C carrier.

Path to Polymerization? SORL1 dimerizes in the endosome. There, scientists propose that the receptor shifts from an elongated to a compact shape, positioning its 3Fn domains (light grey) to form homodimers. Additional dimerization via VPS10p domains (green) could trigger polymerization (bottom), and interaction with retromer. Variants that compromise dimerization, such as Y1816C, get stuck in the endosome, causing AD. [Courtesy of Andersen et al., bioRxiv, 2023.]

How might marooned SORL1 lead to AD? APP is among SORL1’s many cargoes. Its retention in the endosome increases its exposure to amyloidogenic processing by BACE1. This not only ups Aβ production, but also increases β-CTF, a fragment that ramps up endocytosis (Aug 2019 news; Jan 2021 news). Andersen et al. did not investigate how Y1816C-SORL1 affects APP processing, but they did see that iPSC-derived neurons expressing the this mutant had swollen endosomes, an early pathological hallmark of AD (Cataldo et al., 2000, image below).

While Y1816C-SORL1 failed to dimerize in the endosome, another newly identified variant, R953C, didn’t even make it that far. This is according to Young’s study, which posted on BioRxiv July 5. It identified the R953C variant, which Andersen’s compendium ranks as high priority, in a multigenerational family. The mutation tracked with AD or dementia across two generations. Third-generation relatives are in their 40s and early 50s; none have dementia so far.

How does R953C do its dirty work? First author Elnaz Fazeli and colleagues found that, like the Y1816C variant, R953C never reached the cell surface. Unlike Y1816C, R953C was found trapped in the ER, not the endosome. The R953C mutation lies in SORL1’s YWTD domain, which is needed for SORL1's proper folding within the ER. The researchers found mutant SORL1 stuck and misfolded within the ER. This may have led to premature coupling within the ER, blocking SORL1’s maturation and subsequent voyage to the endosome.

What’s more, the researchers think mutant SORL1 might buddy up with wild-type SORL1 within the ER, potentially also exerting a dominant-negative effect. This meshes with the extremely detrimental effects Holstege observed for other YWTD variants within the exome sequencing study. Although the R953C variant did not crop up in that cohort, five AD cases and one control were found with a different mutation—R953H— at the same residue.

A potential dominant-negative effect jibes with what the researchers found when they examined the brains of mutation carriers, Young said. The family’s enrollment at the UW ADRC afforded researchers the rare opportunity to look at how a SORL1 variant influences neuropathology. They performed comprehensive brain autopsies on three of the mutation carriers from the second generation, as well as their mother, who did not carry the variant but nevertheless developed AD at age 78, before her death at age 91. No biological samples were available from their father—a presumed R953C carrier who died with dementia.

Strikingly, the scientists saw severe AD neuropathology, including cerebellum inundated with Aβ plaques, in all three SORL1 R953C mutation carriers. The carriers also had extensive TDP-43 inclusions in the hippocampus and amygdala, indicative of limbic predominant TDP-43 encephalopathy (LATE), as well as varying degrees of Lewy body disease. This severe degree of mixed pathology is typically only found in much older people, Young told Alzforum, yet these SORL1 R953C carriers died between 61 and 75 years of age. Their mother's AD pathology was less severe, and she did not have LATE or DLB.

To Young, this suggests that this particular SORL1 variant wreaks a kind of havoc in the brain that instigates both AD and non-AD pathology. “If this whole endosomal trafficking pathway is gummed up, it could cause accumulation of a bunch of different misfolded proteins,” she said.

Andersen and Holstege are preparing yet more manuscripts describing other detrimental SORL1 mutations in families. These will further support Andersen's SORL1 variant prioritization system, as well as the idea that some SORL1missense variants can cause AD all on their own. Forthcoming data from another family is expected to show an autosomal-dominant inheritance pattern for a missense variant.

With more data than ever implicating SORL1 in AD pathogenesis, and with the Alzforum Mutations dataset as a guide to gauge pathogenicity of individual variants, Andersen, Holstege, Small, and other researchers hope that clinicians will start considering SORL1 alongside APP, PS1, and PS2 when screening patients for familial AD. They could also revisit their hunt for causative variants in families or probands who had previously come up negative for APP or PSEN1/2 mutations.

“The highly dangerous variants should be on a [sequencing] chip,” Andersen said. On their poster at AAIC, the scientists noted that once high-priority missense variants are considered clinically relevant, this will spur clinicians to perform family segregation analyses in carriers, thus providing evidence of autosomal-dominant inheritance patterns for these variants.

Andersen and Holstege urge cell and molecular biologists to use the new tools to pick variants for mechanistic studies.

More broadly, the collective findings clearly point to endosomal recycling as a driving force of AD, emphasized Scott Small of Columbia University in New York. Holstege agreed. She noted that, in her separate genetic studies of centenarians, those who remain sharp into their 100s tend to have endolysosomal genes free of risk variants, and flush with protective ones (Tesi et al., 2023). “It’s a beautiful finding,” Holstege said. “To remain cognitively healthy into old age, you must have a preserved endolysosomal system.”—Jessica Shugart

Comments

1. • Gael Nicolas
     Rouen University Hospital
   • Posted: 21 Jul 2023
   • Paper: The SORL1 p.Y1816C variant causes impaired endosomal dimerization and autosomal dominant Alzheimer's disease

   I think this variant is definitely a strong contributor to AD. However, the pedigrees also show that the patients with DNA available and carrying the variant also carry one APOE4 allele. Actually, APOE4 segregates as well as does SORL1 in these pedigrees. All affected individuals with DNA available are SORL1+/APOE4+. One unaffected individual is SORL1+/APOE4- (family 1) and one unaffected individual is SORL1-/APOE4+ (family 2). To be clear, I have absolutely no doubt of a major role of the SORL1 variant here, but I feel that this is very much consistent with a more complex inheritance and not purely autosomal-dominant, as shown in our penetrance paper (Schramm et al., 2022). Interestingly, we have the same variant in three independent families from France (one of them is mentioned in this preprint). Although there is an obvious aggregation of AD cases in the families, there is a huge diversity of ages of onset and younger cases have a positive family history in both branches, suggesting the contribution of additional factors. Some of them are APOE4+ but not the two youngest probands. This may suggest the contribution of undetected contributing variants along with SORL1.

   Overall, our penetrance paper (Schramm et al., 2022) and many pedigrees suggest a contribution of additional factors with SORL1 variants and that SORL1 alone may not be sufficient/fully penetrant. We have clear evidence for APOE4, as this is a common allele, but we know that there are many other other AD-associated variants, especially rare variants, among known variants (such as families with SORL1+ABCA7 as we previously reported in Campion et al., 2019), and in other papers and, obviously, not yet known variants.

   I thus recommend to use such results with great caution for genetic counseling, as we still don't exactly know how variants in other genes may drastically change an age of onset from 50 to 75-80 for example, or to absence of AD (as also shown for some truncating variants, as in Campion et al., 2019 where a mother transmitted a truncating a truncating variant and was unaffected with AD at age 95 years).

   References:

   Schramm C, Charbonnier C, Zaréa A, Lacour M, Wallon D, CNRMAJ collaborators, Boland A, Deleuze JF, Olaso R, ADES consortium, Alarcon F, Campion D, Nuel G, Nicolas G. Penetrance estimation of Alzheimer disease in SORL1 loss-of-function variant carriers using a family-based strategy and stratification by APOE genotypes. Genome Med. 2022 Jun 28;14(1):69. PubMed. Correction.

   Campion D, Charbonnier C, Nicolas G. SORL1 genetic variants and Alzheimer disease risk: a literature review and meta-analysis of sequencing data. Acta Neuropathol. 2019 Aug;138(2):173-186. Epub 2019 Mar 25 PubMed.

   View all comments by Gael Nicolas
2. • Gael Nicolas
     Rouen University Hospital
   • Posted: 22 Jul 2023
   • Paper: A familial missense variant in the AD gene SORL1 impairs its maturation and endosomal sorting.

   This is another example of a missense variant affecting shedding and maturation of SORL1 in a family, segregating incompletely with AD, and with large diversity of ages of onset. Such diversity, and putative inheritance from the father with dementia at the age of 83, suggests that additional factors may be required to result in AD, at least before the age of 70-75, knowing that LOAD is very common. In my opinion, this variant may be insufficient to cause AD, although it seems to be a strong determinant.

   We also have the same variant as in this work, in one of our unpublished patients (R953C). It is a sporadic case with an age at onset of 56 years, APOE genotype 3/4, unaffected parents at 80, large family, no other case except a paternal grandmother with an age at onset of 80. Unless there is a de novo mutation, which we could not check yet, it is inconsistent with autosomal-dominant inheritance. I hope we can see if one of the parents is a carrier, as this will help us better understand the variant's penetrance.

   This paper adds another variant to the increasing list of variants with a maturation defect. We reported 15 variants with a similar effect in Rovelet-Lecrux et al., 2021 after expression of 70 missense variants in HEK cells. We selected three variants with such an effect, and two without, to further evaluate them in IPSCs after Crispr/cas9 introduction. We found that all three selected missense variants showed a maturation defect, including one with a milder effect. The two variants with the strongest maturation defect showed significantly increased secreted Aβ levels, clearly linking these variants to AD pathophysiology.

   It is, however, unclear why, in the current paper, the authors mention our own paper with the following sentence: "Furthermore, a larger screen of 70 SORL1 coding variants suggested that impaired maturation may be general for dysfunctional proteins although no correlation to AD was established." We only studied SORL1, and we showed a direct link with AD for these specific variants, with the above-mentioned mechanism. We discussed that maturation defect of the encoded protein by a missense variant is a known mechanism for other diseases, but here we studied SORL1 missense variants impairing the maturation of SORL1 protein (this resulting to increased secreted Aβ) and not any other protein. There must be a misunderstanding on this point.

   References:

   Rovelet-Lecrux A, Feuillette S, Miguel L, Schramm C, Pernet S, Quenez O, Ségalas-Milazzo I, Guilhaudis L, Rousseau S, Riou G, Frébourg T, Campion D, Nicolas G, Lecourtois M. Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease. Acta Neuropathol Commun. 2021 Dec 18;9(1):196. PubMed.

   View all comments by Gael Nicolas

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References

News Citations

1. Sorting Out SORL1: 500+ Mutations Mapped, Prioritized in Alzforum Dataset 18 Jul 2023
2. Familial AD Mutations, β-CTF, Spell Trouble for Endosomes 16 Aug 2019
3. Doubling Rab5 in Mice Leads to Neurodegeneration—Without Plaques 9 Jan 2021

Mutations Citations

1. SORL1 Y391C
2. SORL1 Y1816C
3. SORL1 R953C

Paper Citations

1. Holstege H, Hulsman M, Charbonnier C, Grenier-Boley B, Quenez O, Grozeva D, van Rooij JG, Sims R, Ahmad S, Amin N, Norsworthy PJ, Dols-Icardo O, Hummerich H, Kawalia A, Amouyel P, Beecham GW, Berr C, Bis JC, Boland A, Bossù P, Bouwman F, Bras J, Campion D, Cochran JN, Daniele A, Dartigues JF, Debette S, Deleuze JF, Denning N, DeStefano AL, Farrer LA, Fernández MV, Fox NC, Galimberti D, Genin E, Gille JJ, Le Guen Y, Guerreiro R, Haines JL, Holmes C, Ikram MA, Ikram MK, Jansen IE, Kraaij R, Lathrop M, Lemstra AW, Lleó A, Luckcuck L, Mannens MM, Marshall R, Martin ER, Masullo C, Mayeux R, Mecocci P, Meggy A, Mol MO, Morgan K, Myers RM, Nacmias B, Naj AC, Napolioni V, Pasquier F, Pastor P, Pericak-Vance MA, Raybould R, Redon R, Reinders MJ, Richard AC, Riedel-Heller SG, Rivadeneira F, Rousseau S, Ryan NS, Saad S, Sanchez-Juan P, Schellenberg GD, Scheltens P, Schott JM, Seripa D, Seshadri S, Sie D, Sistermans EA, Sorbi S, van Spaendonk R, Spalletta G, Tesi N, Tijms B, Uitterlinden AG, van der Lee SJ, Visser PJ, Wagner M, Wallon D, Wang LS, Zarea A, Clarimon J, van Swieten JC, Greicius MD, Yokoyama JS, Cruchaga C, Hardy J, Ramirez A, Mead S, van der Flier WM, van Duijn CM, Williams J, Nicolas G, Bellenguez C, Lambert JC. Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease. Nat Genet. 2022 Dec;54(12):1786-1794. Epub 2022 Nov 21 PubMed.
2. Jensen AM, Kitago Y, Fazeli E, Vægter CB, Small SA, Petsko GA, Andersen OM. Dimerization of the Alzheimer's disease pathogenic receptor SORLA regulates its association with retromer. Proc Natl Acad Sci U S A. 2023 Jan 24;120(4):e2212180120. Epub 2023 Jan 18 PubMed.
3. Christensen SK, Narimatsu Y, Simoes S, Goth CK, Vaegter CB, Small SA, Clausen H, Andersen OM. Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer's-associated protein sorLA. 2020 Jul 13 10.1101/2020.07.12.199885 (version 1) bioRxiv.
4. Cataldo AM, Peterhoff CM, Troncoso JC, Gomez-Isla T, Hyman BT, Nixon RA. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol. 2000 Jul;157(1):277-86. PubMed.
5. Tesi N, vanderLee SJ, Hulsman M, vanSchoor NM, Huisman M, Pijnenburg Y, vanderFlier WM, Reinders M, Holstege H. Cognitively Healthy Centenarians are genetically protected against Alzheimer's disease specifically in immune and endo-lysosomal systems. 2023 May 17 10.1101/2023.05.16.23290049 (version 1) medRxiv.

Other Citations

1. Mutations

Further Reading

Papers

1. Small SA, Kent K, Pierce A, Leung C, Kang MS, Okada H, Honig L, Vonsattel JP, Kim TW. Model-guided microarray implicates the retromer complex in Alzheimer's disease. Ann Neurol. 2005 Dec;58(6):909-19. PubMed.
2. Vardarajan BN, Zhang Y, Lee JH, Cheng R, Bohm C, Ghani M, Reitz C, Reyes-Dumeyer D, Shen Y, Rogaeva E, St George-Hyslop P, Mayeux R. Coding mutations in SORL1 and Alzheimer disease. Ann Neurol. 2015 Feb;77(2):215-27. PubMed.
3. Campion D, Charbonnier C, Nicolas G. SORL1 genetic variants and Alzheimer disease risk: a literature review and meta-analysis of sequencing data. Acta Neuropathol. 2019 Aug;138(2):173-186. Epub 2019 Mar 25 PubMed.

News

1. Voilà SorLA! Sorting Receptor’s Structure Solved 6 Feb 2015
2. In Mini-Pigs Missing SORL1, Endosomes Swell and Aβ Peptides Rise in CSF 28 Sep 2022