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.
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?
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
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