Narayan P, Sienski G, Bonner JM, Lin YT, Seo J, Baru V, Haque A, Milo B, Akay LA, Graziosi A, Freyzon Y, Landgraf D, Hesse WR, Valastyan J, Barrasa MI, Tsai LH, Lindquist S. PICALM Rescues Endocytic Defects Caused by the Alzheimer's Disease Risk Factor APOE4. Cell Rep. 2020 Oct 6;33(1):108224. PubMed.

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  1. This paper very interestingly reports APOE4-dependent endocytosis defects in astrocytes that can be rescued by CALM overexpression. The use of isogenic cultures of APOE4 astrocytes enabled the controlled analysis of endosomal Rabs, transferrin, and EGF endocytosis—canonical markers of the endocytic pathway.

    The inhibition of endocytosis by astrocytes secreting APOE4 is compelling evidence. However, the rationale behind this inhibition is lacking. Could it be a consequence of reduced APOE4-mediated lipid uptake? The paper’s significance for Alzheimer's disease's could be greater if endocytosis of an AD-relevant cargo, such as β-amyloid oligomers, had been analyzed, defects in which could impact β-amyloid clearance.

    The rescue by CALM overexpression implies that increasing clathrin-mediated endocytosis is a therapeutic target, and its disruption by disease-associated SNPs may be relevant for astrocytes. The impact of the CALM variants in astrocytes should be further explored.

    View all comments by Claudia Almeida

  2. Neuronal early endosome swelling and proliferation, among the earliest specific signs of AD, are linked to multiple downstream pathophysiological consequences (Nixon 2017; Pensalfini et al., 2020) and are driven by APP-dependent and -independent mechanisms, including APOE4 and other genes that increase AD risk.

    This interesting study by Narayan et al. highlights the critical influence of cell type and biological context on regulation of endocytosis and endosomes and responses to these AD risk genes. The authors report that APOE4-expressing IPSC-derived astrocytes contain fewer early endosomes than APOE3 -expressing ones and display an endocytic uptake deficit. By contrast, APOE4-expressing IPSC-derived neurons earlier investigated by the same group exhibited an enlargement of the early endosome compartment (Lin et al., 2018) as seen in neurons in AD brain and in the yeast system studied in their report, which corresponds to an upregulation of endocytosis described in AD and Down’s syndrome models.

    CALM overexpression reversed the endocytic uptake deficit in astrocytes and yeast expressing APOE4 but, in APOE3 cells, it actually induced an APOE4-like endocytic uptake deficit. Cell-type differences in endosomal responses to AD risk factor modulation have been increasingly reported, including in other IPSC-derived models. This underscores the importance of interpreting possible disease-relevant actions on endocytosis for the many GWAS genes affecting this process in relation to the biological context, i.e., cell type, genetic derivation, state of maturation or aging, etc. (Knupp et al., 2020; Fernandez et al., 2020). 

    In this regard, it will be interesting to know how CALM expression impacts the endocytic anomalies in APOE4-expressing IPSC neurons. Also, given that AD-related neuronal endosome dysfunction emerges in adulthood, do astrocytes in adult or aging brains exhibit the same APOE4 effects as in IPSCs? The findings in this report further the case for analysis of endosomal-lysosomal function and dysfunction in single cells or single-cell populations of the cell-heterogeneous brain.

    References:

    Nixon RA. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. FASEB J. 2017 Jul;31(7):2729-2743. PubMed.

    Pensalfini A, Kim S, Subbanna S, Bleiwas C, Goulbourne CN, Stavrides PH, Jiang Y, Lee JH, Darji S, Pawlik M, Huo C, Berg M, Smiley J, Basavarajappa BS, Nixon RA. Endosomal Dysfunction Induced by Directly Over-Activating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease. Cell Reports. 5 January 2020.

    Lin YT, Seo J, Gao F, Feldman HM, Wen HL, Penney J, Cam HP, Gjoneska E, Raja WK, Cheng J, Rueda R, Kritskiy O, Abdurrob F, Peng Z, Milo B, Yu CJ, Elmsaouri S, Dey D, Ko T, Yankner BA, Tsai LH. APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron. 2018 Jun 27;98(6):1141-1154.e7. Epub 2018 May 31 PubMed.

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

    Fernandez CG, Hamby ME, McReynolds ML, Ray WJ. The Role of APOE4 in Disrupting the Homeostatic Functions of Astrocytes and Microglia in Aging and Alzheimer's Disease. Front Aging Neurosci. 2019;11:14. Epub 2019 Feb 11 PubMed.

    View all comments by Ralph Nixon

  3. Narayan et al. provide evidence of APOE4-induced perturbation to endocytosis in iPSC-derived astrocytes. This APOE4-mediated endocytic dysfunction results from a toxic gain of function of the APOE4 isoform and is cell-type-specific.

    They also performed genetic screening in yeast and identified YAP1802 as a genetic modifier of APOE4-associated defects. YAP1802 is the human homolog of the LOAD-associated gene, PICALM. Previous human genetic studies had found that the association of AD risk with PICALM was only significant in APOE ε4 carriers (Jun et al., 2010). PICALM genotype also seems to modulate both brain atrophy and cognitive performance in APOE ε4 carriers (Morgen et al., 2014). PICALM transcripts are both upregulated and downregulated in AD brains (Verheijen and Sleegers, 2018). However, the sentinel GWAS SNP rs3851179 in the PICALM genomic region is associated with decreased AD risk and increased PICALM expression (Parikh et al., 2014). These studies suggest that genetically controlled high levels of PICALM reduce the risk of developing AD in APOE4 carriers. Both APOE and PICALM gene products participate in a common pathogenic pathway leading to AD.

    The authors provide provocative insight into the missing functional link between APOE4 and PICALM through the endocytosis pathway. However, they provide no direct evidence that this functional link between APOE 4 and PICALM participates in the pathogenesis of known players in AD such as amyloid or tau metabolism.

    It is tempting to suggest that the potential link between PICALM and APOE4 in astrocytes could be mediated by effects on cellular cholesterol homeostasis mediated by the LDL receptor (LDLR), which are dysregulated in PICALM-deficient cells (Mercer et al., 2015) and iPSC-derived astrocytes (Lin et al., 2018). LDLR exhibits varying binding affinities for the three APOE isoforms (E4 > E3 >> E2; Yamamoto et al., 2008). This could explain why the PICALM-mediated rescue observed for APOE4 is allele-specific, as reported by Narayan et al.

    The cell-type-specific effect of APOE4 on endocytosis could be a common pathway underlying the mechanisms by which APOE4 affects several neurodegenerative diseases, including AD, PD, and DLB. Modulation of PICALM levels in APOE4 carriers seems to be a novel therapeutic approach. However, PICALM levels seem highly regulated and changes in normal cells could affect the endocytic pathway.

    References:

    Jun G, Naj AC, Beecham GW, Wang LS, Buros J, Gallins PJ, Buxbaum JD, Ertekin-Taner N, Fallin MD, Friedland R, Inzelberg R, Kramer P, Rogaeva E, St George-Hyslop P, Cantwell LB, Dombroski BA, Saykin AJ, Reiman EM, Bennett DA, Morris JC, Lunetta KL, Martin ER, Montine TJ, Goate AM, Blacker D, Tsuang DW, Beekly D, Cupples LA, Hakonarson H, Kukull W, Foroud TM, Haines J, Mayeux R, Farrer LA, Pericak-Vance MA, Schellenberg GD. Meta-analysis confirms CR1, CLU, and PICALM as alzheimer disease risk loci and reveals interactions with APOE genotypes. Arch Neurol. 2010 Dec;67(12):1473-84. PubMed.

    Morgen K, Ramirez A, Frölich L, Tost H, Plichta MM, Kölsch H, Rakebrandt F, Rienhoff O, Jessen F, Peters O, Jahn H, Luckhaus C, Hüll M, Gertz HJ, Schröder J, Hampel H, Teipel SJ, Pantel J, Heuser I, Wiltfang J, Rüther E, Kornhuber J, Maier W, Meyer-Lindenberg A. Genetic interaction of PICALM and APOE is associated with brain atrophy and cognitive impairment in Alzheimer's disease. Alzheimers Dement. 2014 Mar 6; PubMed.

    Verheijen J, Sleegers K. Understanding Alzheimer Disease at the Interface between Genetics and Transcriptomics. Trends Genet. 2018 Jun;34(6):434-447. Epub 2018 Mar 21 PubMed.

    Parikh I, Fardo DW, Estus S. Genetics of PICALM expression and Alzheimer's disease. PLoS One. 2014;9(3):e91242. Epub 2014 Mar 11 PubMed.

    Mercer JL, Argus JP, Crabtree DM, Keenan MM, Wilks MQ, Chi JT, Bensinger SJ, Lavau CP, Wechsler DS. Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis. PLoS One. 2015;10(6):e0129776. Epub 2015 Jun 15 PubMed.

    Lin YT, Seo J, Gao F, Feldman HM, Wen HL, Penney J, Cam HP, Gjoneska E, Raja WK, Cheng J, Rueda R, Kritskiy O, Abdurrob F, Peng Z, Milo B, Yu CJ, Elmsaouri S, Dey D, Ko T, Yankner BA, Tsai LH. APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron. 2018 Jun 27;98(6):1141-1154.e7. Epub 2018 May 31 PubMed.

    View all comments by Bruno Benitez

  4. This paper elegantly combines two different techniques to discover that the AD risk gene PICALM modulates the AD-risk gene APOE4. The paper has a signature from two outstanding labs, combining yeast genetic assays (developed in the late Susan Lindquist’s lab) with isogenic human APOE iPSC-models developed in the lab of Li-Huei Tsai. Together, the techniques make for a powerful combination and provide intriguing new insight on the interplay between two major AD risk factors and their roles in the endosomal system.

    The authors convincingly show that APOE4 has a toxic gain-of-function effect on the endosomal system in yeast, as well as in human iPSC-derived astrocytes. For example, the authors report early endocytic dysfunction in ApoE4/4 iPSC-derived astrocytes, but not in the APOE3/3 or APOE knockout astrocytes. Similarly, APOE4 expression in yeast causes defects in endosomal function and delays growth, defects that are absent in APOE3-overexpressing yeast. PICALM overexpression can rescue these effects in both model systems. Overall, the findings are of great interest to the field as PICALM and APOE4 are both major AD risk genes. In addition, the findings further reinforce the notion that endocytic disruption represents a major pathological event in AD.

    Interestingly, regulation of the early endosome system by ApoE4 seems to be cell-type-specific. Enlarged early endosomes have been observed in APOE4 neurons across model systems (Cataldo et al., 2000; Nuriel et al., 2017; Lin et al., 2018). The current study indicates that in iPSC-derived APOE4 astrocytes, early endosome number is rather decreased and enlargement is not observed. It would be interesting to see if such effects can be observed in astrocytes in other model systems and, if so, what the cell-type specific mechanisms are. That APOE is secreted adds a layer of complexity, as APOE4 generated in one cell type might affect endosomal function in another.

    Importantly, the current work by Narayan and Sienski and colleagues adds further momentum to the already increasing efforts aimed at understanding the cell biology of APOE4 in AD. Efforts such as these will likely be essential for the development of novel therapeutics targeting this major risk factor in the future.

    View all comments by Rik van der Kant

  5. Narayan and colleagues integrate experiments in human astrocytes and yeast models to advance our understanding of endocytic perturbations in Alzheimer’s disease (AD). Based on human genome-wide association studies, a preponderance of AD candidate susceptibility genes impinge on endocytosis and vesicle trafficking pathways. An important next step is to move beyond studying individual genes in isolation, and to determine how they interact with one another in common biological processes.

    This study suggests that the well-known AD risk allele, APOE4, unexpectedly causes a “toxic gain of function” in astrocytes, disrupting normal endocytosis. By performing a targeted genetic modifier screen in budding yeast, the authors discover that the homolog of a second AD susceptibility gene, PICALM, can suppress APOE4 toxicity, and this interaction is conserved in human astrocytes. The study thus shows how two common genetic risk factors, APOE and PICALM, may interact with one another to modulate endocytosis, and further highlights how genetically tractable experimental models, such as yeast, can help us pinpoint other such combinatorial interactions.

    The work also raises a number of intriguing questions, such as how the APOE4-triggered endocytic defect relates to other implicated APOE mechanisms, and their respective contributions to AD risk. Moreover, since APOE is a secreted protein, it would be interesting to determine whether APOE4 secretion is necessary for the endocytic defect to manifest, and further, whether secreted APOE4 may be capable of disrupting endocytosis in other cell types, including neurons and/or microglia.

    View all comments by Joshua Shulman

  6. Endosomal trafficking dysfunction is a recognized pathogenic hub in Alzheimer’s disease. This elegant study provides further evidence for this, using two model systems, hiPSC-derived astrocytes and Baker’s yeast. Using isogenic APOE e3 and e4 human astrocytes, the authors show APOE e4-specific defects in early endosome number and endocytic import. Using Baker’s yeast, they also observed endocytic abnormalities in an APOE e4 yeast model. Taking advantage of this powerful genetic system, they were able to screen for modifiers of the phenotypes and identified the yeast homolog of the human AD risk gene PICALM as a factor that led to reversal of the phenotype. Back in the human system, overexpression of PICALM ameliorated the APOE e4 endocytic deficits in astrocytes.

    These findings solidify that impairment in endosomal network function is a driver of AD pathogenesis and provide mechanistic evidence supporting emerging AD genetic risk factors identified in GWAS. This study also highlights how, while endocytic trafficking is essential to all cells, defects in endosome function have different consequences in the diverse cell types in the brain. This is similar to some of our recent work, where we showed that loss of SORL1, another AD risk gene, resulted in different early endosome phenotypes in hiPSC neurons versus microglia (Knupp et al., 2020, and Alzforum comment).

    Narayan et al.—along with our work and that of others, for example Lin et al., 2018—show that even mild disruptions to this critical cellular pathway change intra- and inter-cellular dynamics, leading to neurodegeneration.

    References:

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

    Lin YT, Seo J, Gao F, Feldman HM, Wen HL, Penney J, Cam HP, Gjoneska E, Raja WK, Cheng J, Rueda R, Kritskiy O, Abdurrob F, Peng Z, Milo B, Yu CJ, Elmsaouri S, Dey D, Ko T, Yankner BA, Tsai LH. APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron. 2018 Jun 27;98(6):1141-1154.e7. Epub 2018 May 31 PubMed.

    View all comments by Jessica Young

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  1. In Astrocytes, ApoE4 Bungles Endocytosis, PICALM Picks Up the Slack