The Role of the Alzheimer's Disease Risk Gene PICALM in Blood Brain Barrier Transport, Alzheimer's Disease and Amyloid Angiopathy

Lead Research Organisation: University of Cambridge
Department Name: Cambridge Institute for Medical Research

Abstract

Recent genetic studies by this and other groups has revealed that PICALM is the third strongest strongest genetic risk factor (after APOE and BIN1) for late onset Alzheimer disease (AD). PICALM's cellular function is to facilitate the formation and intracellular trafficking of a specific subtype of vesicles involved in transporting material from the cell surface to intracellular compartments (termed clathrin-coated vesicles). There are several controversies in the field about how PICALM variants increase risk for AD. Over the last 3 years, we have generated extensive preliminary data which resolve several of these controversies. We have shown that genetic variants in PICALM increase risk both for AD and for pathological deposition of Abeta in blood vessel walls (Cerebral Amyloid Angiopathy - CAA). We have shown that the AD/CAA risk allele increases the production of PICALM protein in cells lining blood vessel walls in the brain (Cerebral Microvascular Endothelial Cells - CMECs). These cells are an important route for transporting proteins such as Abeta into/from the brain. CMECs express high levels of PICALM. We (and others) have shown that in most cells, PICALM has small effects on intracellular trafficking of the amyloid precursor protein (APP) and the presenilin complex, and on the subsequent cleavage of APP by the presenilin complex to generate neurotoxic Abeta peptide. Others have shown that PICALM also plays a role in the formation of autophagosomes, which is involved in in clearance of intracellular protein aggregates. However, the effects of PICALM on these processes are modest. Perturbations in these processes per se are therefore unlikely to account for PICALM-induced increased risk for AD/CAA. Intriguingly, we have recently shown that, modest overexpression of PICALM in cultured human CMECs (similar in degree to that seen in PICALM risk-allele carriers) can: i) dramatically increase the internalisation of Abeta; and ii) increase the subsequent re-externalisation of that Abeta as aggregation-prone seeds which then promote further Abeta aggregation in blood vessel walls. We hypothesize that in CMECs overexpressing PICALM, the increased Abeta uptake and increased subsequent re-secretion of Abeta as fibrillogenic seeds would: i) impair the Abeta across blood vessel walls; and ii) increase the tendency for Abeta aggregation in blood vessel walls. In the present proposal, we will test this hypothesis by employing well-characterised cellular models of the blood brain barrier. These models possess several features not used in prior studies that render our models more realistic models of Abeta transport than those used previously by other groups. We will use these models to work out how PICALM (and its interacting protein partners) affect Abeta trafficking in human CMECs. The experiments proposed here will help identify how PICALM affects amyloid clearance, and may provide targets for novel therapies directed at improving Abeta clearance. Finally, we will seek to acquire data from recent clinical trials of Abeta vaccines to determine whether PICALM genotypes can be used to predict the occurrence of amyloid-associated vascular events (e.g. microhaemorrhages) following treatments directed at Abeta. This proposal will: clarify the role of PICALM in modulating AD/CAA risk; may identify useful targets to manipulate transvascular Abeta clearance; and may reveal whether PICALM genotype predicts risk of complications of anti-Abeta therapies. It will lay the foundation for future work (to be separately funded) in which we will provide in vivo validation of these results.

Technical Summary

We have developed exciting data suggesting that: a) in human brain, PICALM is expressed at highest levels in Cerebral Microvascular Endothelial Cells (CMECs); b) non-coding variants in PICALM are associated with increased risk for AD and Cerebral Amyloid Angiopathy (CAA); c) the AD/CAA risk allele promotes increased PICALM expression in CMECs from human brain; d) equivalent overexpression of PICALM in cultured CMECs modulates several aspects of Abeta biology, but the strongest effect is to enhance uptake and subsequent re-secretion of Abeta as amyloid seeds. To explore the underlying biology we will: 1) Deploy well-characterised models (immortalised human CMECs and polarised, multicellular trans-well culture systems) that possess several advantages over other models; 2) Use super resolution microscopy to characterize the effect of changes in PICALM expression on the quantity and direction of secretion of Abeta seeds (brainvessel/vessel-brain); 3) define the subcellular localization of PICALM in models expressing normal, reduced or increased PICALM; 4) map changes in intracellular distribution of endocytosed Abeta in models expressing normal, reduced or increased PICALM; 5) investigate how changing PICALM expression alters the protein interactome of PICALM; 6) validate which of the changes in the PICALM interactome are necessary and sufficient to alter Abeta uptake and secretion. Our proposal builds upon our expertise in the biology of the BBB (Abbott), bioinformatics (Oliver), intracellular vesicular trafficking (CIMR collaborators), advanced imaging (Kaminski), and human functional genomics (St George-Hyslop). This work will clarify the role of PICALM and may identify potential therapeutic targets to ameliorate vascular clearance of Abeta in AD.

Planned Impact

Impact through innovation/novelty:
There are several potential beneficiaries of this research program. In the immediate timeframe of 1-5 years during the grant, the principal beneficiaries will be academic researchers working on the pathogenesis, diagnosis and treatment of AD. By understanding the role of PICALM, they will be able to: 1) better model this disease; 2) design improved diagnostics (including potential diagnostics for complications for certain types of therapies); and 3) improve therapeutics based on this knowledge. A second group of academics will be those working on blood brain barrier. If our preliminary data indicating that genetic variants in PICALM predict the presence of cerebral amyloid angiopathy is confirmed in clinical trial data, this knowledge will facilitate the design of therapeutic trials with anti-Abeta therapies (all of which are thought to increase the risk of amyloid related microvascular complications). This could lead to cheaper and improved clinical trials directed at the most appropriate genotypically defined subgroup. If these new therapies were successful, they would then bring potential benefits to the well-defined group with low risk of complications. The methods developed by the applicants represent some of the first validated assays permitting measurements of the aggregation of neurotoxic proteins at the blood brain barrier. The Kaminski group in particular has made significant advances in the development of advanced multi-parametric imaging techniques for the study of protein aggregation. With dSTORM we have shown that we obtain structural information of oligomers at a resolution down to 10 nm, in situ, in the environment where amyloids naturally occur..

Impact through collaboration:
Staff working on the project will develop skills in generating comprehensive cellular models of blood brain barrier, a skill which is widely transferable to many aspects of CNS biology including CNS pharmacology. They will also gain skills in cell biology, molecular biology and biochemistry of amyloid and amyloid angiopathy. The applicants have participated in the Neurodegenerative Disease Initiative funded by the Wellcome Trust and the MRC and in the BBB group at Kings. These collaborative work groups will increase the impact of our research. Indeed, by engaging in regular interest group meetings, we have the opportunity to communicate our findings to a multi-disciplinary group of researchers focussing on neurodegenerative diseases.

Impact for Cambridge University:
The expertise acquired through the proposed research project, of these techniques and the optimisation of protocols for sample preparation, will benefit Cambridge University. Indeed, a large number of researchers from the University may take advantage of the presence of platforms developed here in imaging and in blood brain barrier science. All technologies by this group will be made available to all researchers in the University.

Impact for Society:
By understanding the role of PICALM, they will be able to: 1) better model this disease; 2) design improved diagnostics (including potential diagnostics for complications for certain types of therapies); and 3) improve therapeutics based on this knowledge. Over 820,000 people were affected by neurodegenerative diseases in the UK in 2010. This number is forecast to increase rapidly. The annual cost of these age related diseases was over £23 billion in 2009.

Publications

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Johnson DS (2017) Structural and Chemical Biology of Presenilin Complexes. in Cold Spring Harbor perspectives in medicine

 
Description Meetings via the UK Korea Health Innivation Forum with MRC International program (Mark Palmer); UK Ambassador to South Korea; and officials from the S. Korea Ministry of Health and Welfare and Korean Head Industry Develpment Institute
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Medical Research Council
Amount £760,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 02/2016 
End 01/2019
 
Title Method for modulating PICALM expression in brain microvascular endothelial cells 
Description We have developed a method based on anti-sense oligonucleotides to knockdown PICALM expression in the endothelial cells of brain blood vessels, while leaving its expression in brain unchanged. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact We have shown that this method works, causing a 50% knockdown which lasts for many weeks (>2). We are currently assessing whether this will alter brain vascular amyloid loads. 
 
Description Antisense oligonucleotides for manipulation of PICALM expression in cultured human cerebral micro-vascular endothelial cells 
Organisation Tokyo Medical and Dental University
Country Japan 
Sector Academic/University 
PI Contribution We have used these antisense not oligonucleotides to manipulate expression of PICALM in a variety of cell culture models of the human blood brain barrier.
Collaborator Contribution Professor Yokota synthesised and sent us the PICALM antisense oligonucleotides ligated with a targeting agent that would ensure binding to vascular endothelial cells.
Impact This work is still in progress.
Start Year 2016
 
Description In vivo modelling of PICALM in modulating Abeta transfer across the blood brain barrier in a transgenic mouse model of AD 
Organisation University of Toronto
Department Tanz Centre for Research in Neurodegenerative Diseases
Country Canada 
Sector Academic/University 
PI Contribution We have provided extensive cell biology proof of principle that antisense oligonucleotides can manipulate PICALM expression in realistic blood brain barrier cell models. We have provided the antisense oligonucleotides
Collaborator Contribution Joel Watts at the University of Toronto is currently testing whether or not antisense oligonucleotides can be used therapeutically to manipulate PICALM expression in mammalian cerebral microvascular endothelial cells
Impact This work is still in progress
Start Year 2016
 
Description Neurogenetics of PICALM expression in human cerebral microvascular endothelial cells 
Organisation Columbia University
Department Department of Neurology
Country United States 
Sector Academic/University 
PI Contribution We have undertaken genotyping of DNA and RT-PCR quantification of mRNA provided by our collaborators at Columbia extracted from human post-mortem purified cerebral microvascular endothelial cells
Collaborator Contribution Our collaborators at Columbia University have a post-mortem brain tissue bank, operated under ethical approval from Columbia University and the National Institutes of Health. Collaborators have identified frozen normal (non-Alzheimer's disease) control frontal and parietal lobe brain tissue, and extracted DNA and RNA. This has allowed us then to investigate the effect of genotype at PICALM on the mRNA expression of PICALM in human cerebral microvascular endothelial cells
Impact This work is still in progress.
Start Year 2016
 
Description Neuropathology of microvascular changes in PICALM ASO treated and mock treated transgenic mouse models of human cerebrovascular amyloid 
Organisation Newcastle University
Department Institute of Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated a three-way collaboration between the original partners in the current proposal together with a collaboration with Joel Watts at the University of Toronto and with Raj Kalaria at the University of Newcastle to test and validate the utility of antisense oligonucleotides in tissue culture models (us) and then validate it in murine models (University of Toronto and University of Newcastle)
Collaborator Contribution Raj Kalaria at the University of Newcastle has applied his expertise in neuropathology to quantify the effects of PICALM antisense oligonucleotides on cerebrovascular amyloid in a mouse model of cerebral amyloid angiopathy provided by Joel Watts at the University of Toronto
Impact This work is still in progress
Start Year 2016
 
Description Conference Leibniz Institut fur Molekular Pharmakologie in Berlin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using Tau/STORM atLeibniz Institut fur Molekular Pharmakologie in Berlin 17/11/2016 invited talk
Year(s) Of Engagement Activity 2016
 
Description Talk at CNRS-Jacques Monod Conference "Protein misfolding in disease-Toxic aggregation-prone proteins in aging and age-related diseases: from structure to pathology and spreading" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using Tau/STORM at CNRS-Jacques Monod Conference "Protein misfolding in disease-Toxic aggregation-prone proteins in aging and age-related diseases: from structure to pathology and spreading" Tau/STORM 15/09/2016 invited talk
Year(s) Of Engagement Activity 2016
 
Description Episode of Elemental Ideas on Cambridge TV 25 Minute TV show about super-resolution microscopy 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Clemens Kaminski, Gabriele Kaminski Schierle, Colin Hockings, Florian Ströhl, Nathan Curry

Episode of Elemental Ideas on Cambridge TV 25 Minute TV show about super-resolution microscopy
01/07/2016 Explaining research to the public through a video of our group's lab and research work, with interviews of the two group leaders and several postdoctoral research associates and PhD students.
The videos can be accessed for free online.

Elemental Ideas is Cambridge TV's science magazine programme. They've produced more than 50 programmes since August 2015, covering research topics including string theory, diabetes, exoplanets, vulcanology, epigenetics and the wildlife of New Zealand.
Year(s) Of Engagement Activity 2016
URL http://www.cambridge-tv.co.uk/super-resolution-microscopy/
 
Description Explaining research to the public; Demonstrations to primary aged children and their parents about graphene at Graphene MEA  
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Demonstrations to primary aged children and their parents about graphene 6-7 April 2017 Explaining research to the public General Public Graphene MEA
Year(s) Of Engagement Activity 2017
 
Description Graphene Centre Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using Tau/STORM at Graphene Centre Cambridge 27/01/2017 invited talk
Year(s) Of Engagement Activity 2017
 
Description Poster at 13th International Congress on Alzheimer's and Parkinson's disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Amberley Stephens The influence of calcium on the interaction between alpha-synuclein and rat brain synaptic vesicles 29/03/17-02/04/17 13th International Congress on Alzheimer's and Parkinson's disease STED
Year(s) Of Engagement Activity 2017
 
Description Poster at 13th International Congress on Alzheimer's and Parkinson's disease  
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Colin Hockings The Role of Endocytosis in Tau Misfolded State Propagation 29/03/17-02/04/17 13th International Congress on Alzheimer's and Parkinson's disease Tau
Year(s) Of Engagement Activity 2017
 
Description Poster at 20th International Congress of Parkinson's Disease and Movement Disorders 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Janin Lautenschlaeger ALPHA-SYNUCLEIN - SUPER-RESOLUTION MICROSCOPY REVEALS POTENTIAL NEW ROLE AT THE PRE-SYNAPSE 19-23 June 2016 20th International Congress of Parkinson's Disease and Movement Disorders STORM
Year(s) Of Engagement Activity 2016
 
Description Poster at ARUK Research Conference 2016  
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Na Yu Monitoring Tau Aggregation during Neuronal Transport 8-9 March 2016 ARUK Research Conference 2016 Tau, FLIM
Year(s) Of Engagement Activity 2016
 
Description Poster at American Chemical Society Conference 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Suil Collins Identification and development of small molcule inhibitors of the aggregation of amyloid beta April 2-6th 2017 American Chemical Society Conference 2017 "AB, FLIM
"
Year(s) Of Engagement Activity 2017
 
Description Poster at Conferences Jacques-Monod - Protein misfolding disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Janin Lautenschlaeger ALPHA-SYNUCLEIN - SUPER-RESOLUTION MICROSCOPY REVEALS POTENTIAL NEW ROLE AT THE PRE-SYNAPSE 13-16 September 2016 Conferences Jacques-Monod - Protein misfolding disease STORM
Year(s) Of Engagement Activity 2016
 
Description Poster at EMBL Microfluidics Conference 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Suil Collins Identification and development of small molecule inhibitors of the aggregation of amyloid beta 24-26th July 2017 EMBL Microfluidics Conference 2016 "AB, FLIM"
Year(s) Of Engagement Activity 2017
 
Description Poster at Graphene Nownano Summer Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Philippa Hooper Graphene microelectrode array to investigate neuronal function 27-30 June Graphene Nownano Summer Conference Graphene MEA
Year(s) Of Engagement Activity 2016
 
Description Showcasing research to public in a hands on manner at the Cambridge Science Festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Cambridge Science Festival
Showcasing research to public in a hands on manner through various experiments organised in the dept of Chemistry including looking at neurons under a microscope.
Explaining the group's research to the public
Undertaken by several members of the group on a yearly basis since 2016

The Science Festival provides the public with opportunities to explore and discuss issues of scientific interest and concern and to raise aspirations by encouraging young people to consider a career in science, technology, engineering or mathematics.

Each year, the Festival welcomes visitors to hundreds of events and receives extensive national and local media coverage. Over 170 event coordinators organise talks, interactive demonstrations, hands-on activities, film showings and debates with the assistance of around 1,000 staff and students from departments and organisations across the University and research institutions, charities and industry in the eastern region. In addition, over 150 people volunteer their time to act as stewards to ensure visitors have a safe and enjoyable Festival experience.
Year(s) Of Engagement Activity 2016,2017
URL http://www.sciencefestival.cam.ac.uk/
 
Description Talk at GRC on Bioanalytical Sensors  
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using Tau/STORM at GRC on Bioanalytical Sensors 29/06/2016 Gabriele Kaminski invited talk
Year(s) Of Engagement Activity 2016
 
Description Talk 9th UK Korea Neuroscience Synposium in Seoul  
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using Tau/STORM 9th UK Korea Neuroscience Synposium in Seoul 01/09/2016 Gabriele Kaminski invited talk
Year(s) Of Engagement Activity 2016
 
Description Talk at 13th International Conference on Alzheimer's & Parkinson's 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact ALPHA-SYNUCLEIN - SUPER-RESOLUTION MICROSCOPY REVEALS POTENTIAL NEW ROLE AT THE PRE-SYNAPSE at 13th International Conference on Alzheimer's & Parkinson's STORM not yet - 30/03/2016 Janin Lautenschlaeger picked from abstract
Year(s) Of Engagement Activity 2017
 
Description Talk at ARUK 2017 national conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Molecular mechanisms of Tau aggregation and propagation at ARUK 2017 national conference Tau/storm/flim 15/03/17 Claire Michel invited talk
Year(s) Of Engagement Activity 2017
 
Description Talk at CHDI HTT Protein Lifecycle Working Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using seeding/STORM CHDI HTT Protein Lifecycle Working Group 17/03/2016 Gabriele Kaminski invited talk
Year(s) Of Engagement Activity 2016
 
Description Talk at DZNE Tuebingen  
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using Tau/STORM DZNE Tuebingen 09/06/2016 Gabriele Kaminski invited talk
Year(s) Of Engagement Activity 2016
 
Description Talk at NMI Reutlingen 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Towards a molecular understanding of amyloid propagation using super resolution microscopy and multiparametric imaging using seeding/STORM at NMI Reutlingen 15/06/2016 Gabriele Kaminski invited talk
Year(s) Of Engagement Activity 2016
 
Description UK Korea Health Innovation Forum 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Two meetings were held, one in Seoul and one in London. The purpose was to introduce UK and South Korean business and academic units working in brain aging to each other. Attendance was over-subscribed. Arrangements are being made for a follow-up meeting will be held in May 2018 in London.
Year(s) Of Engagement Activity 2017
 
Description poster at Parkinson's UK Research Conference  
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Janin Lautenschlaeger ALPHA-SYNUCLEIN - SUPER-RESOLUTION MICROSCOPY REVEALS POTENTIAL NEW ROLE AT THE PRE-SYNAPSE 7-8 November 2016 Parkinson's UK Research Conference STORM
Year(s) Of Engagement Activity 2016