MEG3 activates necroptosis in human neuron xenografts modeling Alzheimer's disease

1 5 Title: MEG3 activates necroptosis in human neuron xenografts modeling Alzheimer’s disease Authors: Sriram Balusu1,2*, Katrien Horré1,2, Nicola Thrupp1,2, An Snellinx1,2, Lutgarde Serneels1,2, Iordana Chrysidou1,2, Amaia M. Arranz3,4, Annerieke Sierksma1,2, Joel Simrén5,6, Thomas K. Karikari5,6,7, Henrik Zetterberg5,6,8,9,10, Wei-Ting Chen1,2, Dietmar Rudolf Thal11,12, Evgenia Salta13, Mark Fiers1,2,9, Bart De Strooper1,2,9,14* Affiliations: 10 1VIB Center for Brain & Disease Research; 3000 Leuven, Belgium. 2KU Leuven, Leuven Brain Institute ; 3000 Leuven, Belgium. 3Achucarro Basque Center for Neuroscience; 48940 Leioa, Spain. 4Ikerbasque Basque Foundation for Science; 48009 Bilbao, Spain. 5Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital; 431 80 Möndal, 15 Sweden. 6Department of Psychiatry and Neurochemistry, Sahlgrenska Academy at the University of Gothenburg; 431 80 Möndal, Sweden. 7Department of Psychiatry, University of Pittsburgh; Pittsburgh, PA, USA. 8Department of Neurodegenerative Disease, UCL Institute of Neurology; London, WC1N 20 3BG, UK. 9UK Dementia Research Institute at UCL; London, WC1E 6BT, UK. 10Hong Kong Center for Neurodegenerative Diseases; Hong Kong, China. 11Laboratory for Neuropathology, Department of Imaging and Pathology, Leuven Brain Institute (LBI), KU Leuven (University of Leuven); 3000 Leuven, Belgium. 25 12Department of Pathology, University Hospital Leuven; 3000 Leuven, Belgium. 13Laboratory of Neurogenesis and Neurodegeneration, Netherlands Institute for Neuroscience; 1105BA Amsterdam, the Netherlands. 14Lead contact 30 * Corresponding authors: [email protected] or [email protected] 35 2 Abstract: Neuronal cell loss is a defining feature of Alzheimer's disease (AD), but it remains unclear how neurons die and how this relates to other defining characteristics of the disease. Here we demonstrate that human neurons xenografted in mouse brain exposed to amyloid pathology develop sarkosyl-insoluble tau filaments, positive Gallyas silver staining, release phosphorylated 5 tau (p-tau181 and p-tau231) into the blood, and display considerable neuronal cell loss, providing a model for the induction of full Tau pathology by simple exposure to amyloid pathology in AD. The alterations are specific to human neurons and contrast with the mild effects exhibited in the circumventing mouse neurons or in transplanted mouse neurons. A core transcriptional program in the human neurons is characterized by strong upregulation of MEG3, a neuron-specific long 10 noncoding RNA. MEG3 is also upregulated in neurons from AD patients in situ. MEG3 expression alone is sufficient to induce necroptosis in human neurons in vitro. Inhibiting necroptosis using orally administered small molecule receptor-interacting protein (RIP) kinase -1 and -3 inhibitors or RIPK3 knockout rescues neuronal cell loss in this novel AD model. Thus, xenografted human neurons in contrast to mouse neurons are uniquely sensitive to amyloid pathology, recapitulate 15 neuropathological features of AD, and ultimately die by necroptosis. One-Sentence Summary: MEG3 activates neuronal necroptosis and inhibition of the necroptosis pathway rescues neuronal loss in Alzheimer’s disease. 20 3 Main Text: The major question of how the defining hallmarks of Alzheimer’s disease (AD) amyloid-β (Aβ) plaques, neuronal tau tangles, granulovacuolar neurodegeneration and neuronal cell loss relate to each other has never been solved, largely owing to the lack of good models that encompass all neuropathological aspects of the disease. Aβ pathology strongly influences tau pathology kinetics in several mouse models, but only if tau pathology is artificially induced by 5 extra frontotemporal dementia (FTD) causing mutations or by injecting tau-seeds isolated from AD patient brains(1–3). The fundamental unanswered questions remain whether amyloid-β can induce Tau pathology and how neurons die in AD. The many attempts to generate a complete model for AD have failed likely because of (unknown) 10 human-specific features that escape modeling in rodents. Two models have made great strides forward in using human cells to model features of AD, one reconstituting a 3D cell culture based on different types of human cells(4) and one using xenografted human neurons in mouse brain (5). We generated a much-improved mouse model for xenotransplantation of human neurons using a Rag2-/- (Rag2tm1.1Cgn) genetic background and a single AppNL-G-F (Apptm3.1Tcs/Apptm3.1Tcs) knock15 in gene to drive Aβ pathology, instead of the combined APP-PS1 (Tg(Thy1-APP*Swe, Thy1PSEN1*L166P)21Jckr) transgene overexpression used before(6). Human stem cell-derived neuronal progenitor cells (NPCs) transplanted in the control Rag2-/- mice integrate well and develop over time dendritic spines (fig. S1A and B). Two months post-transplantation (2M PT), the xenografted neurons display characteristic mature neuronal (NEUN and MAP2) and cortical 20 markers (CTIP2, SATB2, TBR1, and CUX2). Furthermore, unlike rodent neurons, human neurons display equal 3R and 4R tau splice forms at 6-months post-transplantation (6M PT) (fig. S1C and fig. S5H). Compared to the previously used NOD-SCID animals(6), Rag2-/- animals show a considerably increased life span (>18 months), allowing the study of healthy human neurons during brain aging. 25 We xenografted GFP-labelled H9-derived human cortical neuronal precursor cells (100,000 NPCs/mouse) into Rag2-/-/AppNL-G-F (further referred to as amyloid mice) or Rag2-/-/Appmm/mm (further referred to as control mice). β-sheet staining with dye X34 revealed robust plaque pathology in the amyloid mice (Fig. 1A & fig. S1D-E). RNA sequencing confirmed that the 30 transcriptional profile of the human transplants at 2M in control and amyloid mice are very similar (Fig. 2A). Thus, human neurons integrate and differentiate similarly in control and amyloid mice, in line with the fact that amyloid plaque pathology appears only from 3M onwards in this model. Number of cells are also not significantly different in amyloid and non-amyloid xenografted mice at 2M (fig. S6F). 35 Full blown amyloid plaque pathology is seen at 18M post transplantation. At this late point in time, human neurons in the control brain appear overall healthy, display neuronal projections and dendritic spines intermingled with host microglial (IBA1) and astroglia (GFAP) cells (fig. S1D). In contrast, grafted neurons in the amyloid mice display severe dystrophic neurites near Aβ 40 plaques, associated with microgliosis (6-7 IBA1-positive microglia per Aβ plaque) and astrogliosis 4 (2-3 GFAP-positive astrocytes per plaque) (Fig. 1A, and fig. S2A-B). The glial cell recruitment to plaques is very similar to non-grafted control animals (fig. S3A and B). Immunohistochemistry with phosphorylated tau (P-tau) antibodies, AT8 (P-tau Ser202, Thr205), PHF1 (P-tau Ser396 and Ser404), and MC1 (pathological conformational tau epitope) demonstrate considerable deposition of neuritic plaque tau (NP-tau)(1) in the human neurons of the grafted amyloid mice (Fig. 1C and 5 D) (Surface stained around Aβ plaques (20 µm ring) was ˜2% (AT8), ˜5% (PHF1), and ˜3% (MC1), Fig. 1E-G). Interestingly, both NP-tauand AT8-positive neurons appeared as early as 6-months post-transplantation (fig. S1E), indicating that amyloid deposition drives tau phosphorylation early on in this model. Significant tau pathology is not observed in mouse neurons in the same animal, nor in human grafted control, nor in non-grafted or mouse neurons grafted amyloid mice (Fig. 1C10 D, fig. S1E, fig. S3C, H and fig. S4A-C). Pathological, β-sheet X-34-positive tau-reactivity is seen in the xenografted human neurons (fig. S2C). Furthermore, Gallyas silver staining and tau-immunogold labeling of PHF fibril-like structures extracted with sarkosyl from grafted neurons in the amyloid mice (Fig. 1H and I) 15 confirmed the progression of P-tau into pathological states (> 20 tau-fibrils/EM grid (control n=4, amyloid n=4) (fig. S6F). Such pathology is largely absent in grafted control or non-grafted amyloid mice (Fig. 1H, fig. S6F & fig. S3I-J). Finally, and clinically relevant, we found that p-tau181 and p-tau231(7, 8) is significantly increased in the plasma of the grafted amyloid mice but not in control grafted or non-grafted mice (Fig. 1J-K), reflecting an increased secretion of soluble P-tau 20 species from neurons into the bloodstream in response to amyloid pathology, mimicking what is observed in humans with AD. It is possible to quantify accurately up to 0.1 ng of human DNA in a mixture with 100 ng mouse genomic DNA using qPCR(9) (fig. S6D). Using this assay, we estimate that up to 50% fewer 25 human neurons were present in the amyloid animals compared to control animals (Fig. 1L and fig. S6G) at 6M and 18M post transplantation. There is no significant increase in total number of human cells in control mice between 6M and 18M post transplantation (fig. S6E). The number of mouse neurons was also not significantly affected in the non-grafted control and amyloid mice (fig. S3E-G). Thus, human neurons, but not mouse neurons, develop robust neuropathological A 30 AD-like tau pathology changes including p-tau and tau fibrils upon exposure to amyloid plaques in vivo and degenerate much like their counterparts in the human brain by an unknown death process. Previous work has shown (opposing) effects of Rag2-/- immunodeficiency on amyloid plaque 35 pathology and cellular responses(10, 11). Comparison of soluble Aβ (Aβ40 & Aβ42) and guanidine-extractable Aβ (Aβ40 & Aβ42) revealed no significant differences between Rag2-/- /AppNL-G-F and AppNL-G-F mice (not shown). Bulk RNA sequencing indicated that main aspects of the innate microglia and astroglial cellular transcriptomic responses to amyloid plaques are maintained in the Rag2-/-/AppNL-G-F immune-deficient mouse compared to a previously 40 characterized non-immune suppressed amyloid mouse (APP/PS1)(12, 13) (Fig. 1B) (R2=0.81 for 5 differentially expressed genes). Thus, while we acknowledge that our model does not have an adaptive immune system, key features of the neuropathology of AD such as amyloid accumulation, glial responses, tau aggregation, tau phosphorylation, and neuronal cell loss, are reproduced in transplanted human neurons by simple exposure to amyloid pathology. 5 Transcriptional changes in xenografted neurons reveal induction of necroptosis We isolated the transplanted neuron-positive brain regions guided by the green fluorescent protein (GFP) marker from mice at 2-, 6-, and 18-months post-transplantation and extracted total RNA for sequencing (mean human reads ˜13.3 million) (fig. S5A-B). Mapping to mouse and human reference databases generated species-specific datasets. As expected, upregulation of the mouse 10 genes Axl, B2m, Cst7, Ctss, Itgax, Trem2, Tyrobp, Lyz2, Ccl3, and Ccl4 confirms microglial activation when exposed to amyloid in the Rag2-/-/AppNL-G-F mice (Fig. 1B, fig. S7A-C and Supplementary Table 2)(13). Human samples clustered according to genotype and age (fig. S5C). Differential expression (DE) analysis of the human grafts at two months showed only a few significantly downregulated DE genes, of unknown relevance (Fig. 2A, Supplementary Table 1). 15 In contrast, DE of grafts in amyloid and control conditions at 6M and 18M revealed 916 up and 73 down-regulated genes, and 533 up and 34 down-regulated genes, respectively (Fig. 2B-C, and Supplementary Table 1). Gene alterations of interest include CD74, a marker for tau tanglecontaining cells(14); HBB, a cortical pyramidal neuronal marker(15–17) and A2M, which is associated with neuritic plaques in AD human brains(18) (fig. S5E). In addition to the neuronal 20 signature, in line with our previous findings(19), we also observe astroglial and oligodendrocytic genes, including MOBP, MBP, OPALIN, S100A6, APOE, among others, suggesting that the grafted NPC cells not only generate human neurons but also glia (fig. S5F). The overall expression profiles of the grafts exposed to amyloid plaques at 6M and 18M show strong correlations (R2 = 0.88 for genes differentially expressed at one or both time points), suggesting that the pathological 25 cell states of neurons at 6M and at 18M are similar (fig. S5G). We investigated whether the gene signatures of the human neurons exposed to amyloid in xenografted mice are overlapping with transcriptional changes observed in human AD brains (Supplementary Table 3). We found a remarkable enrichment using gene set enrichment analysis 30 (GSEA) between previously published AD datasets, including the ROSMAP cohort(20) and our data in transplanted neurons at 6M and 18M PT (P.adj <0.05) but not at 2M (fig. S8A-B). Furthermore, GSEA also revealed a striking enrichment in upregulated genes from transplanted neurons and neurons directly reprogrammed from fibroblasts of AD patients (M6: P.adj=1.64e-05, M18: P.adj=2.19e-04). These data confirm that our transplanted neurons capture AD-relevant 35 transcriptional signatures(21). Functional gene ontology (GO) enrichment analysis of significantly upregulated genes (P.adj <0.05) at 6M using DAVID and REVIGO covered semantic space around positive regulation of transcription, protein phosphorylation, positive regulation of MAPK cascade, inflammatory 40 responses, including TNF and interferon signaling, cell proliferation, aging, tissue regeneration, 6 and myelination. (Fig. 2D and Supplementary Table 1). Some reports have suggested that neurons in AD display signatures of hypo-maturity, dedifferentiation, and cell cycle re-entry(21–24). Using previously published datasets (Supplementary Table 1) and Gene set enrichment analysis (GSEA), we find that cellular signatures of hypo-maturity, dedifferentiation, P53, HIF signaling, NF-kB, Myc, TGFβ-signaling, regulation of cell cycle (positive and negative), and cell-cycle re-entry are 5 indeed enriched in the 6M and 18M grafts but not at 2M (Fig. 2E). Conversely, mature neuronal marker pathways, such as synaptic plasticity, synaptic transmission, long-term potentiation, and axon guidance were not significantly enriched (Fig. 2E). How neurons die in AD remains a highly controversial discussion(25, 26). As shown in Fig. 1L and fig. S6G, about ˜50% of xenografted human neurons are lost in the amyloid mice. We did not 10 observe any significant alterations in the expression of genes associated with cell death mechanisms such as apoptosis or ferroptosis, but found significant upregulation of MLKL, the gene encoding the executor protein of necroptosis (Fig. 2B-C, fig. S2E & fig. S6A-C). This is not observed in the transcriptome of the host tissue (fig. S7A-C). We used pRIPK1 (Ser 166), pRIPK3 (Ser 227), and pMLKL (Ser 358) specific antibodies to stain brain tissue from 18M grafted and 15 non-grafted mice. This resulted in intense punctuate staining with a vesicular pattern in the soma of the neurons in grafted amyloid mice. These vesicular structures co-stained with casein kinase 1 delta (CK1), a marker for granulovacuolar degeneration (Fig. 2L and fig. S6I). Non-grafted control and amyloid animals or mouse neurons derived from NPC and similarly transplanted into mice did not display these pathologies (fig. S3D and fig. S4D). 20 We validated this observation by qPCR on RNA extracted from the temporal gyrus of AD and age-matched control brain samples, which revealed significant upregulation of MLKL and RIPK3, an upstream kinase in the necroptosis pathway in the human brain (Fig. 2F-H). Based on initial observations made in the xenograft model described here, we have already extensively confirmed the presence of necroptosis markers in granulovacuolar degeneration in neurons of AD patients in 25 a previous publication(27). Thus, our model has predictive value for the neuropathology of AD. MEG3 modulates the neuronal necroptosis pathway We identified 36 up and 21 down-regulated long noncoding RNAs (lncRNA) in the grafts of 6month-old animals (Supplementary Table 1). Some of these non-coding RNAs (e.g., NEAT1) have 30 been implicated in AD(28), but overall it is unclear whether and how they contribute to pathogenesis. In our data, the long non-coding RNA MEG3 (Maternally Expressed 3) was the most strongly (˜10 fold) upregulated gene in human neurons exposed to amyloid pathology (Fig. 2B-C & fig. S2D), but not in the host mouse neurons (fig. S7D-F). MEG3 has been linked to cell death pathways(29) via p53(30), is involved in the TGFβ pathway(31), and has been associated with 35 Huntington’s disease(32). In a cerebral ischemia-reperfusion injury model, repression of MEG3 has beneficial effects(33). 7 While MEG3 has not been previously implicated in AD, we found upregulation of MEG3 in AD patient brains in a single nucleus transcriptomic database(34). We confirmed a 2-3-fold upregulation of MEG3 in RNA extracted from the temporal gyrus of human AD brains (Supplementary Table 5) using qPCR (Fig. 2I). MEG3 in situ hybridization combined with cellspecific immunohistochemical markers NeuN (neurons), IBA1 (microglia), and GFAP 5 (astrocytes) demonstrated its exclusive expression in the nucleus of neurons (fig. S9A-B) and its strong enrichment in AD brains (2-3 puncta/nucleus in control and 8-10 puncta/nucleus in AD brain (P= <0.0001), (Fig. 2J-K). In AD, MEG3-expressing neuronal nuclei appeared blotched with reduced DAPI intensity, and MEG3-positive neurons also displayed high levels of the necroptosis marker pMLKL (fig. S9C). 10 MEG3 has 15 known transcriptional variants in humans(35). We performed Sanger sequencing on brain cDNA and found that MEG3 transcript variant-1 (accession number NR_002766.2) is most abundantly expressed in the human adult brain. A lentivirus was used to express MEG3 V1 in H9derived mature cortical neurons (Fig. 3A-B). Expression of MEG3 in the neurons resulted in a strong reduction of cell viability at 9 days after transduction compared with control-transduced 15 human neurons (Fig. 3C). The MEG3 induced cell loss can be rescued by the necroptosis inhibitors ponatinib, dabrafenib or necrosulfonamide (NSA) or by CRISPR mediated deletion of RIPK1, RIPK3, and MLKL in vitro (Fig. 3I-J and fig. S10H-J). Immunohistochemistry analysis revealed the presence of activated necroptotic markers, pRIPK1 (S166), pRIPK3 (S227), and pMLKL (S358) (Fig. 3D-F). Necroptosis-positive neurons displayed a reduced amount of cytoskeletal 20 filament neurofilament-H (NF-H). Western blot analysis confirmed increased pRIPK1 (S166) levels (Fig. 3G), showing that the neurons were dying from necroptosis. Next, to understand to what extent the MEG3 expression contributes to transcriptional changes identified in the xenografted neurons, we performed RNA sequencing of MEG3 transduced neurons 7-days post-transduction. To compare transcriptomic signatures of the MEG3 transduced 25 neurons with those identified in xenografted neurons, we performed gene set enrichment analysis (gene set enrichment analysis). We ranked the genes according to their log fold change differential expression between amyloid-exposed and non-exposed xenografted neurons (at M6, see Fig. 2C), and compared those to the top genes upregulated in response to MEG3 transduction in neurons in vitro. We found a significant enrichment (NES=1.5, P.adj=1.2e-05) (Fig. 3H and Supplementary 30 Table 4). This suggests that some transcriptional changes in the xenografted neurons might be secondary to the upregulation of MEG3. DE analysis of MEG3 transduced neurons revealed no changes in necroptosis genes (fig. S10A-C) but DAVID Gene Ontology (GO) analysis of leading edge genes from GSEA on upregulated genes from 6M transplanted human neurons and in vitro MEG3 expressing neurons revealed signatures of NF-kB and TNF signaling which have been 35 related to necroptosis induction(36). In addition, signatures of lipid transport, interferon- signaling, positive regulation of peptidyl-tyrosine phosphorylation, and apolipoprotein-L signaling are observed (fig. S10D and Supplementary Table 4). 8 Inhibition of necroptosis by small molecules or ablation of RIPK3 blocks human neuronal loss in the xenografted mice We first used a previously characterized shRNA antisense construct (PMID: 31656536 and PMID: 32201430) against MEG3 to confirm the direct role of MEG3 expression in the transplanted neurons when exposed to amyloid plaques. Downregulation of MEG3 significantly improved the 5 neuronal survival and was associated with downregulated expression of necrosome proteins (Fig. 3K-L and fig. S10E-G). It remains however unknown whether the neurons that degenerate during AD or in our neuronal transplants are all dying by necroptosis or whether necroptosis markers are limited to the neurons that are remaining at the stage of pathological analysis. We thus treated xenografted mice (3 groups of n=5) from 2M until 6M post-transplantation with the orally 10 available necroptosis kinase inhibitors, ponatinib (30 mg/kg), and dabrafenib (50 mg/kg). Ponatinib inhibits RIPK1(37) and RIPK3(38) and is an FDA-approved drug for the treatment of acute lymphoid leukemia (ALL) and chronic myeloid leukemia (CML). Dabrafenib is a more specific inhibitor of RIPK3 (39). Immunostaining at six months revealed a striking reduction in the levels of pRIPK1, pRIPK3, and pMLKL in both treatment groups compared to untreated mice 15 (Fig. 4A-D), without altering the glial response to amyloid (fig. S9D). qPCR analysis of neuronal cell numbers revealed a significant increase in neurons in the treated groups compared to control groups (Fig. 4E). In further experiments, we have transplanted RIPK1 or RIPK3 knockout human stem cells generated using CRISPR/Cas9 similar to the ones used in the in vitro experiments above. In contrast to the in vitro experiments, none of the neurons transduced with guide RNA against 20 RIPK1 survived, indicating that knock out of RIPK1 is lethal in vivo. Previous work has indeed shown that constitutive deletion of RIPK1 in mice leads to embryonic lethality (PMID: 27819682). In contrast, deletion of the RIPK3 gene with gRNA significantly improved neuronal survival (Fig. 4 F-H). Immunofluorescence staining confirmed the significant decrease of necroptosome signals in RIPK3 deficient human neurons. 25 9 Conclusion Our results demonstrate that amyloid pathology is sufficient to induce full hallmark neuropathological features of AD in non-genetically manipulated human neurons, including dystrophic neurites, tau fibrils, granulovacuolar degeneration, cell loss, and increased plasma ptau181 and -231 in the grafted mice. One of the major unsolved questions in AD research is how 5 neurons die. Some reports have proposed apoptotic mechanisms, but it has never been demonstrated convincingly that this drives neuronal cell loss in the disease(40). While previous publications suggested activated necroptosis in the AD brain(27, 41), we demonstrate here that a necroptosis mediated neuronal loss in the xenograft model can be rescued by treatment with clinically relevant necroptosis inhibitors or by ablating the RIPK3 gene in the transplanted 10 neurons(39). Thus, we suggest that neuronal death in AD is largely driven by necroptosis, linking the loss of neurons to inflammatory processes that are upstream of this well-studied death pathway(13, 42, 43). Therefore, therapies that prevent neuronal cell loss, in combination with more mainstream Aβ and tau targeted interventions, might be useful additions to the current efforts to develop disease-modifying strategies for AD(44–46). Our data suggest that necroptosis is 15 downstream of the accumulation of pathological tau and is induced by the upregulation of the noncoding RNA MEG3 possibly via TNF-inflammatory pathway signaling. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and influence a variety of biological processes, including brain aging and neurodegenerative disease(47). The large number of noncoding RNAs that are differentially expressed in our novel AD model warrant further 20 investigation. It is deeply intriguing that human transplanted neurons display an AD phenotype, while mouse neurons interspersed within the graft or NPCs derived from mouse neurons transplanted in a similar way as their human counterparts, do not display such signs. This strongly suggests that yet unknown human-specific features define the sensitivity of the neurons to amyloid pathology. We 25 notice that transcriptional analysis of the transplanted human neurons indicates signatures of downregulation of mature neuronal properties and upregulation of immature signaling pathways. This agrees with observations in a recent study that analyzed the transcriptional profiles in neurons directly derived from fibroblasts of AD patients (iNs)(21). The neurons in that study did not show tangles, amyloid, or necroptosis markers typical of AD, and it was not clear how those observations 30 relate to the classical hallmarks of AD. While the contribution of these immature signatures to the disease process in our model needs further investigation, our data indicate that necroptosis is an important contributor to cell death in Alzheimer’s Disease. Necroptosis is an active area of drug development in cancer (ref) and ALS (ref), and we therefore suggest that it is worthwhile to explore this pathway further for potential drug development in AD as well. 35 Finally, it is of crucial importance to better understand what precisely makes that transplanted human neurons become diseased when exposed to amyloid pathology, while mouse neurons remain preserved. Understanding the resilience of mouse neurons to amyloid pathology will not Figure 2: A B C F G H Control AD DAPI/MEG3 J KL Control AD 0 10 20 30 40 MEG3 puncta/nucleus ✱✱✱✱ P= <0.0001 ControlAmyloid GFP/pRIPK1 GFP/pRIPK3 GFP/pMLKL Amyloid vs Control 2M (human) Amyloid vs Control 6M (human) Amyloid vs Control 18M (human) D E I Mature neuronal gene sets Neuronal dedifferentiation gene sets M2 M6 M18 Aging Tissue regeneration Myelination Positive regulation of gene expression Positive regulation of transcription Peptidyl-tyrosine phosphorylation Positive regulation of MAPK cascade Negative regulation of cell population Cell proliferation and growth Cellular response to interferon-β Cellular response to tumor necrosis factor Cellular response to growth factors Immune response Antigen processing and presentation Response to hypoxia Response to interferon-γ Control AD 0 1 2 3 4 Relative RIPK1 expression ns P= 0.0947 Control AD -1 0 1 2 3 4 Relative RIPK3 expression ✱✱ P= 0.0076 Control AD -1 0 1 2 3 4 Relative MLKL expression ✱✱ P= 0.0043 Control AD 0 1 2 3 4 Relative MEG3 expression ✱ P= 0.0043 Figure 2: Transcriptional changes in xenografted neurons. Bland-Altman MA plot showing differential expression of genes from RNA sequencing of the human grafts isolated from control and amyloid mice at (A) 2months (control n=5, amyloid n=7), (B) 6 months (control n=5, amyloid n=5), and (C) 18months (control n=4, amyloid n=3) posttransplantation.Red, significantly upregulated genes.Blue, significantly downregulated genes (FDR < 0.05). FC=fold change, CPM=counts per million.Comparisons of logFC at month 6and month 18 are shown in Extended data figure 5g. (D) Gene Ontology (GO) analysis showing terms associated with genes upregulated in amyloid mice at 6M. Point size represents the fold enrichment of upregulated genes in the term, and color represents the –log10FDR.Only terms with FDR < 0.1 are shown.The xand yaxes represent the 'semantic space' -that is, how similar two terms are to each other (calculated by Revigo,PMID: 21789182). (E) Heatmap of normalised enrichment scores (NES) in neuronal dedifferentiation gene sets ranked along the differentially expressed genes in the xenografts (amyloid vs control) shown in (Fig. 2a-c). Positive enrichments are shown in red, negative enrichments are shown in blue.Significant FDR values (P.adj < 0.01)are shown as numbers. (F) Analysis of RIPK1gene expression using qRT-PCR on AD (n=11)and control (n=10)post-mortem human brain samples. (G) Analysis of RIPK3gene expression using qRT-PCR on AD (n=11)and control (n=10)post-mortem human brain samples. (H) Analysis of MLKL gene expression using qRT-PCR on AD (n=11)and control (n=10)post-mortem human brain samples. (I) Analysis of MEG3gene expression using qRT-PCR on AD (n=11)and control (n=10)post-mortem human brain samples. (J) Representative confocal images showing MEG3RNAScope in temporal gyrus of AD (n=3) and control (n=2) post-mortem human brain samples.Nucleus (DAPI, blue), MEG3(red).Scalebar 10 µM. (K) Quantitative representation of the number of MEG3puncta per nucleus in control (>100 nuclei/sample, n=2), and AD (>100 nuclei/sample, n=3) post-mortem human brain samples. (L) Representative confocal images showing the expression of activated necroptosis pathway markers in 18-months old human neurons in control (n=4) and amyloid (n=4) mice.Grafted human neurons (GFP, green), pRIPK1, pRIPK3or pMLKL (red).White arrows indicate pRIPK1, pRIPK3, or pMLKL positive cells.Scalebar 30 µm. Values are presented as mean ±SEM.Student’s t-test in (F-I, and K) to measure the statistical significance. Figure 3: D E A B C G F pRIPK1 Merge MEG3 LV Control LV pRIPK3 Merge MEG3 LV Control LV MEG3 LV Control LV pMLKL Merge H Control LV MEG3 LV -15 -10 -5 0 5 ΔΔCT values ✱✱ P=0,0043 Day 7 Day 9 Day 11 0 50 100 150 % Cell survival ns ✱✱ ✱✱✱ Control LV (n=8) MEG3 LV (n=8) P=0,0016 P=0,0001 I J DAPI pRIPK1GFP HUNU Merge MEG3 shRNA Control LV K L 105 106 107 Number of human cells Total numbaer of cells MEG3 shRNA_Titin ✱✱ 0.0094 Control MEG3 shRNA 0 50 100 150 200 Cell survival (%) Control MEG3 ✱✱ ns ns ns 0.0099 DMSO Pona. Dabr. NSA Figure 3: Long noncoding RNA MEG3induces necroptosis in human neurons. (A) Schematic representation of the MEG3expression strategy using lentiviral vectors (LV) in H9derived human neurons. (B) Analysis of the MEG3expression in H9-derived neurons at DIV75,seven days post-transduction with either control LV (n=5) or MEG3LV (n=6). (C) Analysis of neuronal cell survival using CellTiter-Glo reagent after transducing with either control LV (n=8) or MEG3LV (n=8) on day-7, day-9, and day-11 post-transduction. (D) Representative confocal image showing activated necroptotic marker (pRIPK1, Ser 166)in H9derived neurons at DIV75 transduced with either control LV (n=3) or the MEG3LV (n=3). Nucleus (DAPI, blue), pRIPK1(grey, indicated with red arrows), transduced neurons (GFP, green), neurons (NF-H, red).Scalebar 30 µm. (E) Representative confocal image showing activated necroptotic marker (pRIPK3, Ser 227)in H9derived neurons at DIV75 transduced with either control LV (n=3) or the MEG3LV (n=3). Nucleus (DAPI, blue), pRIPK3(grey, indicated with red arrows), transduced neurons (GFP, green), neurons (NF-H, red).Scalebar 30 µm. (F) Representative confocal image showing activated necroptotic marker (pMLKL,Ser 358)in H9derived neurons at DIV75 transduced with either control LV (n=3) or the MEG3LV (n=3). Nucleus (DAPI, blue), pMLKL (grey, indicated with red arrows), transduced neurons (GFP, green), neurons (NF-H, red).Scalebar 30 µm. (G) Immunoblot analysis of pRIPK1(Ser 166)levels.Proteins were extracted seven days posttransduction from H9-derived neurons at DIV75 transduced with either control LV (n=4) or MEG3LV (n=8). GAPDH is used as the loading control.Two independent differentiations (Diff). (H) The same Bland-Altman MA plot showing differential expression of bulk RNA sequencing of human grafts from 6months as in Fig. 2b. Genes highlighted in red are the leading-edge genes identified in agene-set enrichment analysis (GSEA), taking the top 400 upregulated genes from the bulk sequencing of the primary neurons expressing MEG3(Extended Data Fig. 10) and plotting them against the amyloid vs control fold changes of the human grafts (FDR<0.05). FC=fold change, CPM=counts per million. (I) Schematic representation of the necroptosis inhibition in vitro using H9-derived human neurons and ponatinib (0.5 µM), dabrafenib (0.9 µM) and necrosulfonamide (0.5 µM). (J) Analysis of neuronal cell survival using CellTiter-Glo reagent.Experiment was performed in triplicates with 6technical replicates for each replicate. (K) Immunohistochemistry analysis of pRIPK1levels in samples obtained from the 6-month-old transplanted animals transplanted with control LV (n=5) or the MEG3shRNA (n=5) transduced human NPCs.Nuclei were stained with DAPI (blue), GFP was represented in green, pRIPK1was represented in red, and human nuclei were depicted in grey.White arrows indicate pRIPK1positive cells.Scalebar 30 µm. (L) The human cell number was estimated from 6-month-old xenografted mice from control (n=5) and MEG3shRNA (n=12)using q-PCR.Values are presented as mean ±SEM.Student’s t-test in (B and L), One-way ANOVA with Tukey’s post hoc test for multiple comparisons was used in (C and J)to measure the statistical significance. Figure 4: X34/GFP/pMLKL/HUNU X34/GFP/pRIPK1/HUNU Control Ponatinib Dabrafinib X34/GFP/pRIPK3/HUNU Amyloid mice A B C D E Control Ponatinib Dabrafenib 0 20 40 60 % pRIPK1 positive cells ✱✱✱✱ ✱✱✱ ✱ Control Ponatinib Dabrafenib 0 10 20 30 40 % pRIPK3 positive cells ✱ ✱✱✱ ns Control Ponatinib Dabrafenib 0 10 20 30 40 % pMLKL positive cells ✱✱✱ ✱✱✱ ns Control Ponatinib Dabrafenib 0 1×107 2×107 3×107 Tot a l n um be r o f h um an ce ll s ns ✱✱✱✱ ✱✱ F G 0 1×107 2×107 3×107 Number of human cells ✱✱ ✱✱ Cas9 - + + RIPK1 sgRNA + + - RIPK3 sgRNA + + - 0.0042 0.0016 H -Cas9 DAPI/GFP/pRIPK3/HUNU + Cas9 DAPI/GFP/pRIPK1/HUNU Figure 4: Inhibition of necroptosis prevents human neuronal cell loss in vivo. (A) Representative confocal images showing the expression of the activated necroptotic markers pRIPK1, pRIPK3, and pMLKL in amyloid animals (n=5), amyloid animals treated with either ponatinib (n=5) or dabrafenib (n=6). Amyloid (X34,blue), human neurons (GFP, green), pRIPK1, pRIPK3, and pMLKL (red), human nuclei (HUNU, grey).White arrows indicate necroptosis marker positive neurons. Scale bar 30 µm. (B) Quantitative representation of the percent number of human cells showing immunoreactivity to pRIPK1in control (n=5), ponatinib (n=5), and dabrafenib (n=6) treated animals. (C) Quantitative representation of the percent number of human cells showing immunoreactivity to pRIPK3in control (n=5), ponatinib (n=5), and dabrafenib (n=6) treated animals. (D) Quantitative representation of the percent number of human cells showing immunoreactivity to pMLKL in control (n=5), ponatinib (n=5), and dabrafenib (n=6) treated animals. (E) Quantitative estimation of the number of human cells present in control (n=5), ponatinib (n=5), and dabrafenib (n=6) treated grafted mice at 6months post-transplantation. (F) Schematic representation of the RIPK1and RIPK3KO generation and transplantation.NPCs derived from the H9-inducible Cas9line are transduced with the sgRNAs corresponding to either RIPK1or RIPK3. 100,000 cells were grafted into each mouse at P1-P2 stage for each condition. (G) Quantitative representation of the number of neurons surviving from 6months old mice transplanted with RIPK1sgRNA without doxycycline (n=7) or RIPK1sgRNA with doxycycline (n=7) or RIPK3sgRNA with doxycycline (n=10)using qPCR. (H) Representative immunofluorescence analysis of phosphorylated necroptosis markers pRIPK1(n=3) pRIPK3(n=3) from 6months old mice transplanted with RIPK1sgRNA without Cas9or RIPK3sgRNA with Cas9.White arrows indicates either pRIPK1or pRIPK3positive cells.Scalebar 30µM.Values are presented as mean ±SEM.One-way ANOVA with Tukey’s post hoc test for multiple comparisons was used in B-E and Gto measure the statistical significance. A B C D GFP (human graft) GFP (human graft) Supplementary Figure 1: X34 GFP IBA1 GFAP Merge ControlAmyloid Control 3R 4R MergeGFP Amyloid Amyloid Magnified X34 GFP AT8 TOPRO3 Merge E Supplementary Figure 1: Integration of the transplanted neurons in the Rag2-/- animals. (A) Coronal section of 18-months old grafted mouse brain showingthe graft location (green, indicated with white arrows). Scale bar 1000 µm. (B) Higher magnification image of neuronal process displaying dendrites and mature spines (right boxes).White arrows indicates dendritic spines.Scale bar 10 µm. (C) Representative confocal images of grafted neurons 6months post-transplantation stained with 3R (magenta) and 4R (red) TAU isoforms (examples of human neurons indicated with white arrows). Scale bar 30 µm. (D) Representative confocal images showing glial responses at 18 months post-transplantation. Amyloid (X34 in blue), human neurons in green (GFP), GFAP (grey), IBA1(red).Scale bar 30 µm. (E) Representative confocal images 6-months old grafted mice showing dystrophic neurites and neuritic plaque tau (NP-tau) pathology (AT8) around the X34 plaques 6months post-transplantation. The top panel shows NP-tau, indicated with white arrows.The bottom panel shows amagnified region of one plaque showing dystrophic neurites and NP-tau.Amyloid (X34 in blue), human neurons in green (GFP), P-tau (AT8, red), TOPRO3(nucleus, magenta).Scale bar 30 µm. Supplementary Figure 2: A B C D E 3D segmentation X34/AT8/HUNU X34/PHF1/HUNU X34/MC1/HUNU 0 10 20 30 Number'of'microglia'in' 20'µm'X34'ring N.D Control Amyloid 0 2 4 6 8 10 Number'of'astroglia'in' 20'µm'X34'ring N.D Control Amyloid Control Amyloid 0.0 0.5 1.0 1.5 Normalised log MEG3 expression ns Control Amyloid 0.0 0.5 1.0 1.5 Normalised log MLKL expression ns Control Amyloid 0 2 4 6 8 Normalised log MEG3 expression P adj.=5.38e-136 Control Amyloid 0 1 2 3 4 Normalised log MLKL expression P adj. =7.88e-13 Control Amyloid 0 2 4 6 8 Normalised log MEG3 expression P adj.=4.16e-181 Control Amyloid 0 1 2 3 4 Normalised log MLKL expression P adj.=2.22e-05 2 months 6 months 18 months 2 months 6 months 18 months Supplementary Figure 2: Characterization of the 18 months old grafted animals. (A) Quantification of the number of host microglia around the Aβ plaque, within 20 microns diameter (n=4, >100 plaques/mice). (B) Quantification of the number of host astrocytes around the Aβ plaque, within 20 microns diameter (n=4, >100 plaques/mice). (C) Neurons immunoreactive to AT8, PHF1or MC1are segmented in Imaris software and rendered to reveal intracellular X34 staining (β-sheet fibrillary structures), an indicative of the intracellular tau fibrils.Extracellular X34 (blue) staining represents Aβ plaques where as intracellular X34 (blue) represents tau β-sheet structures.White arrows indicate X34 staining in neuronal somas.Amyloid (X34,blue), P-tau (red), human nucleus (HUNU, grey).Scale bar 5µm. (D) Human MEG3expression from 2-months (control n=5, amyloid n=7),6-months (control n=5, amyloid n=5), and 18-months (control n=4, amyloid n=3) post-transplantation. (E) Human MLKL expression from 2-months (control n=5, amyloid n=7),6-months (control n=5, amyloid n=5), and 18-months (control n=4, amyloid n=3) post-transplantation. A Supplementary Figure 6: E B C D 012345 20 22 24 26 28 30 huTitin region qPCR standard curve log number of human/mouse cells Ct value human+mouse mix gDNA input human alone gDNA input Amyloid vs Control (6M) Amyloid vs Control (6M) Amyloid Amyloid magnified GFP pMLKL CK1𝛅Merge I Control Amyloid 0 10 20 30 Number of fibrils/EM grid Control Amyloid 0 1×107 2×107 3×107 4×107 Tot al n um be r of hu ma n ce ll s ✱ P=0.0153 F G Control (6M) Control (18M) 0 1×107 2×107 3×107 4×107 Total nu mb er o f hu ma n ce ll s ns Control Amyloid 0 50 100 150 200 250 2 months TITIN nomalized (%) % Human cells ns H Supplementary Figure 6: Neuronal cell death analysis in transplanted human neurons. (A) Ingenuity pathway analysis (IPA) of canonical necroptosis pathway overlaid with differentially expressed gene from the 6-months old neuronal xenografts.Red indicates upregulation, and green indicates downregulation. (B) Bland-Altman MA plot showing expression of apoptosis pathway genes in the 6month old neuronal xenografts. (C) Bland-Altman MA plot showing expression of ferroptosis pathway genes in the 6month old neuronal xenografts. (D) Establishment of the standard curve for qPCR using TITIN with human genomic DNA (red line) and human plus mouse genomic DNA (blue line). (E) Quantitative representation of the human neurons in control mice (6Mand 18M)(n=6) using qPCR. (F) Quantification of the number of immunogold positive fibrils identified in the grafted control (n=4) and amyloid mice (n=4). (G) Quantitative representation of the human neurons at 18Mpost-transplantation in control (n=5) and amyloid (n=6) mice using qPCR. (H) Quantitative representation of the human neurons at 2M post-transplantation in control (n=6) and amyloid (n=6) mice using qPCR. (I)Representative confocal images showing co-localization of pMLKL with granulovacuolar degeneration (GVD) marker casein kinase 1delta (CK1𝛅)in 18Mold amyloid mice.Human graft (GFP in green), pMLKL (red), CK1𝛅(grey).Scale bar 30 µM. Amyloid vs Control 2M (mouse) Amyloid vs Control 6M (mouse) Amyloid vs Control 18M (mouse) A B C Supplementary Figure 7: Supplementary Figure 7: Gene expression profiles of mouse host tissue from grafted mice at 2, 6 and 18 months. (A) Bland-Altman MA plot showing the differential gene expression of mouse transcriptome at 2-months (control n=5, amyloid n=7), (B), 6-months (control n=5, amyloid n=5), and (C), 18-months (control n=4, amyloid n=3) old mice.Red, significantly upregulated genes.Blue, significantly downregulated genes (FDR < 0.05). Selected microglial activation genes are labelled.FC=fold change, CPM=counts per million.Analysis of mouse Meg3expression 2- (D), 6- (E) and 18 (F) months post transplantation.Bulk RNA sequencing reads from the RNA sequencing is extracted from the 2-, 6-, and 18-months old animals and presented as normalized counts (log scale) from either control or amyloid animals.ns, not significant. Control Amyloid 0 5 10 15 Normalised log Meg3 expression ns Control Amyloid 0 5 10 15 Normalised log Meg3 expression ns Control Amyloid 0 5 10 15 Normalised log Meg3 expression ns D E F Supplementary Figure 8: A B Supplementary Figure 8: Xenografted neurons recapitulate signatures identified in human postmortem tissue and other xenografted neurons. (A) Heatmap of normalized enrichment scores (NES) in indicated gene sets ranked along the human graft fold changes (upregulated genes, amyloid vs control) shown in figure 2a-c. Positive enrichments are shown in red, negative enrichments are shown in blue.Significant FDR values (p.adj < 0.01)are shown as text in the boxes. M2, upregulated genes in human neurons 2months posttransplantation (PT). M6, upregulated genes in human neurons 6M PT. M18,upregulated genes in human neurons 18MPT. (B) Heatmap of normalised enrichment scores (NES) in indicated gene sets ranked along the human graft fold changes (downregulated genes, amyloid vs control) shown in figure 2a-c. Positive enrichments are shown in red, negative enrichments are shown in blue.Significant FDR values (p.adj < 0.05)are shown as text. M2, downregulated genes in human neurons 2months posttransplantation (PT). M6, downregulated genes in human neurons 6M PT. M18,downregulated genes in human neurons 18MPT. Datasets used in the above comparison: Espuny-Camacho et al., 2017 xN AD young, DE upregulated genes form human neurons 4months post-transplantation (PT).Espuny-Camacho et al., 2017 xN AD old, 6M-8M PT.(PMID:28238547). Martens et al., 2021 iN AD, DE genes from neurons directly reprogrammed from the fibroblastderived from AD patients compared to neurons reprogrammed from control fibroblasts. (PMID:33910058). Annese (2018)HC AD, DE genes from Late-Onset AD (LOAD) compared to controls from the hippocampus.(PMID:29523845) Blalock (2011)LC NFT, DE genes from laser captured P-tau positive neurons.(PMID:21756998) Blalock (2004)MMSE, DE genes from MiniMental Status Examination (MMSE>20)from AD brains. (PMID:14769913). Mostafavi 2018 DLPFC, DE genes from dorsolateral prefrontal cortex from AD (PMID:29802388). DAPI GFAP MEG3 NEUN Merge As Nu Nu As Nu Nu As Nu Nu As Nu Nu As Nu Nu DAPI IBA1 MEG3 NEUN Merge Mg Nu Nu Nu Mg Nu Nu Nu Mg Nu Nu Nu Mg Nu Nu Nu Mg Nu Nu Nu Supplementary Figure 9: A B C DAPI/pMLKL/MEG3/NeuN Control AD Vehicle Ponatinib Dabrafinib X34/IBA1/GFAP D Supplementary Figure 9: MEG3is selectively expressed in neurons. (A) Representative confocal images from FFPE fixed human hippocampal brain samples stained with MEG3(RNA scope, red) and combined with immunostaining for GFAP (green), NeuN (grey), and DAPI (blue).Nu=Neuron, As=Astrocytes.Scale bar 30 µm. (B) FFPE fixed human hippocampal brain samples were used for MEG3RNA scope (red) and combined with immunostaining for IBA1(green), NeuN (grey), and DAPI (blue).Nu=Neuron, Mg=microglia.Scale bar 30 µm. (C) Representative confocal images showing the colocalization of pMLKL (green) in the MEG3 positive neurons in AD (n=3) and control (n=3) hippocampal brain samples.Scale bar 30 µm. (D) Analysis of the glial cell after from the mice administered with control diet (n=5) or diet mixed with Ponatinib (n=5) or Dabarafenib (n=6). Mice treated with the indicated drug from 2months to 6 months added to the regular mouse maintenance diet.Brain samples were isolated after perfusion with PBS followed by 4% PFA post fixation overnight.Vibratome sections of 40 microns are made and stained with a X34 (blue, amyloid plaques), IBA1(red, microglia), and GFAP (gray,astrocytes). Upper panel is alarger field of view from the cortex and the lower panel is amagnified field. Scalebar 30 um. A C D Supplementary Figure 10: Leading edge (human graft) MEG3 upregulated genes B Control MEG3 shRNA 0 1 2 3 MEG3 expression in vivo Fold MEG3 expression ✱✱✱ 0.0002 Conrol LV GFP MEG3 (RNASCOPE) Merge MEG3 shRNA E GF 0 50 100 150 200 %cell survial RIPK1 sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ✱ ns ✱ 0 100 200 300 %cell survial RIPK3 sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ns ns ✱✱ 0 50 100 150 %cell survial MLKL sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ✱✱✱ ns ✱✱✱ 0 50 100 150 200 %cell survial RIPK1 sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ✱ ns ✱ 0 100 200 300 %cell survial RIPK3 sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ns ns ✱✱ 0 50 100 150 %cell survial MLKL sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ✱✱✱ ns ✱✱✱ 0 50 100 150 200 %cell survial RIPK1 sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ✱ ns ✱ 0 100 200 300 %cell survial RIPK3 sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ns ns ✱✱ 0 50 100 150 %cell survial MLKL sgRNA + + + + Cas9 - + - + Control LV + + - + MEG3 LV - - + + ✱✱✱ ns ✱✱✱ H JI Supplementary Figure 10: RNA sequencing of the MEG3expressing neurons. (A) Barplots of the total number of reads per sample obtained from RNA sequencing of MEG3oexpression (OE, control n=6, overexpression n=6). (B) Multi-dimensional scaling (MDS) of the RNA sequencing samples of MEG3expression (OE).Neurons derived from two independent differentiations (Diff), control LV (n=6), MEG3LV (n=6). (C) Bland-Altman MA plot showing differential expression of bulk RNA sequencing of MEG3expression (control LC n=6, MEG3LV n=6). Significantly upregulated genes are shown in red and downregulated genes in blue (FDR < 0.05). Full differential expression results are shown in Supplementary Table 6. (D) DAVID gene ontology analysis of leading-edge upregulated genes from the GSEA analysis (indicated with red color in fig.3h). Top 10 GO terms were displayed. -ve=negative, +ve=positive, reg.=regulation ,TF=transcription factor, TNF=tumor necrosis factor, APP=antigen process and presentation. (E) Schematic representation of the MEG3knockdown approach using MEG3shRNA.Lentiviral vectors containing the MEG3shRNA sequence were cloned into the lentiviral backbone and packaged. H9-derived NPCs were transduced with either the control GFP lentivirus or the MEG3shRNA, followed by puromycin selection to select positively transduced cells.Transduced cells were then transplanted into amyloid mice. (F) qRT-PCR analysis was conducted to assess human MEG3expression using RNA extracted from xenografted mouse brains at six months of age (control n=5; MEG3shRNA n=13). (G) MEG3knockdown validation was performed using RNA Scope analysis on samples obtained from transplanted animals at six months of age.The white arrow indicates the MEG3RNA Scope puncta (red). Analysis of necroptosis inhibition in vitro using the RIPK1, RIPK3, and MLKL knock-out cells.Neuroprogenitor cells (NPCs), derived from the H9 stem cell line transduced with RIPK1(H),RIPK3(I),and MLKL (J),were plated in flatbottom black 96-well plates.Cells were treated with doxycycline to induce the Cas9in the indicated conditions. On approximately day 60 in vitro (~DIV60), the cells were transduced with either acontrol lentivirus or MEG3 lentivirus and cells were maintained in the NMM until the CellTiter-Glo® assay.Ten days post transduction for the MEG3CellTiter-Glo® luminescent cell viability assay was performed to measure cell viability.The assay was conducted in triplicates with six technical replicates for each replicate.The data from the control wells were normalized, and the results are presented as the percentage of cell survival between the control and treated conditions.Bonferroni multiple comparison test us used for measuring the statistical significance.