29 Sep 2022

There's a new animal model of AD in town, and it’s not a worm, fly, or rodent. It’s a wee little pig. As described in their paper published September 20 in Cell Reports Medicine, researchers led by Charlotte Sørensen of Aarhus University in Denmark used CRISPR to take down a copy of SORL1—in Göttingen mini-pigs. In this porcine model, endosomes swelled within cells of the brain and Aβ peptides and total tau increased in the cerebrospinal fluid. Unlike mice, mini-pigs are large enough to allow for easy collection of CSF. Alas, there was no sign of amyloid plaques or tau tangles in 2½-year-olds, but the researchers think that these may have developed as the mini-pigs aged.

“This is a very elegant model that represents presymptomatic conditions driven by SORL1 loss of function, something that is difficult to study in human carriers,” commented Jessica Young of the University of Washington in Seattle. “This work highlights that biomarkers can reflect early cellular dysfunction and thus provide an early timepoint for potential therapeutic intervention.”

To create these animals, first author Olav Andersen and colleagues used CRISPR to knock out one or both copies of the SORL1, which may rank as an autosomal-dominant AD gene (June 2018 news; Alvarez-Mora et al., 2022). They chose pigs not only for the volume of their CSF, but because their brains fold into gyri and sulci, just like human brains do, and they share more ultra-conserved genomic regions with humans than do mice. After some setbacks—including one litter in which most of the piglets died a few days after birth—the researchers had one SORL1 homozygous knock-out, eight SORL1 heterozygotes, and 10 wild-type mini-pigs for the study.  

What did they find? First of all, the SORL1-deficient pigs (both homo- and heterozygotes) had 70 percent more Aβ38, 49 percent more Aβ40, and 69 percent more Aβ42 in their CSF than did control animals. Their CSF also had more sAPPβ, which is generated by endosomal cleavage of APP by β-secretase. However, judging by pig PiB-PET (try saying that three times fast), the SORL1-deficient mini-pigs had no amyloid plaques. This fits with the uptick of CSF Aβ peptides, which plummet in human CSF as amyloid plaques ensnare the peptides in the brain.

Measuring tau turned out to be a trickier endeavor. In contrast to Aβ, where the mini-pig and human sequences match perfectly, porcine tau shares only 89 percent sequence homology with human tau. The researchers found more total tau in the CSF of SORL1-deficient mini-pigs than wild-type controls. However, when they tried to measure phospho-tau using antibodies, which typically pick up the human form, including AT8, they got weak signals, suggesting that phospho-tau was either not present in the porcine CSF or that the antibodies could not detect it.

Gaël Nicolas of Rouen University Hospital in France thinks that the CSF findings provide further support for the central role of Ab peptides in AD pathophysiology, because increased Aβ peptides were associated with increased tau levels in the CSF. This pattern also occurs in asymptomatic people who carry APP, PSEN1, or PSEN2 mutations. 

The researchers also examined the brains of four SORL1 heterozygotes and four wild-type mini-pigs that they sacrificed between 24-30 months of age. They found no evidence of amyloid plaques, tangles, brain atrophy, or erosion of white-matter tracts. The researchers think that had the pigs lived longer, these pathological hallmarks of AD would have emerged. Mini-pigs that overexpresses pathogenic variants of PSEN1 and APP also have no plaques or tangles at 18 months (Jakobsen et al., 2016). Mini-pigs can live for about 15 years, so these 1½- and 2-year-olds would be considered young adults.

Preclinical AD for Pigs? Compared to wild-type mini-pigs, SORL1-deficient mini-pigs had elevated Aβ and tau in their CSF, and enlarged endosomes. They did not have Aβ plaques or signs of neurodegeneration. [Courtesy of Sørensen et al., Cell Reports Medicine, 2022.]

One striking phenotype did stand out from the postmortem analysis. Enlarged endosomes crowded neurons in the cortices of SORL1-deficient, but not wild-type, mini-pigs. These endosomes were more than twice the size of those in wild-type mini-pigs. This makes sense given SORL1’s role in recycling endosomes. Without the sorting protein, endosomes linger and swell (Jun 2020 news). The researchers proposed that the endosomal traffic jam likely explains the elevated Aβ peptides in the CSF, since previous studies found that SORL1 deficiency slows the recycling of APP out of endosomes, where β-secretase activity is at its highest (Mishra et al., 2022).

Nicolas wasn’t so sure about that, writing that the role of enlarged endosomes, both in cases of SORL1 inactivation and in APP/PSEN mutations, is difficult to interpret. “Whether or not this phenomenon plays a direct pathophysiological role, or happens in parallel to increased Aβ secretion, remains to be determined,” he wrote.

The findings dovetail with other studies implicating endolysosomal dysfunction as an early event in AD, even among people without disease-associated variants in SORL1 (Cataldo et al., 2000). Notably, endosomal woes can be triggered by increases in β-CTF—a fragment produced by BACE1 cleavage of APP—which revs up endocytosis through its recruitment of Rab5 (Jan 2010 news; Aug 2019 news). Doubling Rab5 in transgenic mice produced a similar effect (Jan 2021 news).

“The mini pig should also be instrumental in establishing whether the CSF biomarkers currently useful in AD diagnosis are equally useful as a window on the most important disease-triggering events in AD, which increasingly implicate endosomal-lysosomal dysfunction as well as multiple APP metabolites,” commented Ralph Nixon of New York University School of Medicine.—Jessica Shugart

Comments

1. • Jessica Young
     University of Washington
   • Posted: 28 Sep 2022

   Genetic evidence suggests that truncating SORL1 mutations are causative for AD. Several in vitro models have shown that one consequence of loss of SORL1 function is impairment in endosomal pathways, such as recycling (Hung et al., 2021; Mishra et al., 2022). In this study, Andersen et al. use Gottingen mini-pigs to model SORL1 deficiency in vivo. They used CRISPR/Cas9 to generate SORL1+/- pigs and observed CSF biomarkers that are consistent with both early AD phenotypes and endosomal dysfunction: increases in Aβ and tau, and a reduction in soluble SORLA protein. When mini-pig brains were examined, they found an increase in the size of early endosomes. Swollen early endosomes, indicative of cellular traffic jams, are an early cytopathology in AD and have also been observed in vitro in neuronal models (for example: Knupp et al., 2020). However, the mini-pig brains did not show the classic neuropathologies of AD, amyloid plaques and tau tangles, nor did they show evidence of overt neurodegeneration at the time point analyzed.

   All told, this is a very elegant model that represents presymptomatic conditions driven by SORL1 loss of function, something that is difficult to study in human carriers. This work highlights that biomarkers can reflect early cellular dysfunction and thus identify an early timepoint for potential therapeutic intervention. Furthermore, since endosomal traffic jams have now been observed across multiple SORL1 models, both in vivo and in vitro, this work is further confirmation that the primary driver for SORL1 loss of function leading to AD is through disruptions in the endosomal network.

   This study introduces a powerful preclinical model and future experiments detailing AD-related phenotypes as SORL1-deficient mini-pigs age will be very interesting and informative for the field.

   References:

   Hung C, Tuck E, Stubbs V, van der Lee SJ, Aalfs C, van Spaendonk R, Scheltens P, Hardy J, Holstege H, Livesey FJ. SORL1 deficiency in human excitatory neurons causes APP-dependent defects in the endolysosome-autophagy network. Cell Rep. 2021 Jun 15;35(11):109259. PubMed.

   Mishra S, Knupp A, Szabo MP, Williams CA, Kinoshita C, Hailey DW, Wang Y, Andersen OM, Young JE. The Alzheimer's gene SORL1 is a regulator of endosomal traffic and recycling in human neurons. Cell Mol Life Sci. 2022 Feb 28;79(3):162. PubMed.

   Knupp A, Mishra S, Martinez R, Braggin JE, Szabo M, Kinoshita C, Hailey DW, Small SA, Jayadev S, Young JE. Depletion of the AD Risk Gene SORL1 Selectively Impairs Neuronal Endosomal Traffic Independent of Amyloidogenic APP Processing. Cell Rep. 2020 Jun 2;31(9):107719. PubMed.

   View all comments by Jessica Young

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References

News Citations

1. Gaining Notoriety, SORL1 Claims Spot Among Top Alzheimer’s Genes 7 Jun 2018
2. Without SORL1, Endosomes Swell in Neurons but not Microglia 2 Jun 2020
3. APP in Pieces: βCTF implicated in Endosome Dysfunction 6 Jan 2010
4. Familial AD Mutations, β-CTF, Spell Trouble for Endosomes 16 Aug 2019
5. Doubling Rab5 in Mice Leads to Neurodegeneration—Without Plaques 9 Jan 2021

Paper Citations

1. Alvarez-Mora MI, Blanco-Palmero VA, Quesada-Espinosa JF, Arteche-Lopez AR, Llamas-Velasco S, Palma Milla C, Lezana Rosales JM, Gomez-Manjon I, Hernandez-Lain A, Jimenez Almonacid J, Gil-Fournier B, Ramiro-León S, González-Sánchez M, Herrero-San Martín AO, Pérez-Martínez DA, Gómez-Tortosa E, Carro E, Bartolomé F, Gomez-Rodriguez MJ, Sanchez-Calvin MT, Villarejo-Galende A, Moreno-Garcia M. Heterozygous and Homozygous Variants in SORL1 Gene in Alzheimer's Disease Patients: Clinical, Neuroimaging and Neuropathological Findings. Int J Mol Sci. 2022 Apr 11;23(8) PubMed.
2. Jakobsen JE, Johansen MG, Schmidt M, Liu Y, Li R, Callesen H, Melnikova M, Habekost M, Matrone C, Bouter Y, Bayer TA, Nielsen AL, Duthie M, Fraser PE, Holm IE, Jørgensen AL. Expression of the Alzheimer's Disease Mutations AβPP695sw and PSEN1M146I in Double-Transgenic Göttingen Minipigs. J Alzheimers Dis. 2016 Jul 14;53(4):1617-30. PubMed.
3. Mishra S, Knupp A, Szabo MP, Williams CA, Kinoshita C, Hailey DW, Wang Y, Andersen OM, Young JE. The Alzheimer's gene SORL1 is a regulator of endosomal traffic and recycling in human neurons. Cell Mol Life Sci. 2022 Feb 28;79(3):162. PubMed.
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.

Further Reading

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