Presenilin Mutations Stall Endosomal Transport, Swell Axons
Dysfunctional presenilin 1, infamous for misprocessing the amyloid precursor protein in familial Alzheimer’s disease, isn’t a one-trick pony. PS1 variants also weaken lysosomes, slow endosomal trafficking, and bloat axons. How? By pumping the molecular brake on motor proteins, say researchers led by Ralph Nixon, New York University. In Science Advances on April 29, they reported that in PS1 knockout neurons, dysfunctional endosomes wedged open cation channel TRPML1. Calcium then flooded out, activating kinases that phosphorylated and stalled protein motors that transport endosomes to the soma.
- In PS1 variant mice, an overactive calcium channel starts a harmful cascade.
- Endosome transport stalls, axons swell.
- Blocking the channel restored normal endosome transport and axons.
Blocking the calcium release, or the kinases, restored endosome trafficking. This scenario also played out in tissue slices from old mice carrying familial AD (FAD) PS1 variants. These findings support previous work suggesting that presenilin 1 moonlights to manage calcium balance and lysosomal function.
“This research is a major advance in understanding the underlying mechanisms of Alzheimer’s by providing a connection between PS1 mutations and impaired axonal transport,” Claire Mitchell, University of Pennsylvania, Philadelphia, told Alzforum. Grace Stutzmann, Rosalind Franklin University, North Chicago, agreed. “It stands out as a needed piece of the cell biology puzzle of AD,” she wrote (comment below). “The field pulls heavily from genetics and end-point proteinopathy studies, but would greatly benefit from more functional and mechanistic studies, such as this, to delineate how gene-level changes directly contribute to AD phenotypes.”
In FAD, variants of PS1, the proteolytic subunit of the γ-secretase complex, overproduce long, toxic Aβ peptides that form amyloid plaques (Apr 2022 news). However, Nixon and others had reported that PS1 helps acidify lysosomes, and that PS1 variants fail in this respect (Jun 2010 news; Coffey et al., 2014). Lysosomes rely on low pH to degrade unwanted proteins, and Nixon found that blocking this proteolysis stalled axonal transport of autophagic vacuoles and late endosomes, causing dystrophic neurites that looked uncannily like those found in the AD brain (May 2011 news). In PS1 knockout cells, lysosomes with high pH dumped massive amounts of calcium into the cytosol via the TRPML1 channel (Lee et al., 2015). This cation channel is important for lysosomal transport. Mutations in the gene cause trafficking defects and the lysosomal storage disorder, type IV mucolipidosis (reviewed by Wang et al., 2014). Could this channel link faulty PS1 to wonky axonal transport?
To find out, co-first authors Pearl Lie and Lang Yoo used cultured cortical neurons from PS1 knockout mice. They measured the pH of late endosomes (LE) with an acid-sensitive dye, and tracked the organelles’ movements along axons. Normally, LE pH drops as they trek down the axon to the soma en route to fusing with lysosomes; however, in the KO neurons, their pH was abnormally high. Compared to LEs in healthy neurons, about half as many of the KO LEs traversed in retrograde along the axon, stalling more often and for longer periods.
A TRPML1 agonist similarly slowed LE transport to a slog. In these neurons, and in PS1 KOs, the lethargic organelles ballooned along with the axons. In contrast, when the researchers treated KO neurons with a TRPML1 channel blocker, endosomes motored along axons akin to those in control neurons. The authors concluded that the cation channel caused the traffic jam.
The scientists suspected motor proteins might be lost or malfunctioning. To test the former, they isolated LE-rich cellular fractions of PS1 KO neurons and measured the amounts of kinesin, dynactin, and dynein isoforms. KOs had the same amount, or even more, of the motor proteins than did control cells.
Were these motors working properly? KO neurons contained two-thirds more phosphorylated dynein intermediate chain (p-DIC) than controls. Endosomes bind p-DIC, which can only hitch a ride on dynactin when it has been dephosphorylated (Mitchell et al., 2012; Jie et al., 2017; Vaughan et al., 2001). Could promiscuous phosphorylation be to blame for stalling transport?
To test this, the researchers co-immunostained and tracked LEs with antibodies that specifically bind either DIC or p-DIC. Endosomes in PS1 KO neurons bound more p-DIC and trekked a shorter distance than those in control cells. Likewise, KO neurons expressing a phosphomimetic mutant of DIC had swollen, sluggish LEs, while cells expressing a non-phosphorylatable mutant had normal endosome shape and retrograde speed, even though PS1 was absent.
The authors think that hyperphosphorylation of DIC hinders its ability to shove LEs along. In a series of cell culture experiments, they found that calcium released through TRPML1 activated c-Jun N-terminal kinase, which in turn phosphorylated DIC. Previous reports had shown that knocking out PS1 rendered JNK hyperactive, and that the overactive kinase hampers retrograde transport (Kim et al., 2001; Drerup and Nechiporuk, 2013). Blocking JNK improved retrograde endosome transport and reduced the number of dystrophic neurites.
All told, the findings indicate that when PS1 variants raise the pH in late endosomes and lysosomes, calcium released by TRPML1 riles up JNK to hyperphosphorylate DIC, which stalls vesicle transport (see image below). “Each of these points in the path can further amplify and spin off additional pathogenic cascades, thus creating a network of seemingly disparate features that share common upstream sources,” Stutzmann wrote.
Acid Trip? Early endosomes (EE) in dendrites migrate down the axon (green arrows) becoming more acidic as they go, maturing into late endosomes (LE) and eventually morphing into lysosomes (top). When LEs are acidic enough, they have a good trip (bottom left). When they are not, overactive TRPML1 (blue knob) pumps out excess calcium, which activates JNK to phosphorylate DIC (red circle), hampering dynein movement (bottom right). [Courtesy of Lie et al., Science Advances, 2022.]
Could this string of events hold true in vivo? The researchers used PS1 knock-in mice expressing human presenilin 1 with the M146V FAD variant. LE acidification and axonal transport appeared normal in cortical slices from 6-month-old mice, but as they got older, axonal transport declined. In cortical slices from 13-month-old mice, LEs were less acidic than in young controls. By 22 months, mice had dystrophic neurites stuffed with LEs, p-DIC, and p-JNK.
“Faulty PS1, compounded with lysosome acidification issues caused by aging, allows retrograde transport problems and dystrophic neurites to appear only in older mice, mimicking sporadic AD,” Nixon said. He noted that many factors might corrupt lysosomal acidification.
Mitchell agreed. “There may be an age-dependent increase in lysosomal pH in people who do not have a PS1 mutation, so this dysfunction and dystrophic neurite formation may be relevant to many more people with AD, regardless of how the organelles fail to acidify,” she said. For this reason, Hagit Elda-Finkelman, Tel Aviv University, Israel, thinks the work has broader implications for neurodegenerative disorders (comment below).
However, Ilya Bezprozvanny, University of Texas Southwestern Medical Center, Dallas, cautioned that it is hard to tell which event truly kicks off the catastrophic cascade in age-related diseases. “While the authors suggest that abnormal lysosomal acidification is the most upstream pathogenic event, it is also possible that other signaling events, such as dysregulation of endoplasmic reticulum calcium signaling or accumulation of Aβ oligomers, drive the pathology in parallel,” he said (Jun 2010 news).
As for the role of TRPML1, it has caught the eyes of pharma companies. In 2019, Merck acquired the rights to small molecule agonists against the channel (company press release). Mitchell cautioned that too much calcium can be detrimental. “Scientists must be aware of potential complications from overstimulating this channel,” she said.—Chelsea Weidman Burke
Mutation Interactive Images Citations
- Ratio of Short to Long Aβ Peptides: Better Handle on Alzheimer's than Aβ42/40?
- Death of the Neatnik: Neurons Perish When Trash Clutters Their Space?
- Lysosomal Block Clogs Transport, Swells Neurites
- Perplexing Presenilins: New Evidence for Calcium Leak Channels
Research Models Citations
- Coffey EE, Beckel JM, Laties AM, Mitchell CH. Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer's disease-linked presenilin 1 A246E mutation can be reversed with cAMP. Neuroscience. 2014 Mar 28;263:111-24. Epub 2014 Jan 10 PubMed.
- Lee JH, McBrayer MK, Wolfe DM, Haslett LJ, Kumar A, Sato Y, Lie PP, Mohan P, Coffey EE, Kompella U, Mitchell CH, Lloyd-Evans E, Nixon RA. Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification. Cell Rep. 2015 Sep 1;12(9):1430-44. Epub 2015 Aug 20 PubMed.
- Wang W, Zhang X, Gao Q, Xu H. TRPML1: an ion channel in the lysosome. Handb Exp Pharmacol. 2014;222:631-45. PubMed.
- Mitchell DJ, Blasier KR, Jeffery ED, Ross MW, Pullikuth AK, Suo D, Park J, Smiley WR, Lo KW, Shabanowitz J, Deppmann CD, Trinidad JC, Hunt DF, Catling AD, Pfister KK. Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport. J Neurosci. 2012 Oct 31;32(44):15495-510. PubMed.
- Jie J, Löhr F, Barbar E. Dynein Binding of Competitive Regulators Dynactin and NudE Involves Novel Interplay between Phosphorylation Site and Disordered Spliced Linkers. Structure. 2017 Mar 7;25(3):421-433. Epub 2017 Feb 2 PubMed.
- Vaughan PS, Leszyk JD, Vaughan KT. Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin. J Biol Chem. 2001 Jul 13;276(28):26171-9. Epub 2001 May 4 PubMed.
- Kim JW, Chang TS, Lee JE, Huh SH, Yeon SW, Yang WS, Joe CO, Mook-Jung I, Tanzi RE, Kim TW, Choi EJ. Negative regulation of the SAPK/JNK signaling pathway by presenilin 1. J Cell Biol. 2001 Apr 30;153(3):457-63. PubMed.
- Drerup CM, Nechiporuk AV. JNK-interacting protein 3 mediates the retrograde transport of activated c-Jun N-terminal kinase and lysosomes. PLoS Genet. 2013;9(2):e1003303. Epub 2013 Feb 28 PubMed.
- Lie PP, Yoo L, Goulbourne CN, Berg MJ, Stavrides P, Huo C, Lee JH, Nixon RA. Axonal transport of late endosomes and amphisomes is selectively modulated by local Ca2+ efflux and disrupted by PSEN1 loss of function. Sci Adv. 2022 Apr 29;8(17):eabj5716. PubMed.
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Rosalind Franklin University/The Chicago Medical School
This study is an important extension in a series of mechanistic studies from the Nixon lab detailing how misregulation of lysosome through PS or Ca2+ deficits contribute to key features of AD. The value of this study lies in the intricate linkage of PS deficits, either in model cells or AD mouse models, which impedes proper construction of the vATPase proton pump, and thus impairs proper acidification of lysosomes.
This result in deficits in autophagosome degradation and excess calcium release from lysosome through pH-regulated TRPML1 channels. The authors revealed that the excess calcium-triggered kinase activity hyperphosprylated dynein components and slowed, or interfered with, retrograde transport, contributing to the dystrophic neurites that are characteristic of AD cellular pathology.
This study stands out as a needed piece in the cell biology puzzle of AD, a field which pulls heavily from genetics and end-point proteinopathy studies, but would greatly benefit from more functional and mechanistic studies such as this to delineate how these gene-level changes directly contribute to AD phenotypes, and specifically how the defining protein aggregates and cellular pathophysiology arise.
During this exciting time of deep transcriptomics and revelations in gene-level changes in AD, this series of studies from the Nixon lab has ticked many of these boxes, tracing the pathway from PS mutations or loss of function, to lysosome de-acidification, calcium dysregulation, protein mishandling and trafficking deficits. Each of these points in the path can further amplify and spin off additional pathogenic cascades, thus creating a network of seemingly disparate features that share common upstream sources.
It would further solidify the study to know more regarding the nature, role, and status of the endogenous activators of TRPML1, as well as additional consequences of the altered calcium release, but given the depth and compelling detail of the current findings, these remaining questions would likely fill another complete study.
Tel Aviv University
The mechanisms by which Presenilin mutations lead to Alzheimer’s disease pathogenesis remains somewhat elusive. In previous work, Ralph Nixon’s group showed that lack of PSEN1 in neurons impairs lysosomal acidification resulting in deleterious effects produced by uncontrolled calcium efflux through hyperactivation of TRMP1, an ion channel in the lysosome.
In this new paper, the group further investigated the molecular mechanism underling axon degeneration mediated by impaired acidification of the lysosomal organelles, and its potential relation to PSEN1. Apparently, reduced acidification of late endosomes/amphisomes disturbs their retrograde cellular transport mediated by the motor protein dynein. In this signaling cascade scenario, de-acidification results in “inside-out” activation of TRMP1, leading to persistent calcium efflux, activation of c-Jun N-terminal kinase (JNK), and aberrant phosphorylation of dynein that slows down retrograde transport.
Lack of PSEN1 in PSEN1 knockout neurons copied theses effects, thus implicating PSEN1 dysfunction in impaired retrograde motility of de-acidified organelles. Hence, insufficient transport of the lysosomal system vesicles represents a major cause of axon degeneration.
However, the use of PSEN1 KO systems in most of their studies raises a concern of the relevance of these results to AD that rather express PSEN1mutants. The authors addressed this question by using a mouse model expressing familial AD PSEN1 mutant. A similar phenomenon was observed in the mouse brains, which exhibited dystrophic neurites, de-acidified lysosomes, and accumulated degradative organelles, although these observations were age-dependent.
An important aspect of this work is its broad implications to neurodegenerative disorders, linking acidification failure in the lysosome pathway with axonal dystrophy. An interesting question in this respect is whether de-acidification of lysosomes imposed by other signaling components would result in a similar signaling cascade. For example, the kinase, GSK-3, an important drug discovery target in AD, has been shown to impair lysosomal acidification via the mTORC1/autophagy pathways. It will be interesting to examine if hyperactive GSK-3, often seen in AD conditions, impairs lysosome-organelles cellular traffic via its ability to de-acidify lysosomes.
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