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. Correction.

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  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:

    Blondeau F, Ritter B, Allaire PD, Wasiak S, Girard M, Hussain NK, Angers A, Legendre-Guillemin V, Roy L, Boismenu D, Kearney RE, Bell AW, Bergeron JJ, McPherson PS. 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.

    Koo SJ, Markovic S, Puchkov D, Mahrenholz CC, Beceren-Braun F, Maritzen T, Dernedde J, Volkmer R, Oschkinat H, Haucke V. 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.

    Moreau K, Fleming A, Imarisio S, Lopez Ramirez A, Mercer JL, Jimenez-Sanchez M, Bento CF, Puri C, Zavodszky E, Siddiqi F, Lavau CP, Betton M, O'Kane CJ, Wechsler DS, Rubinsztein DC. PICALM modulates autophagy activity and tau accumulation. Nat Commun. 2014 Sep 22;5:4998. PubMed.

    Miller SE, Sahlender DA, Graham SC, Höning S, Robinson MS, Peden AA, Owen DJ. The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM. Cell. 2011 Nov 23;147(5):1118-31. PubMed.

    Miller SE, Mathiasen S, Bright NA, Pierre F, Kelly BT, Kladt N, Schauss A, Merrifield CJ, Stamou D, Höning S, Owen DJ. 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.

    View all comments by Taisuke Tomita

  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:

    Harold D, Abraham R, Hollingworth P, Sims R, Gerrish A, Hamshere ML, Pahwa JS, Moskvina V, Dowzell K, Williams A, Jones N, Thomas C, Stretton A, Morgan AR, Lovestone S, Powell J, Proitsi P, Lupton MK, Brayne C, Rubinsztein DC, Gill M, Lawlor B, Lynch A, Morgan K, Brown KS, Passmore PA, Craig D, McGuinness B, Todd S, Holmes C, Mann D, Smith AD, Love S, Kehoe PG, Hardy J, Mead S, Fox N, Rossor M, Collinge J, Maier W, Jessen F, Schürmann B, van den Bussche H, Heuser I, Kornhuber J, Wiltfang J, Dichgans M, Frölich L, Hampel H, Hüll M, Rujescu D, Goate AM, Kauwe JS, Cruchaga C, Nowotny P, Morris JC, Mayo K, Sleegers K, Bettens K, Engelborghs S, De Deyn PP, Van Broeckhoven C, Livingston G, Bass NJ, Gurling H, McQuillin A, Gwilliam R, Deloukas P, Al-Chalabi A, Shaw CE, Tsolaki M, Singleton AB, Guerreiro R, Mühleisen TW, Nöthen MM, Moebus S, Jöckel KH, Klopp N, Wichmann HE, Carrasquillo MM, Pankratz VS, Younkin SG, Holmans PA, O'Donovan M, Owen MJ, Williams J. Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease. Nat Genet. 2009 Oct;41(10):1088-93. 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.

    Parikh I, Medway C, Younkin S, Fardo DW, Estus S. An intronic PICALM polymorphism, rs588076, is associated with allelic expression of a PICALM isoform. Mol Neurodegener. 2014 Aug 29;9:32. PubMed.

    Ando K, Brion JP, Stygelbout V, Suain V, Authelet M, Dedecker R, Chanut A, Lacor P, Lavaur J, Sazdovitch V, Rogaeva E, Potier MC, Duyckaerts C. 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.

    Moreau K, Fleming A, Imarisio S, Lopez Ramirez A, Mercer JL, Jimenez-Sanchez M, Bento CF, Puri C, Zavodszky E, Siddiqi F, Lavau CP, Betton M, O'Kane CJ, Wechsler DS, Rubinsztein DC. PICALM modulates autophagy activity and tau accumulation. Nat Commun. 2014 Sep 22;5:4998. PubMed.

    Chapuis J, Hansmannel F, Gistelinck M, Mounier A, Van Cauwenberghe C, Kolen KV, Geller F, Sottejeau Y, Harold D, Dourlen P, Grenier-Boley B, Kamatani Y, Delepine B, Demiautte F, Zelenika D, Zommer N, Hamdane M, Bellenguez C, Dartigues JF, Hauw JJ, Letronne F, Ayral AM, Sleegers K, Schellens A, Broeck LV, Engelborghs S, De Deyn PP, Vandenberghe R, O'Donovan M, Owen M, Epelbaum J, Mercken M, Karran E, Bantscheff M, Drewes G, Joberty G, Campion D, Octave JN, Berr C, Lathrop M, Callaerts P, Mann D, Williams J, Buée L, Dewachter I, Van Broeckhoven C, Amouyel P, Moechars D, Dermaut B, Lambert JC, GERAD consortium. Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology. Mol Psychiatry. 2013 Nov;18(11):1225-34. Epub 2013 Feb 12 PubMed.

    Zhou Y, Hayashi I, Wong J, Tugusheva K, Renger JJ, Zerbinatti C. 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.

    Strittmatter WJ, Saunders AM, Goedert M, Weisgraber KH, Dong LM, Jakes R, Huang DY, Pericak-Vance M, Schmechel D, Roses AD. 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.

    Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW. 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.

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

    Rudinskiy N, Grishchuk Y, Vaslin A, Puyal J, Delacourte A, Hirling H, Clarke PG, Luthi-Carter R. 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.

    Cho MH, Cho K, Kang HJ, Jeon EY, Kim HS, Kwon HJ, Kim HM, Kim DH, Yoon SY. 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.

    View all comments by Jean-Pierre Brion

  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:

    Treusch S, Hamamichi S, Goodman JL, Matlack KE, Chung CY, Baru V, Shulman JM, Parrado A, Bevis BJ, Valastyan JS, Han H, Lindhagen-Persson M, Reiman EM, Evans DA, Bennett DA, Olofsson A, DeJager PL, Tanzi RE, Caldwell KA, Caldwell GA, Lindquist S. Functional links between Aβ toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast. Science. 2011 Dec 2;334(6060):1241-5. PubMed.

    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.

    Moreau K, Fleming A, Imarisio S, Lopez Ramirez A, Mercer JL, Jimenez-Sanchez M, Bento CF, Puri C, Zavodszky E, Siddiqi F, Lavau CP, Betton M, O'Kane CJ, Wechsler DS, Rubinsztein DC. PICALM modulates autophagy activity and tau accumulation. Nat Commun. 2014 Sep 22;5:4998. PubMed.

    View all comments by Zhen Zhao

  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:

    Leinenga G, Götz J. 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.

    View all comments by Jürgen Götz

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