When Faced with Amyloidosis, Human Transplants Die by Necroptosis
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When they find themselves trapped in an amyloid-ridden mouse brain, transplanted human neurons meet an untimely, necrotic demise. While this had been known before, a new-and-improved xenograft model has unveiled some of the dirty details behind this cascade. On September 15 in Science, researchers led by Bart De Strooper of the U.K. Dementia Research Institute, London, reported that, as amyloid accumulates in APP knock-in mice, pathological forms of tau start to amass inside the xenografted human neurons, ultimately forming neurofibrillary tangles.
- Human neurons xenografted into APPNL-G-F mice develop tau pathology, including tangles.
- Half of human neurons died within six months of transplant; likely killed by necroptosis.
- A steep rise in the lncRNA MEG3 might have tripped off the death cascade.
Half of the transplanted neurons die—via a cell death pathway called necroptosis—already during the early stages of amyloid deposition. The researchers tied the mayhem to MEG3—a long noncoding RNA that skyrocketed in the human cells in response to amyloid build-up, setting off the deadly cascade. Putting the kibosh on this particular flavor of cell death spared the human transplants. The findings illuminate an amyloid-instigated form of neurodegeneration that appears specific to human neurons.
“The findings suggest an explanation for why it has been challenging to produce Aβ-dependent tau pathology and neurodegeneration in mouse models,” wrote Daniel Sirkis and Jennifer Yokoyama of the University of California, San Francisco, in an editorial in Science. “The work also provides evidence for the importance of a cell-death pathway in AD that may be therapeutically tractable.”
“The paper takes us a step closer to understanding the mechanism(s) involved in neuronal cell loss in AD,” wrote Domenico Praticò of Temple University in Philadelphia.
Death by Amyloid. Human neural progenitor cells transplanted into a mouse model of amyloidosis ultimately develop tau pathology and succumb to necroptosis, while their murine neighbors survive. [Courtesy of Sirkis and Yokoyama, Science Perspectives, 2023.]
Mouse cells don’t express the isoforms of tau that tangle in human cells, nor do they succumb to neurodegeneration in mouse models of amyloidosis. To model these aspects of AD, researchers have devised ways to incorporate human cells into their studies, for example by using ever-more elaborate three-dimensional co-culture systems of human-stem-cell-derived neurons and glia (Sep 2023 news). Another approach is to transplant human cells into the mouse brain. De Strooper’s group has been honing this chimeric mouse model approach, previously reporting that human neural progenitor cells transplanted into an APP/PS1 transgenic mouse died in apparently gruesome, necrotic fashion (Feb 2017 news). Before they died, the transplants had started to accumulate phosphorylated tau, although full-fledged neurofibrillary tangles never formed. This could be because the APP/PS1 mice used in the experiments were bred on a NOD-SCID background, a highly immunosuppressed strain that succumbs to tumors by 9 months of age. The researchers speculated that had the mice lived longer, perhaps a fuller spectrum of AD pathology would have unfolded.
To find out, first author Sriram Balusu and colleagues used Rag2 knockout mice—a less-immunosuppressed strain that lacks B and T cells but still churns out other types of immune cells and can live up to two years. The scientists also switched to a more physiological model of amyloidosis, crossing the Rag2 knockouts to APPNL-G-F knock-ins. Shortly after the mice were born, the researchers transplanted 100,000 human cortical neural progenitor cells (NPCs) into the brains of both APPNL-G-F Rag2 KO mice, as well as into Rag2 KO controls. Two months later, the transplanted cells appeared to have made themselves at home, forming healthy connections with their mouse neural neighbors.
Four months later, once amyloid deposition was underway, the situation was dramatically different. While the transplants still appeared healthy in control mice, roughly half had perished in the APP knock-in hosts. As plaques formed around them, both mouse neurons and the surviving human neurons became “disheveled,” with dystrophic neurites, amidst a hotbed of activated microglia and astrocytes. In the human, but not mouse neurons, aggregates of phosphorylated tau started to accumulate.
By 18 months, amyloidosis had overtaken the brain, and the surviving human neurons contained full-fledged neurofibrillary tangles of tau. Relative to xenografted control mice, or to ungrafted APP-KI mice, the APP-KI mice hosting human neurons had elevated p-tau181 and p-tau231 in their plasma. This suggested that, when faced with amyloidosis, the human neurons developed tau pathology and also churned out phosphorylated tau, akin to neurons in people with AD.
Amyloid Triggers Tau. Only when exposed to amyloid (blue, bottom), did transplanted human neurons (green) express markers of pathological tau (red). [Courtesy of Balusu et al., Science, 2023.]
To search for clues about how the xenografted neurons were degenerating, the scientists performed transcriptomics at two, six, and 18 months after transplantation. As amyloidosis developed, the transplanted neurons assumed a gene-expression profile that resembled that reported in postmortem brain samples from people with AD. This included an uptick in expression of genes involved in MAPK signaling, TNF-α and interferon signaling, cell proliferation, aging, tissue regeneration, and myelination.
Curiously, the amyloid-exposed human neurons also shifted into an immature state, ramping up expression of genes involved in cell cycle re-entry, and turning down mature neuronal pathways. This hypomature phenotype had also been reported in human AD brain samples (Jun 2021 news).
Another finding stood out in the transcriptomes of amyloid-exposed human neurons. MLKL, the so-called executor of necroptosis, was a dramatically upregulated. Immunostaining the mouse brains with necroptosis markers p-RIPK1, p-RIPK3, and p-MLKL revealed that, in 18-month-olds, the death cascade was in full swing in transplanted human neurons but not in their murine neighbors.
Specifically, the researchers spotted the necroptotic protein trio huddled into punctate, vesicular structures called necrosomes. In some cases, casein kinase 1 δ, a marker of granulovacuolar degeneration bodies, mingled within these structures. GVBs are vesicles of autolysosomal origin loaded with granules of densely packed proteins. They have been tied to tau and α-synuclein toxicity (Wiersma and Scheper, 2020; Jorge-Oliva et al., 2022). De Strooper and colleagues previously found these structures in neurons from AD brain samples, where they are thought to reflect a last-ditch effort to survive by sequestering the death proteins (Jan 2020 news).
Wiep Scheper of Amsterdam University Medical Center in the Netherlands, whose work has explored the link between GVBs and tau pathology, called the findings from the xenograft model impressive. However, she noted that the partial colocalization between CK1δ and necrosome structures in the study appeared to be different from GVBs in the human brain, and that more work is needed to confirm that the punctate structures observed in the transplanted human neurons are bona fide GVBs. That can be done by staining for a wider array of GVB core as well as lysosomal membrane markers, and in more cells, she said.
Notably, researchers spotted the GVB-like structures in some neurons with, and some without, tau tangles. To Balusu, this supports the idea that tangles are not the toxic form of tau pathology. He believes other types of tau aggregate, such as oligomers, might be involved in setting off GVD. This idea jibes with new findings from Scheper’s lab, which tied GVBs to shorter tau filaments within the neuronal soma (Jorge-Oliva et al., 2023).
Finally, the researchers hunted for culprits that may have instigated the necroptotic cascade in the human transplants. They zeroed in on MEG3, a long noncoding RNA that was ramped up by a full order of magnitude in the amyloid-exposed human, but not mouse, neurons, and two- to threefold in human AD brain samples. This lncRNA has been implicated in cell death pathways and neurodegeneration (Jiang et al., 2021; Chanda et al., 2018). Overexpressing MEG3 in cultured human neurons hastened their death by necroptosis, a fate that could be avoided by treating the cells with necroptosis inhibitors.
Strikingly, counteracting the necroptosis cascade in the human transplanted neurons, either by knocking down their expression of MEG3 or RIPK-3 prior to xenografting, or by treating the mice with necroptosis inhibitors, significantly improved their survival within the amyloid-ravaged hosts.
Scheper looks forward to future studies with the model that might disentangle the relationship between tau pathology and necroptosis. In particular, she asked, does blocking necroptosis also get rid of tau pathology, and/or GVB-like structures? Sirkis and Yokoyama made a similar point in their editorial: “Another open question is whether the improved neuronal survival that results from reduction of MEG3 expression occurs by modulating the emergence of tau pathology, or despite such pathology.”
Taking a step even further back, Praticò noted that the question of whether tau pathology even triggers the MEG3-necroptotic cascade remains unsettled.—Jessica Shugart
References
News Citations
- In 3D Cell Model of AD, Microglia and CD8+ T Cells Gang Up on Neurons
- Chimeric AD Model Shows Human Neurons Are Uniquely Vulnerable to Aβ
- Neurons Made from Fibroblasts Keep Imprint of Alzheimer's, Aging
- Does Tau Kill Neurons by Way of Necroptosis?
Paper Citations
- Wiersma VI, Scheper W. Granulovacuolar degeneration bodies: red alert for neurons with MAPT/tau pathology. Autophagy. 2020 Jan;16(1):173-175. Epub 2019 Oct 23 PubMed.
- Jorge-Oliva M, Smits JF, Wiersma VI, Hoozemans JJ, Scheper W. Granulovacuolar degeneration bodies are independently induced by tau and α-synuclein pathology. Alzheimers Res Ther. 2022 Dec 14;14(1):187. PubMed.
- Jorge-Oliva M, van Weering JR, Scheper W. Structurally and Morphologically Distinct Pathological Tau Assemblies Differentially Affect GVB Accumulation. Int J Mol Sci. 2023 Jun 29;24(13) PubMed.
- Jiang N, Zhang X, Gu X, Li X, Shang L. Progress in understanding the role of lncRNA in programmed cell death. Cell Death Discov. 2021 Feb 8;7(1):30. PubMed.
- Chanda K, Das S, Chakraborty J, Bucha S, Maitra A, Chatterjee R, Mukhopadhyay D, Bhattacharyya NP. Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington's Disease. RNA Biol. 2018;15(10):1348-1363. Epub 2018 Oct 26 PubMed.
Further Reading
Papers
- Sirkis DW, Yokoyama JS. Role for cell death pathway in Alzheimer's disease. Science. 2023 Sep 15;381(6663):1156-1157. Epub 2023 Sep 14 PubMed.
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Temple University, School of Medicine
Described for the first time more than 100 years ago, brain shrinkage secondary to cell loss together with Aβ plaques and tau tangles is one of the three major features of Alzheimer's disease pathology. While in the last 25 years we have been able to recreate the brain deposits by inserting mutant human genes into rodent DNA, reproducing the cell loss has always been a challenge. Thus, despite the rodent brain becoming filled with both deposits, in most cases cell loss is not significant and common. This fact has hampered our understanding on how neuronal cells die in AD.
This paper takes us a step closer to understanding the mechanism(s) involved in neuronal cell loss in AD. By performing a series of elegant studies, they provide strong data supporting the hypothesis that necroptosis is at least in part responsible for the death of neurons in AD.
To reach this goal they implemented a model in which human or mouse neurons are transplanted in APP knock-in mice, a model of brain amyloidosis. After six months, they observed that human neurons developed immunoreactivities for phosphorylated tau (p-tau) epitopes and tau fibril-like structures, suggesting a progression of p-tau into pathological tau. Additionally, they found that the same animals had an increase in circulating plasma levels of p-tau181 and p-tau231, which would mimic what is seen in human AD. The tau post-translational modifications and peripheral changes were absent in grafted controls and non-grafted mice. However, they did not find any difference in the glial cell’s response to the plaques between transplanted and non-transplanted control APP mice.
Interestingly, about half of these neurons were also lost in the amyloid-bearing animals when compared with controls. In search for mechanism(s) responsible for it, while they did not find changes in the expression of genes associated with apoptosis or ferroptosis, they observed a significant upregulation of MLK1, a gene involved in necroptosis. They went on to show that the long noncoding RNA MEG3 was upregulated 10-fold in neurons transplanted in the APP knock-in mice but not in host mouse neurons. MEG3 alone was able to induce necroptosis in vitro, whereas inhibition of necroptosis or down-regulation of MEG3 rescued neuronal loss in transplanted human neurons. The authors conclude that brain amyloidosis is sufficient to induce AD-like tau neuropathology, and that neuronal death is largely driven by necroptosis.
I find this paper important and very interesting, its results highly intriguing. While the authors properly attribute all the tau changes in the transplanted neurons to the amyloidotic phenotype of the APP mice used, we cannot completely exclude that other “hits” could also be responsible.
It would have been interesting to know whether the same neurons that develop the p-tau had an increase in their Aβ levels. Studies have shown that intracellular Aβ accumulates early in the course of the disease and is also harmful (Gouras et al., 2005; Palmqvist et al., 2017; Huang et al., 2020). On the same note, what about plasma levels of Aβ in these mice? The authors speculate that because neuronal death in AD is largely driven by necroptosis, this fact could link neuronal loss to upstream inflammatory processes. However, no changes in glial responses were observed in these animals.
Finally, while it is evident that necroptosis is the downstream event triggered by MEG3 upregulation, the unanswered questions are: Is tau pathology triggering MEG3 which then initiates necroptosis? What happened to tau pathology if necroptosis is blocked?
Future studies will certainly address these additional questions, which will take us even closer to solve the puzzle on how Aβ and tau relate to each other, and how neurons die in AD.
References:
Gouras GK, Almeida CG, Takahashi RH. Intraneuronal Abeta accumulation and origin of plaques in Alzheimer's disease. Neurobiol Aging. 2005 Oct;26(9):1235-44. PubMed.
Palmqvist S, Schöll M, Strandberg O, Mattsson N, Stomrud E, Zetterberg H, Blennow K, Landau S, Jagust W, Hansson O. Earliest accumulation of β-amyloid occurs within the default-mode network and concurrently affects brain connectivity. Nat Commun. 2017 Oct 31;8(1):1214. PubMed.
Huang L, McClatchy DB, Maher P, Liang Z, Diedrich JK, Soriano-Castell D, Goldberg J, Shokhirev M, Yates JR 3rd, Schubert D, Currais A. Intracellular amyloid toxicity induces oxytosis/ferroptosis regulated cell death. Cell Death Dis. 2020 Oct 6;11(10):828. PubMed.
The Hebrew University of Jerusalem
I found this study to be of major interest, for several reasons. The de Strooper team implanted female H9 stem cell-originated neurons in the brains of AD mouse models, which displayed multiple AD biomarkers and overexpression of the longest splice variant of the noncoding MEG transcript, known from a recent Nature Medicine study to associate with social stress responses. That study is not mentioned here, probably due to the limited space; but social stress is known in AD. Aging involves declined cholinergic pathways, decreased cognition and elevated inflammation, given the loss of the cholinergic blockade of inflammatory pathways. Together, this accelerates ferroptosis, which combines cell death with inflammation and provides hope for novel medications, though sadly as a long-term hope.
That female-originated neurons were involved fits with our recent discovery of cognition loss related decline of cholinergic pathways in AD female brain regions due to depleted mitochondrial-originated transfer RNA fragments that explained the female-accelerated demise of AD cognitive functions (Shulman et al., 2023). One wonders if the de Strooper findings will differ in neurons derived from male-originated stem cells.
References:
Shulman D, Dubnov S, Zorbaz T, Madrer N, Paldor I, Bennett DA, Seshadri S, Mufson EJ, Greenberg DS, Loewenstein Y, Soreq H. Sex-specific declines in cholinergic-targeting tRNA fragments in the nucleus accumbens in Alzheimer's disease. Alzheimers Dement. 2023 May 9; PubMed.
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