Genome-wide association studies have fingered PICALM as a consistent link to Alzheimer’s disease. The protein promotes clathrin-mediated endocytosis and has been tied to APP processing and increased Aβ production in neurons. In the May 25 Nature Neuroscience, researchers led by Berislav Zlokovic at the University of Southern California in Los Angeles propose a completely different mechanism for how PICALM influences Alzheimer’s risk. In transgenic mice, this assembly protein helped internalize Aβ into endothelial cells and accompanied it to the bloodstream, thus clearing amyloid from the brain. In endothelial cell cultures that model the blood-brain barrier, a single nucleotide polymorphism associated with lower AD risk doubled PICALM expression and Aβ transport, providing a possible functional mechanism for this GWAS hit. Moreover, people who died of late-onset Alzheimer’s had less PICALM in their endotheliums than age-matched controls.
Costantino Iadecola at Weill Cornell Medical College, New York, praised the broad spectrum of experiments, which he said convincingly linked PICALM to Aβ clearance. “This paper reinforces the idea that blood vessels play a key role in regulating the homeostasis of Aβ in the brain. It suggests that selectively upregulating PICALM in endothelial cells could be a therapeutic strategy,” Iadecola told Alzforum. He was not involved in the research.
PICALM brings together clathrin and adaptor protein complex 2, key components of the coated endocytosis pits on the cell membrane. In neurons, PICALM facilitates Aβ production by internalizing APP or γ-secretase (see Xiao et al., 2012; Kanatsu et al., 2014; Oct 2011 news). However, endothelial cells in brain capillaries express more PICALM than neurons do, suggesting that the protein might do something special there (see Baig et al., 2010).
Since endothelial cells transport Aβ from the brain to the bloodstream, the authors wondered if PICALM might aid in this process. Joint first authors Zhen Zhao, Abhay Sagare, and Qingyi Ma injected Aβ40 and Aβ42 into the brains of 3-month-old PICALM heterozygous knockout mice; homozygous knockouts die in utero. After 30 minutes, about a third more of the peptide remained stuck in the brains of the heterozygous knockouts than in the brains of their littermate controls. The authors then crossed the PICALM heterozygotes with Tg2576 mice, which overexpress APP with the Swedish mutation and accumulate plaques by 1 year of age. At 3 months of age, the offspring had more than twice as much soluble extracellular Aβ as Tg2576 controls, and by 9 months, almost four times as much amyloid plaque. The mice struggled to build nests, burrow, and recognize new objects. Viral delivery of the PICALM gene to hippocampal brain endothelium at 5 months cut amyloid load in half and improved behavior by 6 months of age, confirming that lack of PICALM caused these deficits.
To decipher the mechanism, the authors used primary cultures of human brain endothelial cells that model blood-brain barrier properties (see Zhu et al., 2010). The group previously reported that extracellular Aβ must bind to the low-density lipoprotein receptor related protein 1 (LRP1) on brain capillary endothelial cells to be transported through the cell to the bloodstream (see Deane et al., 2004). In the present study, the authors found that PICALM fastened onto the Aβ/LRP1 complex within 30 seconds of adding Aβ to the basolateral membrane. PICALM remained bound as Aβ traveled across the cell in vesicles, a journey known as transcytosis (see image below). The complex also transiently associated with the two endosomal trafficking proteins Rab5, found in early endosomes, and Rab11, which regulates transport of vesicles across the cell to the luminal membrane. Transcytosis took about five minutes. Knockdown of PICALM slashed internalization of Aβ and transcytosis by about 90 percent. Knockdown of LRP1, clathrin, Rab5, and Rab11 each had a similar effect, demonstrating the role of these endocytic and trafficking molecules.
“We know Aβ is cleared through transcytosis, but no one knew exactly how this happened. This paper refines the molecular mechanisms of endothelial Aβ trafficking,” Iadecola noted.
How do these cell and animal studies relate to what happens in people? The authors report that cortical microvessels from 30 postmortem brains at advanced stages of AD had about half the level of PICALM as the same vessels from 20 age-matched controls. People with the least PICALM had the most amyloid and performed worst on cognitive tests. To test if Aβ clearance was compromised in patients, the authors cultured endothelial cells taken from AD brains shortly after death. In these cultures, PICALM levels were down by a third and transcytosis by half, compared with age-matched controls. LRP1 was also suppressed. Adding back PICALM and LRP1 using a viral vector restored transcytosis to nearly normal levels, the authors reported.
It is unknown why PICALM is low in sporadic AD. PICALM expression is normal in APP transgenic mice, hence the authors conclude this is likely not caused by Aβ. Other factors, such as inflammation or hypoxia, might be responsible, they speculate. In people who carry PICALM variants, however, the data are clearer. The minor allele of the rs3851179 SNP has been repeatedly found to protect against Alzheimer’s. This SNP occurs upstream of the coding region and has been associated with increased expression (see Parikh et al., 2014). The authors confirmed that cultured endothelial cells with the protective variant doubled their PICALM production and shunted twice as much Aβ through their cell bodies as those carrying the major allele. The finding may help explain why an SNP that pumps up PICALM expression protects against disease, in contrast to some mouse studies that suggested more PICALM would worsen pathology.
Lars Bertram at the University of Lübeck, Germany, said the new findings agree with other human studies that tie PICALM to Aβ levels in cerebrospinal fluid. “These data fit nicely with earlier work from our group, where we found a dosage-dependent correlation between lower CSF Aβ40 levels and the PICALM risk SNP rs541458 identified in AD patients and controls from Germany (see Schjeide et al., 2011). This relationship is similar to what has long been established for the ApoE4 allele,” Bertram wrote to Alzforum.
The data raise the question of whether upregulating PICALM in endothelium could be therapeutic. The authors plan to screen for drugs that can do this. Iadecola said that because endothelial cells are more accessible than brain cells, it might be possible to selectively target them. He added that transcytosis is a major conduit for Aβ removal, responsible for dumping about a quarter of total peptide levels, with another quarter flushed through CSF (see Roberts et al., 2014).
Selective targeting might be important, because PICALM acts differently in neurons. Taisuke Tomita at the University of Tokyo recently reported that less PICALM dampens Aβ production in neurons, resulting in less amyloid deposition (see Apr 2015 conference news). This is the reverse of Zlokovic’s results, where less PICALM worsened amyloid. The discrepancy may arise in part from the mouse models used, Tomita suggested. His studies involved wild-type mice or transgenics with low APP expression, where clearance of Aβ may be a lesser factor. “An excessive amount of Aβ, either administered by injection, or expressed from an APP transgene, could saturate Aβ clearance mechanisms, which might be suppressed by deletion of PICALM, leading to Aβ accumulation,” he wrote to Alzforum (see full comment below).
If PICALM promotes Aβ production in neurons while enhancing clearance by endothelium, would more PICALM be good or bad for people? While the answer is unknown, Iadecola pointed out that clearance, not production, is the main problem in late-onset AD (see Dec 2010 news).—Madolyn Bowman Rogers
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Research Models Citations
- Xiao Q, Gil SC, Yan P, Wang Y, Han S, Gonzales E, Perez R, Cirrito JR, Lee JM. Role of Phosphatidylinositol Clathrin Assembly Lymphoid-Myeloid Leukemia (PICALM) in Intracellular Amyloid Precursor Protein (APP) Processing and Amyloid Plaque Pathogenesis. J Biol Chem. 2012 Jun 15;287(25):21279-89. PubMed.
- Kanatsu K, Morohashi Y, Suzuki M, Kuroda H, Watanabe T, Tomita T, Iwatsubo T. Decreased CALM expression reduces Aβ42 to total Aβ ratio through clathrin-mediated endocytosis of γ-secretase. Nat Commun. 2014 Feb 28;5:3386. PubMed.
- Baig S, Joseph SA, Tayler H, Abraham R, Owen MJ, Williams J, Kehoe PG, Love S. Distribution and expression of picalm in Alzheimer disease. J Neuropathol Exp Neurol. 2010 Oct;69(10):1071-7. PubMed.
- Zhu D, Wang Y, Singh I, Bell RD, Deane R, Zhong Z, Sagare A, Winkler EA, Zlokovic BV. Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor. Blood. 2010 Jun 10;115(23):4963-72. Epub 2010 Mar 26 PubMed.
- Deane R, Wu Z, Sagare A, Davis J, Du Yan S, Hamm K, Xu F, Parisi M, LaRue B, Hu HW, Spijkers P, Guo H, Song X, Lenting PJ, Van Nostrand WE, Zlokovic BV. LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms. Neuron. 2004 Aug 5;43(3):333-44. 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.
- Schjeide BM, Schnack C, Lambert JC, Lill CM, Kirchheiner J, Tumani H, Otto M, Tanzi RE, Lehrach H, Amouyel P, von Arnim CA, Bertram L. The role of clusterin, complement receptor 1, and phosphatidylinositol binding clathrin assembly protein in Alzheimer disease risk and cerebrospinal fluid biomarker levels. Arch Gen Psychiatry. 2011 Feb;68(2):207-13. PubMed.
- Roberts KF, Elbert DL, Kasten TP, Patterson BW, Sigurdson WC, Connors RE, Ovod V, Munsell LY, Mawuenyega KG, Miller-Thomas MM, Moran CJ, Cross DT 3rd, Derdeyn CP, Bateman RJ. Amyloid-β efflux from the central nervous system into the plasma. Ann Neurol. 2014 Dec;76(6):837-44. Epub 2014 Oct 24 PubMed.
- Zhao Z, Sagare AP, Ma Q, Halliday MR, Kong P, Kisler K, Winkler EA, Ramanathan A, Kanekiyo T, Bu G, Owens NC, Rege SV, Si G, Ahuja A, Zhu D, Miller CA, Schneider JA, Maeda M, Maeda T, Sugawara T, Ichida JK, Zlokovic BV. Central role for PICALM in amyloid-β blood-brain barrier transcytosis and clearance. Nat Neurosci. 2015 Jul;18(7):978-87. Epub 2015 May 25 PubMed.