Clogged endosomal traffic is a feature of Alzheimer’s and, according to a paper in the October 6 Cell Reports, the two AD risk genes ApoE4 and PICALM normally toggle its ebb and flow. Researchers led by Li-Huei Tsai and inspired by the late Susan Lindquist at Massachusetts Institute of Technology in Cambridge report that in human astrocytes expressing ApoE4, endosomal trafficking falters. Using yeast to identify modifiers of this deficit, the researchers realized that PICALM, another AD risk gene, restored trafficking in these astrocytes. The finding places endosomal function front and center in AD pathogenesis.

  • Endocytosis waned in human astrocytes expressing ApoE4.
  • A yeast screen pegged PICALM as playing a role this deficit.
  • Boosting PICALM restored endocytosis in the astrocytes.

“Overall, the findings are of great interest to the field, as PICALM and APOE4 are both major AD risk genes,” wrote Rik van der Kant of Vrije Universiteit Amsterdam. Jessica Young, University of Washington, Seattle, agreed. “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,” she wrote to Alzforum.

From the distended endosomes spotted in the brains of people with Alzheimer’s to the numerous endosomal trafficking genes among GWAS hits, it is clear that vesicular woes are part and parcel of the disease (Nixon 2005; Oct 2013 news). Some researchers have reported that endocytosis goes awry in the earliest preclinical stages of AD, before amyloidosis takes off, and that ApoE4 carriers are particularly prone to these early deficits (Cataldo et al., 2000). As the strongest genetic risk factor for late-onset AD, ApoE4 wreaks havoc in the brain via several mechanisms, some dependent on, and some independent of, Aβ. Previously, Tsai’s group reported that in iPSC-derived neurons, ApoE4 appeared to rev up endocytosis, leading to swollen endosomes (Lin et al., 2018). 

For the current study, co-first authors Priyanka Narayan and Grzegorz Sienski and colleagues explored what ApoE4 does to endocytosis in astrocytes, the predominant cellular suppliers of ApoE in the brain. Starting with iPSCs from a healthy E3/E3 donor, the researchers used CRISPR gene editing to generate an isogenic E4/E4 line, then differentiated both into astrocytes. Compared to their E3 counterparts, E4 astrocytes had fewer early endosomes, as gauged by approximately 80 and 60 percent fewer Rab5+ and EEA-1+ puncta, respectively. Both had equally many recycling endosomes, while E4 astrocytes had slightly fewer Rab7+ late endosomes.

Encumbered Endocytosis. Astrocytes expressing ApoE3 (top) internalized more fluorescently tagged transferrin than their ApoE4-expressing counterparts (bottom). [Courtesy of Narayan et al., Cell Reports, 2020.]

The E4-astrocytes were sluggish at gobbling up the fluorescently tagged endocytosis ligands EGF and transferrin. An isogenic ApoE knockout line had no such deficit, suggesting that the endocytic malfunction in E4-astrocytes was borne of a toxic gain, rather than loss, of function. The researchers confirmed the ApoE4-driven deficit in endocytosis in a second pair of isogenic lines—this time starting with an E4/E4 parental line and using CRISPR to introduce E3.

To find potential modifiers of this endocytic defect, the researchers used baker’s yeast. Compared with yeast expressing human ApoE3, those expressing ApoE4 had abnormal early endosomes. However, unlike the human astrocytes, the E4-yeast had a great many more early endosomes than E3-yeast. Indeed, this odd overabundance was similar to what the researchers had previously observed in iPSC-derived neurons. Although the type of defect was different than the endosomal loss seen in E4-astrocytes, the outcome was similar: It led to defective internalization of endocytic ligands.

ApoE4 expression also slowed down the yeasts’ growth, and the scientists exploited having such an overt phenotype to screen for endosomal genes that would rescue it. Among a panel of 34 genes involved in endocytosis, Yap1802 emerged as the savior. Yap1802 encodes an adaptor that promotes budding of endocytic vesicles, and when overexpressed in E4-yeast, it corrected the endocytosis defect. Might it do the same in human astrocytes?

PICALM Restores Astrocyte Appetite. ApoE4 erodes endocytosis in astrocytes, but overexpression of PICALM counteracts this deficit. [Courtesy of Narayan et al., Cell Reports, 2020.]

The human version of Yap1802 is none other than PICALM, an AD risk gene involved in clathrin-dependent endocytosis (Harold et al., 2009; May 2015 news). While risk variants in PICALM sap its expression, protective variants appear to boost it (Parikh et al., 2014). 

This isn’t the first time PICALM and ApoE4 have been uttered in the same breath—previous studies have found that PICALM risk variants are detrimental only in ApoE4 carriers (Jun et al., 2010; Morgen et al., 2014). Might upping PICALM counteract the E4-induced endosomal defect in astrocytes? Indeed, the researchers found that overexpression of PICALM by way of a lentivirus corrected the endocytosis defect in E4 astrocytes.

Curiously, more PICALM triggered an endosomal defect in E3-astrocytes, in agreement with a previous study calling too much PICALM detrimental in healthy cells (Tebar et al., 1999). 

While it is clear that PICALM figures in clathrin-dependent endocytosis, how this counteracts ApoE4 remains to be seen. Indeed, exactly how ApoE4 affects endocytosis is unclear, and appears to differ markedly among cell types, Julia TCW of the Icahn School of Medicine in New York told Alzforum. Another factor to consider is how genetic background might sway interactions between ApoE4 and PICALM. To address it, TCW is comparing ApoE effects in iPSC-derived cells from multiple donors (Jul 2018 conference news). 

Tsai thinks the findings support the idea that ApoE4 does different things to different cell types. For example, it swells endosomes in neurons, but reduces their number in astrocytes. In microglia, ApoE4 expression ramps up with age and skews inflammatory responses. Defects in lipid metabolism caused by ApoE4 may underlie these cell-type specific phenotypes, Tsai said.

Ralph Nixon of New York University thinks it’s important to study how PICALM influences distinct ApoE4-driven defects in different cell types, especially in neurons. Nixon questioned how the iPSC-based findings would translate to human brain, where defects in endosomal function emerge with age. “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,” he wrote.

Josh Shulman of Baylor College of Medicine in Houston, Texas, wondered how the endocytic defect of ApoE4 relates to its other modi operandi, and their respective contributions to AD risk. “Since APOE is a secreted protein, it would be interesting to determine whether APOE4 secretion is necessary for manifestation of the endocytic defect, and further, whether secreted APOE4 may be capable of disrupting endocytosis in other cell types, including neurons and/or microglia,” he wrote.

Claudia Almeida of the University of Lisbon, Portugal, asks why ApoE4 was derailing astrocytic endocytosis. “Could it be a consequence of reduced APOE4-mediated lipid uptake?” she asked. Almeida noted that the findings would be more salient to AD if endocytosis of relevant cargo, such as Aβ oligomers, had been analyzed.

Bruno Benitez of Washington University, St. Louis, sees relevance beyond AD. “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,” he wrote.—Jessica Shugart

Comments

  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.

  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:

    . 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.

    . Endosomal Dysfunction Induced by Directly Over-Activating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease. Cell Reports. 5 January 2020.

    . 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.

    . 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.

    . 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.

  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:

    . 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.

    . Genetic interaction of PICALM and APOE is associated with brain atrophy and cognitive impairment in Alzheimer's disease. Alzheimers Dement. 2014 Mar 6; PubMed.

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

    . Genetics of PICALM expression and Alzheimer's disease. PLoS One. 2014;9(3):e91242. Epub 2014 Mar 11 PubMed.

    . Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis. PLoS One. 2015;10(6):e0129776. Epub 2015 Jun 15 PubMed.

    . 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.

  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.

  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.

  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:

    . 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.

    . 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.

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References

News Citations

  1. Paper Alert: New Alzheimer’s Genes Published
  2. New Role For PICALM: Flushing Aβ From the Brain
  3. ApoE Has Hand in Alzheimer’s Beyond Aβ, Beyond the Brain

Paper Citations

  1. . Endosome function and dysfunction in Alzheimer's disease and other neurodegenerative diseases. Neurobiol Aging. 2005 Mar;26(3):373-82. PubMed.
  2. . Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol. 2000 Jul;157(1):277-86. PubMed.
  3. . 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.
  4. . Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease. Nat Genet. 2009 Oct;41(10):1088-93. PubMed.
  5. . Genetics of PICALM expression and Alzheimer's disease. PLoS One. 2014;9(3):e91242. Epub 2014 Mar 11 PubMed.
  6. . 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.
  7. . Genetic interaction of PICALM and APOE is associated with brain atrophy and cognitive impairment in Alzheimer's disease. Alzheimers Dement. 2014 Mar 6; PubMed.
  8. . Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic. Mol Biol Cell. 1999 Aug;10(8):2687-702. PubMed.

Further Reading

Papers

  1. . Stuck in traffic: an emerging theme in diseases of the nervous system. Trends Neurosci. 2014 Feb;37(2):66-76. Epub 2014 Jan 8 PubMed.
  2. . The Endosomal-Lysosomal Pathway Is Dysregulated by APOE4 Expression in Vivo. Front Neurosci. 2017;11:702. Epub 2017 Dec 12 PubMed.

Primary Papers

  1. . PICALM Rescues Endocytic Defects Caused by the Alzheimer's Disease Risk Factor APOE4. Cell Rep. 2020 Oct 6;33(1):108224. PubMed.