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., 2012Kanatsu et al., 2014Oct 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).

Protective PICALM.

Compared to mice with normal PICALM levels (top panel), almost four times as much amyloid plaque (green) accumulates in the hippocampi of 9-month-old APP mice lacking PICALM (bottom). [Image courtesy of Zhao et al., Nature Neuroscience.]

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.

Bosom Buddies.

Aβ (purple), PICALM (red), and LRP1 (green) remain tightly associated (overlay appears white) as they travel across an endothelial cell (nucleus in blue). Inset blown up in upper right corner. [Image courtesy of Zhao et al., Nature Neuroscience.]

“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

Comments

  1. CALM/PICALM is one of the most abundant clathrin adaptors in endocytic clathrin-coated vesicles and regulates the endocytic process at presynaptic active zones of neurons (Blondeau et al., 2004; Koo et al., 2011). Regarding the pathological aspect of PICALM gene variants in AD, we and others have reported that PICALM regulates Aβ production via endocytosis of APP and γ-secretase, presumably in neurons. Also, other researchers have reported that PICALM plays an important role in autophagy and tau clearance (Moreau et al., 2014), suggesting that PICALM is a multifunctional protein. Here, with an impressive array of methodologies and mouse models, Zlokovic’s group clearly indicate that PICALM functions as an adaptor protein for trans-endocytosis of an Aβ-LRP complex in endothelial cells. This is consistent with recent results by David Owen’s lab, since PICALM directly regulates the endocytosis rate of clathrin-coated vesicles (Miller et al., 2011; Miller et al., 2015).

    The biggest difference with our results is the effect of reduced expression of PICALM. We found this reduces Aβ levels and attenuates amyloid deposition, whereas Zlokovic and colleagues report that it impairs Aβ efflux from the brain and exacerbates the plaque load. One possible explanation is that we used wild-type and A7 mice, the latter expressing only very low amounts of the APP transgene. 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.

    It would be interesting to know the correlation between the amyloid pathology and genetic status of PICALM in AD patients.

    References:

    . Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc Natl Acad Sci U S A. 2004 Mar 16;101(11):3833-8. Epub 2004 Mar 8 PubMed.

    . SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses. Proc Natl Acad Sci U S A. 2011 Aug 16;108(33):13540-5. Epub 2011 Aug 1 PubMed.

    . PICALM modulates autophagy activity and tau accumulation. Nat Commun. 2014 Sep 22;5:4998. PubMed.

    . The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM. Cell. 2011 Nov 23;147(5):1118-31. PubMed.

    . CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature. Dev Cell. 2015 Apr 20;33(2):163-75. PubMed.

  2. This study by Zhao et al. elegantly demonstrates that PICALM is involved in transcytosis of Aβ through the blood-brain barrier and plays a role in Aβ clearance. The PICALM gene encodes a protein (phosphatidyliniositol-binding clathrin assembly protein) involved in clathrin-dependent endocytosis. GWAS studies have demonstrated that some PICALM allelic variants (in non-coding sequences) are genetic risks factors for late-onset Alzheimer's disease (LOAD) (Harold et al., 2009). The exact cellular mechanisms of this genetic susceptibility are still poorly understood, although previous reports on the expression of PICALM by endothelial cells (Baig et al., 2010; Parikh et al., 2014) suggested that reduction of Aβ clearance might be such a mechanism.

    A critical finding of  Zhao et al. is that they observed that human endothelial cells derived from individuals with a protective PICALM allele have higher PICALM levels and increased Aβ clearance.

    In addition, Zhao et al. reported a decrease of PICALM level in AD brain in the microvessel fraction. This decrease confirms our previous report of a reduced PICALM in AD brain homogenates (Ando et al., 2013). In addition to this decrease, we observed that at least a fragment of PICALM co-localized with tangles in neurons in situ in LOAD, in familial AD, and in Down’s syndrome. PICALM also co-immunoprecipated with tau from human brain (Ando et al., 2013). Interestingly, Moreau et al. (2014) observed that downregulation of PICALM impaired tau clearance by autophagy and induced accumulation of phosphorylated tau. PICALM was not observed in Aβ deposits or non-tau aggregates (Ando et al., 2013). Thus, in addition to its role in Aβ transport in brain endothelial cells, there is also evidence that PICALM might affect tau processing, as it is the case for some other GWAS genes like Bin1 (Chapuis et al., 2013), Clusterin (Zhou et al., 2014), and APOE (Strittmatter et al., 1994; Huang et al., 2001). PICALM is expressed in many cell types, including in neurons and in microglial cells. Interestingly, we observed, as also reported by Zhao et al., an increased PICALM immunoreactivity in neurons in AD (Ando et al., 2013). This may be related to the report of increased expression of PICALM inducing tangle-like structures in the photoreceptor cell layer in zebrafish (Moreau et al., 2014). We also reported a significant increase of PICALM immunoreactivities in microglial cells, which might be related to a previous report of increased level of PICALM transcripts in AD brain (Parikh et al., 2014). 

    PICALM is a substrate of calpains and caspases (Ando et al., 2013; Kim and Kim, 2001; Rudinskiy  et al., 2009) and is cleaved in AD brains. Taken together, these observations suggest that AD might induce distinct dysregulation of PICALM expression depending on cell types.

    One potential confounding effect of assessing the effect of Picalm deletion in Picalm +/- mice is that the reduced expression of Picalm in this model happens in several cell types, not only endothelial cells. This makes it more difficult to interpret changes in Aβ levels, or other molecules, in APP mice crossed with Picalm +/- mice, as reported by Zhao et al. Haploinsufficiency of Picalm could also affect autophagosome formation and modulate autophagy (Chapuis et al., 2013), and might affect both extracellular (Cho et al., 2014) and intracellular Aβ levels.

    Future studies will be needed to clarify what the consequences of changes of PICALM expression are for Aβ and tau processing in non-endothelial cells. Altogether, studies reported so far point to PICALM as a modulator of both Aβ and tau clearance.

    References:

    . Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease. Nat Genet. 2009 Oct;41(10):1088-93. PubMed.

    . Distribution and expression of picalm in Alzheimer disease. J Neuropathol Exp Neurol. 2010 Oct;69(10):1071-7. PubMed.

    . An intronic PICALM polymorphism, rs588076, is associated with allelic expression of a PICALM isoform. Mol Neurodegener. 2014 Aug 29;9:32. PubMed.

    . Clathrin adaptor CALM/PICALM is associated with neurofibrillary tangles and is cleaved in Alzheimer's brains. Acta Neuropathol. 2013 Jun;125(6):861-78. PubMed.

    . PICALM modulates autophagy activity and tau accumulation. Nat Commun. 2014 Sep 22;5:4998. PubMed.

    . Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology. Mol Psychiatry. 2013 Feb 12; PubMed.

    . Intracellular clusterin interacts with brain isoforms of the bridging integrator 1 and with the microtubule-associated protein Tau in Alzheimer's disease. PLoS One. 2014;9(7):e103187. Epub 2014 Jul 22 PubMed.

    . Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: implications for Alzheimer disease. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11183-6. PubMed.

    . Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8838-43. PubMed.

    . Cleavage of purified neuronal clathrin assembly protein (CALM) by caspase 3 and calpain. Exp Mol Med. 2001 Dec 31;33(4):245-50. PubMed.

    . Calpain hydrolysis of alpha- and beta2-adaptins decreases clathrin-dependent endocytosis and may promote neurodegeneration. J Biol Chem. 2009 May 1;284(18):12447-58. PubMed.

    . Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome. Autophagy. 2014 Oct 1;10(10):1761-75. Epub 2014 Jul 22 PubMed.

  3. We really appreciate Dr. Ando and colleagues' comments.

    Without any doubt, PICALM has multiple functions besides Aβ clearance via the blood brain barrier that can be linked to AD, such as mitigating Aβ toxicity in neurons (Treusch et al., 2011), modulating Aβ production (Xiao et al., 2012), controlling  the balance of different Aβ species (Kanatsu et al., 2014), and preventing Tau accumulation (Moreau et al., 2014), as shown by different groups. These distinct functions are likely achieved through PICALM’s differential expression and signaling in different cell types, as Dr. Ando has pointed out. Therefore, it is of great importance to investigate PICALM’s functions in other cell types in relation to AD pathogenesis, particularly neurons and microglia.

    In addition, we would like to point out the rescue experiment reported in our paper Cre-dependent AAV re-expression of PICALM in the endothelium rescued Aβ clearance defects in the Picalm+/- mice, indicating an endothelial specific effect rather than global haploinsufficiency.

    References:

    . Functional links between Aβ toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast. Science. 2011 Dec 2;334(6060):1241-5. PubMed.

    . 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.

    . Decreased CALM expression reduces Aβ42 to total Aβ ratio through clathrin-mediated endocytosis of γ-secretase. Nat Commun. 2014 Feb 28;5:3386. PubMed.

    . PICALM modulates autophagy activity and tau accumulation. Nat Commun. 2014 Sep 22;5:4998. PubMed.

  4. This is a nice study illustrating the point that assisting in the clearance of Aβ, in addition to preventing its formation, presents a suitable avenue for therapeutic interventions. Together with a study we have published recently (Leinenga and Götz, 2015), it further demonstrates that there are several mechanisms of Aβ clearance that might be exploited in combination.

    References:

    . Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer's disease mouse model. Sci Transl Med. 2015 Mar 11;7(278):278ra33. PubMed.

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Traffic Control—Curb Endocytosis to Curb AD Pathogenesis?
  2. The Feud, Act II: Do Alzheimer’s Genes Affect Amyloid or Tau?
  3. Paper Alert: In Vivo Human Data Shows Reduced Aβ Clearance in AD

Research Models Citations

  1. Tg2576

Paper Citations

  1. . 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.
  2. . Decreased CALM expression reduces Aβ42 to total Aβ ratio through clathrin-mediated endocytosis of γ-secretase. Nat Commun. 2014 Feb 28;5:3386. PubMed.
  3. . Distribution and expression of picalm in Alzheimer disease. J Neuropathol Exp Neurol. 2010 Oct;69(10):1071-7. PubMed.
  4. . 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.
  5. . LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms. Neuron. 2004 Aug 5;43(3):333-44. PubMed.
  6. . Genetics of PICALM expression and Alzheimer's disease. PLoS One. 2014;9(3):e91242. Epub 2014 Mar 11 PubMed.
  7. . 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.
  8. . Amyloid-β efflux from the central nervous system into the plasma. Ann Neurol. 2014 Dec;76(6):837-44. Epub 2014 Oct 24 PubMed.

Further Reading

Primary Papers

  1. . 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.