Van Acker ZP, Perdok A, Hellemans R, North K, Vorsters I, Cappel C, Dehairs J, Swinnen JV, Sannerud R, Bretou M, Damme M, Annaert W. Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism. Nat Commun. 2023 May 24;14(1):2847. PubMed.
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University of Adelaide
This paper is an important step forward in understanding lysosomal dysfunction in the context of PLD3 deficiency. The study examines the consequences of dysfunction of a lysosomal enzyme—and Alzheimer’s disease (AD) genetic risk factor—at a level of detail that has seldom been attempted.
This study contends that PLD3 degrades mitochondrial DNA to keep lysosomes in good working order. When this doesn’t happen, as appears to be the case with AD-linked PLD3 variants, lysosomal dysfunction ensues that culminates in lysosomal leakage of mitochondrial DNA, which spurs cGAS-STING signaling and APP fragment accumulation.
To me, this is a story about lysosomal degradative capacity and autophagic stress, i.e., the accumulation of autophagic cargo when lysosomal degradative capacity is surpassed by incoming autophagic cargo flow. Last year, Ralph Nixon showed that autophagic stress drives amyloid plaque formation (Lee et al., 2022). This study further builds our knowledge of fundamental mechanisms in AD pathogenesis. This is done by examining the molecular consequences of autophagic stress by using an AD-relevant model—modification of PLD3 in cells.
Van Acker et al. also focus our attention on the finer points of how to tackle the lysosomal system as a therapeutic target for AD. Once again, we are confronted by data that illustrate why solely increasing autophagy will likely fail as a therapeutic avenue in established disease. Increasing the flow of molecular waste into a struggling lysosome that has succumbed to AD-related processes, or has been dealt a bad genetic hand, will likely make the situation worse. This concept was demonstrated nicely in this study by toggling autophagic waste flow through experimental manipulation of PINK1. The authors therefore emphasize the importance of targeting lysosomal degradative capacity, and not just “autophagy” in general, as is so often glossed over in the wider literature.
There are specific open questions that would be important to follow up on. For example, what makes lysosomes leaky in this study? Lysosomal rupture is an important part of the mechanism presented in Van Acker et al., however, the mechanisms remain obscure. This is an exceptionally difficult question to address, but it is a key missing piece of the puzzle.
Accumulation of mitochondrial DNA is posited as a key element of lysosomal failure here. One peculiarity in this study is that rescue of PLD3 KO cells with the M6R PLD3 variant, which shows hyperfunction with regard to clearance of mitochondrial DNA from the lysosome in the same study, causes increased autophagic stress and decreased mitochondrial function commensurate with hypofunctional PLD3 alleles. This would seem to run against the hypothesis that lysosomal accumulation of mitochondrial DNA is solely responsible for downstream organellar dysfunction and hints at broader mechanisms related to lysosomal hydrolytic failure.
Further research should follow up on how generalizable these findings are to other models of AD pathogenesis. There are some amazing results in this paper—one I found particularly striking was the accumulation of mitochondrial DNA inside lysosomes in the absence of PLD3. This work was performed in cell lines, but are analogous changes present in mouse models of amyloidosis, or sections from human brain? Finally, it’s always worth asking whether modelling environmental factors that sway AD risk in humans impinge on the same biology.
References:
Lee JH, Yang DS, Goulbourne CN, Im E, Stavrides P, Pensalfini A, Chan H, Bouchet-Marquis C, Bleiwas C, Berg MJ, Huo C, Peddy J, Pawlik M, Levy E, Rao M, Staufenbiel M, Nixon RA. Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. Nat Neurosci. 2022 Jun;25(6):688-701. Epub 2022 Jun 2 PubMed.
Lund University
This is an excellent paper on PLD3’s role in Alzheimer’s disease by Van Acker and colleagues from the Annaert lab in Leuven. What makes this work fascinating, beyond the high-level cell biology work, is that it elegantly ties together dysfunction of lysosomes to mitochondria, autophagy, lipids, and APP. Although I have no expertise on the genetics of PLD3 in AD, and am aware of the mixed literature on PLD3 in AD, e.g., on APP processing, I found it very interesting how PLD3 mutations, with either more or less mtDNA exonuclease activity to mtDNA, impaired lysosomes, and, among other changes, altered mitochondrial respiration, induced mitophagy, and promoted accumulation of cholesterol/cholesterol esters. It was also fascinating that alterations from PLD3 mutations were reduced in APP knockout cells, and that APP C-terminal fragments are involved in effects of the PLD3 mutants.
The origins of AD lay in how cellular systems are affected early on, even prior to the appearance of the characteristic amyloid plaques and neurofibrillary tangles. The current work shows how PLD3 mutations can negatively impact cellular functions. How aging induces such PLD3 mutations to augment cellular dysfunctions that eventually lead to AD will require more work to elucidate, but likely relates to prior evidence for dysfunctions due to the age of cellular systems (mitochondria, lysosomes).
One minor caveat is that this work was all done in human neuroblastoma cells. The marked induction of autophagic organelles is particularly pronounced in neuronal dystrophies in AD, the cell biology of which might differ somewhat. The authors mention the possibility that an analogous lysosomal backup might also affect microglia in AD.
University Kiel
A tour de force, this work analyzes the function of the lysosomal phospholipase D3 enzyme, which has been identified as an AD risk gene, although the genetic link is under debate. From in vitro studies it was known that PLD3 acts as a 5′-3′ exonuclase, but its natural substrates remained enigmatic. The authors now identified mitochondrial DNA as being utilized by PLD3. Mitochondria are delivered to lysosomes mainly by mitophagy, a specialized autophagy pathway.
Using PLD3-knockout neuroblastoma cells, followed by rescue experiments with wild-type and AD-associated PLD3 mutants, it could be revealed that impaired mtDNA hydrolyzing function due to PLD3 misfunction caused an increase in lysosomal nucleotide content, an altered catabolic activity of lysosomes, less pronounced lysosome-mitochondria contact sites associated with disrupted mitochondrial fitness, and reduced clearance by mitophagy. The study suggests that these effects induce (transient) lysosomal ruptures, where mtDNA can escape and activate signaling through cGAS/STING. This pathway senses DNA, enabling induction of innate immune defense mechanisms. Here, the authors try to link cGAS-STING activation with defective APP metabolism, i.e., an accumulation of lysosomal cholesterol and C-terminal APP fragments. Importantly, depleting APP in mice reduced the STING activation. Direct inhibition of STING also normalized APP-CTF levels, suggesting feedback loops between lysosomal nucleotide metabolism, cGAS/STING signaling, and lysosomal APP metabolite degradation.
The study is another wonderful example of why endosomes/lysosomes and their dysfunction, caused by altered activities of lysosomal enzymes/proteins, is linked to a complex pathology culminating in neuroinflammation and neurodegeneration, as frequently reported for AD, PD, and other neuropathologies. The specific case of PLD3 and its role as a newly discovered lysosomal mitochondrial exonuclease further illustrate this essential link.
The study is, as usual from the lab of these authors, extremely well and carefully performed. The series of elegant experiments provide compelling evidence that mitochondrial DNA represents a natural PLD3 substrate. How PLD3 differentiates between nuclear and mitochondrial DNA substrates is an open question. In the case of PLD3 deficiency or PLD3 mutations, mtDNA can accumulate within lysosomes.
Surprisingly, a lysosomal storage disease with PLD3-deficiency has not yet been described in people. The pathological accumulation of mtDNA most likely also impairs other lysosomal hydrolase activities, including cholesterol-handling proteins and proteolytic activities. The latter are required for efficient degradation of membrane-bound C-terminal APP fragments. Interestingly, despite the rather pronounced lysosomal phenotype, no effect on lysosomal pH was revealed, a factor which has been recently linked to the development of neurodegeneration in AD.
More importantly, the primary and secondary effects of PLD3 dysfunction lead to a, in my view, still-unclear impairment of lysosomal integrity or, in other words, ruptures where intraluminal DNA may enter the cytosol, causing TLR9 and cGas/STING signaling. Here, it is of note that TLR9 and STING are activated differently: TLR9 through luminal buildup of lysosomal DNA, STING through cytosolic DNA from mitochondria, as shown in this study. When autophagy was inhibited in PLD3-deficient cells by downregulation of PINK1, TLR9 signaling was suppressed.
To what extent the APP-CTF metabolic effect and the cGas/STING signaling events are causally linked remains unclear. It could be that STING induces autophagy that further leads to a buildup of APP-CTFs in autophagosomes. Inhibiting STING decreases the buildup of autophagolysosomes, making APP-CTF accessible to processing. Interestingly, depletion of APP also partially rescues STING activation, an exciting link which is not yet fully understood. Lysosomes seem less leaky so that less DNA can escape and activate STING. Could the normalization of PLD3-knockout lysosomes by depletion of APP be linked to the fact that APP-CTF increases SREBP2, and thus cholesterol synthesis, maybe leading to more rigid/leaky lysosomes?
Despite these open questions, APP-CTF metabolism and the cGas/STING signaling pathway may contribute to our understanding of the pathological cascades observed in the progression of Alzheimer’s disease related to APP metabolism and neuroinflammation. Here also, the role of the structurally related lysosomal PLD4 nuclease requires attention, since both PLD3 and 4 may act in concert and in a cell-type-specific manner, i.e., PLD4 seems more relevant in immune cells, such as microglia.
University College London
Wim Annaert and colleagues have carried out a very thorough study of the functions of PLD3, combining detailed biochemical and cell biology studies in cell lines. Previous work from a number of labs has found that PLD3 is a lysosomal exonuclease, and that it is found within accumulations of lysosomes in dystrophic neurites around amyloid plaques in human postmortem brain. Similarly, there have been several reports, including the original genetics paper from Cruchaga, Goate, and colleagues, that increasing and decreasing PLD3 expression results in reciprocal changes in Aβ peptide production. With the ongoing debate around whether PLD3 variants are truly pathogenic, and the increasing interest in endolysosomal dysfunction as a pathogenic mechanism in Alzheimer’s disease, this is a timely study.
Here, the authors go beyond those earlier studies to analyze the function of proposed pathogenic variants of PLD3 in neuroblastoma cells (SH-SY5Y), beginning with PLD3 exonuclease activity, which they demonstrate is particularly targeted to mitochondrial DNA. The results are fascinating, but not straightforward, as some variants demonstrate increased, and others decreased, exonuclease activity. However, all variants appear to lead to lysosome and autophagy dysfunction, which the authors interpret as simply that any change in lysosomal degradation of mtDNA leads to lysosome dysfunction.
The alternative hypothesis is that lysosome dysfunction is independent of the exonuclease activity—which would be inconsistent with the linear model proposed here for how PLD3 variants might contribute to elevated AD risk. In essence, the authors argue that PLD3 variants alter lysosomal degradation of mtDNA, and any change in this process results in lysosome dysfunction, which, in turn enables release of single-stranded DNA from the lysosome to the cytoplasm. This in turn activates the cGAS-STRING pathway, activating autophagy, and leading to altered APP processing and Aβ production, further disrupting endolysosome and autophagy function.
While an attractive model, much of this is necessarily speculative at this stage, and many of the details require fleshing out. It will be very interesting to see how this model applies in neurons, with their complex vesicular trafficking networks, and in microglia, which have all the same cellular machinery and express PLD3. As to the pathogenicity of PLD3 coding variants, this paper is an important contribution to the debate, but on its own can’t provide a definitive answer.
NOVA Medical School
The authors use a replacement/rescue strategy to determine the impact of late-onset AD variants in SHSY5Y cells knocked out for PLD3, a lysosomal exonuclease. They carefully demonstrated that mitochondrial DNA is a PLD3 substrate, and that PLD3 mutants cause defects in mitochondria degradation and function, leading to the appearance of morphologically altered endolysosomes. They further dissected downstream nucleotide sensing pathways linked to inflammation. The authors also found that PLD3 KO- and PLD3 variant-dependent endosomal dysfunction potentiated cholesterol accumulation and APP processing, leading to increased APP-C-terminal fragments (CTFs) and Aβ secretion. How the exonuclease activity and undigested mitochondria increase APP endosomal processing was left unexplored.
It was surprising that APP KO rescued PLD3 KO and variant phenotypes, including impaired autophagy and mitochondria degradation. Although other authors have established connections between autophagy and APP, and we have established that Aβ affects the endolysosomal system, it remains to be demonstrated how the absence of APP rescues PLD3 function. The authors suggest it may be via accumulation of toxic APP-CTFs at PLD3 abnormal endosomal compartments. This is an exciting hypothesis that needs to be tested.
The GWAS identification of several genes in the endolysosomal system potentiated this study on PLD3, as it did ours on Bin1 and CD2AP, and others’ on CALM and Sorla (reviewed by Guimas et al., 2018). These results add to the idea that endosomal dysfunction is determinant in the control of Aβ production.
Interestingly, the authors go further. By linking lysosomal dysfunction to mitochondria, the cellular bioenergetic capacity, and lipid metabolism, they confirm the endolysosomal system as an essential hub of cellular homeostasis in general. Importantly, these results should be confirmed in polarized and synaptically connected neurons that have differentiated to control cellular homeostasis to cope with synaptic activity.
References:
Guimas Almeida C, Sadat Mirfakhar F, Perdigão C, Burrinha T. Impact of late-onset Alzheimer's genetic risk factors on beta-amyloid endocytic production. Cell Mol Life Sci. 2018 Jul;75(14):2577-2589. Epub 2018 Apr 27 PubMed.
University of Washington
GWAS and other studies have consistently linked genes in pathways related to vesicular trafficking, neuroinflammation, and cholesterol metabolism, as high risk for late-onset AD. It is quite likely that the genes that function in these pathways are not mutually exclusive. This concept is shown nicely in this exciting work from the Annaert lab. Here they dissect the functions of PDL3, a gene associated with LOAD risk. Surprisingly, they pinpoint the function of PDL3 as primarily degrading mitochondrial DNA (mtDNA). Furthermore, the data suggest that LOAD variants in PDL3 induce a pathogenic feed-forward loop involving endo-lysosome leakiness and autophagic degradative capacity deficiencies, activation of immune pathways, and dysregulated cholesterol metabolism.
This work has the potential to classify variants in PDL3 based on the phenotypes described in this paper, which will give new insight into pathogenicity and help dissect how different LOAD-associated variants affect the function of the protein. Hopefully, future work will examine if and how impaired autophagy and lysosome degradative capacity also lead to tau pathology. Finally, this work further solidifies that deficiencies in endo-lysosomal processes are key contributors to neuronal dysfunction in AD.
New York University School of Medicine/Nathan Kline Institute
This paper adds interesting new insight into the functioning and malfunctioning of PLD3 within lysosomes in a neuroblastoma cell model. Van Acker et al. demonstrate a normal role in lysosomes for PLD3 in degrading mitochondrial DNA, and confirm in this model that PLD3 deficiency alters lysosomal morphology and activities. There is no full consensus on the genetic associations of PLD3 polymorphisms with late-onset AD. Even so, the lysosome phenotype generated in PLD3 mouse models does recapitulate the autophagy-lysosome abnormalities that are reported in Yuan et al. (2022) and are also seen in neurons in mouse/cell models of Alzheimer’s disease (AD) and Down's syndrome (DS) and in AD/DS brains (Jiang et al., 2010; Jiang et al., 2016; Jiang et al., 2019; Lee et al., 2015; Lee et al., 2022), which reflect disruption of lysosomal proteolysis. That lysosomal membrane damage and leakage of mtDNA leads to activation of the cGAS-STING pathway is intriguing and may well be part of the explanation for the seemingly counterproductive induction of autophagy in AD that is sustained, paradoxically, in the face of declining lysosome efficiency (Bordi et al., 2016).
The parallels between the phenotype in this cell model and the endolysosome-autophagy phenotype we have described in mouse familial AD models, DS patient cells, and mouse models support mounting evidence, in both early onset and late-onset AD and related disorders, that lysosomal dysfunction driving autophagy failure is a key shared pathogenic mechanism across all types of AD.
Van Acker et al. describe additional details of the lysosome dysfunction in their PLD3 model that implicate a key role for APP-CTF in driving this dysfunction. These include general proteolytic impairment, altered TRPML1 channel-mediated calcium alterations, and lysosomal membrane damage, all seeming to stem from lysosomal accumulation of APP-βCTF. This is welcome data, adding to the mounting evidence supporting the pathogenic significance of βCTF in AD pathogenesis (Jiang et al., 2010; Jiang et al., 2016; Jiang et al., 2019; Lauritzen et al., 2019; Tamayev et al., 2012).
As previously reported, we documented a similar constellation of lysosomal-related abnormalities as being among the varied consequences of elevated βCTF levels in our models, and showed their reversal by lowering βCTF levels (Jiang et al., 2019; Lee et al., 2015; Lee et al., 2022; Bordi et al., 2016; Lauritzen et al., 2019; Tamayev et al., 2012; Im et al., 2023). The effects of βCTF combine with the evidence of direct βCTF action on endosome dynamics/signaling in very early stage AD mediated by aberrantly activating rab5. Among other pathogenic effects, rab5 hyper-activation blunts nerve growth factor trafficking that supports cholinergic neuron survival (Kim et al., 2016; Salehi et al., 2006; Xu et al., 2016). Although lysosomal pH was not investigated in detail in the van Acker study, βCTF that accumulates in lysosomes in AD models directly interacts with, and inhibits, lysosomal vATPase and lysosomal acidification (Im et al., 2023).
Besides being able to account for the aforementioned lysosomal impairments, a generalized inhibition of substrate hydrolysis in lysosomes expected due to substrate storage and pH elevation in the PLD3 model also may well have a major impact on the degradation of mtDNA.
References:
Yuan P, Zhang M, Tong L, Morse TM, McDougal RA, Ding H, Chan D, Cai Y, Grutzendler J. PLD3 affects axonal spheroids and network defects in Alzheimer's disease. Nature. 2022 Dec;612(7939):328-337. Epub 2022 Nov 30 PubMed.
Jiang Y, Mullaney KA, Peterhoff CM, Che S, Schmidt SD, Boyer-Boiteau A, Ginsberg SD, Cataldo AM, Mathews PM, Nixon RA. Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition. Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1630-5. Epub 2009 Dec 28 PubMed.
Jiang Y, Rigoglioso A, Peterhoff CM, Pawlik M, Sato Y, Bleiwas C, Stavrides P, Smiley JF, Ginsberg SD, Mathews PM, Levy E, Nixon RA. Partial BACE1 reduction in a Down syndrome mouse model blocks Alzheimer-related endosomal anomalies and cholinergic neurodegeneration: role of APP-CTF. Neurobiol Aging. 2016 Mar;39:90-8. Epub 2015 Dec 2 PubMed.
Jiang Y, Sato Y, Im E, Berg M, Bordi M, Darji S, Kumar A, Mohan PS, Bandyopadhyay U, Diaz A, Cuervo AM, Nixon RA. Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99). J Neurosci. 2019 Jul 3;39(27):5255-5268. Epub 2019 May 1 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.
Lee JH, Wolfe DM, Darji S, McBrayer MK, Colacurcio DJ, Kumar A, Stavrides P, Mohan PS, Nixon RA. β2-adrenergic Agonists Rescue Lysosome Acidification and Function in PSEN1 Deficiency by Reversing Defective ER-to-lysosome Delivery of ClC-7. J Mol Biol. 2020 Apr 3;432(8):2633-2650. Epub 2020 Feb 24 PubMed.
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Bordi M, Berg MJ, Mohan PS, Peterhoff CM, Alldred MJ, Che S, Ginsberg SD, Nixon RA. Autophagy flux in CA1 neurons of Alzheimer hippocampus: Increased induction overburdens failing lysosomes to propel neuritic dystrophy. Autophagy. 2016 Dec;12(12):2467-2483. Epub 2016 Nov 4 PubMed.
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