Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model
2022; Springer Nature; Volume: 15; Issue: 2 Linguagem: Inglês
10.15252/emmm.202216556
ISSN1757-4684
AutoresDaniel V. Oliveira, Kirsten Coupland, Wenchao Shao, Shaobo Jin, Francesca Del Gaudio, Sailan Wang, Rhys Fox, Julie W. Rutten, Johan Sandin, Henrik Zetterberg, Johan Lundkvist, Saskia A.J. Lesnik Oberstein, Urban Lendahl, Helena Karlström,
Tópico(s)S100 Proteins and Annexins
ResumoArticle16 December 2022Open Access Source DataTransparent process Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model Daniel V Oliveira Daniel V Oliveira orcid.org/0000-0003-0622-2934 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic Contribution: Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Kirsten G Coupland Kirsten G Coupland orcid.org/0000-0002-4320-1043 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Wenchao Shao Wenchao Shao Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Shaobo Jin Shaobo Jin orcid.org/0000-0002-9064-9246 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Francesca Del Gaudio Francesca Del Gaudio orcid.org/0000-0001-8342-1377 Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Sailan Wang Sailan Wang Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Rhys Fox Rhys Fox Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Investigation, Writing - review & editing Search for more papers by this author Julie W Rutten Julie W Rutten Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands Contribution: Methodology, Writing - review & editing Search for more papers by this author Johan Sandin Johan Sandin Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Alzecure Foundation, Huddinge, Sweden Alzecure Pharma, Huddinge, Sweden Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Henrik Zetterberg Henrik Zetterberg Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK UK Dementia Research Institute at UCL, London, UK Hong Kong Center for Neurodegenerative Diseases, Clear Water Bay, Hong Kong, China Contribution: Resources, Formal analysis, Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Johan Lundkvist Johan Lundkvist Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Alzecure Foundation, Huddinge, Sweden Sinfonia Biotherapeutics, Huddinge, Sweden Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Saskia AJ Lesnik Oberstein Saskia AJ Lesnik Oberstein orcid.org/0000-0002-1268-8995 Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands Contribution: Resources, Validation, Methodology, Writing - review & editing Search for more papers by this author Urban Lendahl Corresponding Author Urban Lendahl [email protected] orcid.org/0000-0001-9543-8141 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Helena Karlström Corresponding Author Helena Karlström [email protected] orcid.org/0000-0002-0498-2473 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Daniel V Oliveira Daniel V Oliveira orcid.org/0000-0003-0622-2934 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic Contribution: Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Kirsten G Coupland Kirsten G Coupland orcid.org/0000-0002-4320-1043 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Wenchao Shao Wenchao Shao Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Shaobo Jin Shaobo Jin orcid.org/0000-0002-9064-9246 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Francesca Del Gaudio Francesca Del Gaudio orcid.org/0000-0001-8342-1377 Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Sailan Wang Sailan Wang Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Rhys Fox Rhys Fox Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Investigation, Writing - review & editing Search for more papers by this author Julie W Rutten Julie W Rutten Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands Contribution: Methodology, Writing - review & editing Search for more papers by this author Johan Sandin Johan Sandin Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Alzecure Foundation, Huddinge, Sweden Alzecure Pharma, Huddinge, Sweden Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Henrik Zetterberg Henrik Zetterberg Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK UK Dementia Research Institute at UCL, London, UK Hong Kong Center for Neurodegenerative Diseases, Clear Water Bay, Hong Kong, China Contribution: Resources, Formal analysis, Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Johan Lundkvist Johan Lundkvist Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Alzecure Foundation, Huddinge, Sweden Sinfonia Biotherapeutics, Huddinge, Sweden Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Saskia AJ Lesnik Oberstein Saskia AJ Lesnik Oberstein orcid.org/0000-0002-1268-8995 Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands Contribution: Resources, Validation, Methodology, Writing - review & editing Search for more papers by this author Urban Lendahl Corresponding Author Urban Lendahl [email protected] orcid.org/0000-0001-9543-8141 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Helena Karlström Corresponding Author Helena Karlström [email protected] orcid.org/0000-0002-0498-2473 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Daniel V Oliveira1,2,†, Kirsten G Coupland1,†, Wenchao Shao1, Shaobo Jin1,3, Francesca Del Gaudio3, Sailan Wang1, Rhys Fox1,3, Julie W Rutten4, Johan Sandin1,5,6, Henrik Zetterberg7,8,9,10,11, Johan Lundkvist1,5,12, Saskia AJ Lesnik Oberstein4, Urban Lendahl *,1,3 and Helena Karlström *,1 1Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden 2Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic 3Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden 4Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands 5Alzecure Foundation, Huddinge, Sweden 6Alzecure Pharma, Huddinge, Sweden 7Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden 8Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden 9Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK 10UK Dementia Research Institute at UCL, London, UK 11Hong Kong Center for Neurodegenerative Diseases, Clear Water Bay, Hong Kong, China 12Sinfonia Biotherapeutics, Huddinge, Sweden † These authors contributed equally to this work as first authors *Corresponding author. Tel: +46 (0)8 524 873 23; E-mail: [email protected] *Corresponding author. Tel: +46 (0)8 524 835 48; E-mail: [email protected] EMBO Mol Med (2023)15:e16556https://doi.org/10.15252/emmm.202216556 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; no preventive or curative therapy is available. CADASIL is caused by mutations in the NOTCH3 gene, resulting in a mutated NOTCH3 receptor, with aggregation of the NOTCH3 extracellular domain (ECD) around vascular smooth muscle cells. In this study, we have developed a novel active immunization therapy specifically targeting CADASIL-like aggregated NOTCH3 ECD. Immunizing CADASIL TgN3R182C150 mice with aggregates composed of CADASIL-R133C mutated and wild-type EGF1–5 repeats for a total of 4 months resulted in a marked reduction (38–48%) in NOTCH3 deposition around brain capillaries, increased microglia activation and lowered serum levels of NOTCH3 ECD. Active immunization did not impact body weight, general behavior, the number and integrity of vascular smooth muscle cells in the retina, neuronal survival, or inflammation or the renal system, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of NOTCH3 accumulation in a CADASIL mouse model supporting further development towards clinical application for the benefit of CADASIL patients. Synopsis The disease CADASIL, an inherited stroke and dementia syndrome affecting the brain vasculature, is currently incurable and with no treatment options. We present data in a CADASIL mouse model that active immunization may represent a novel treatment strategy with no observed adverse side effects. Active immunization with a mutated NOTCH3 peptide led to reduced NOTCH3 extracellular domain deposition around brain capillaries in a transgenic CADASIL mouse model. Active immunization furthermore led to reduced levels of NOTCH3 extracellular domain in the blood. Active immunization resulted in increased numbers of activated microglial cells. No loss of vascular smooth muscle cells nor kidney pathology was observed in the immunized mice, indicating that the active immunization is tolerable from a toxicity perspective. The paper explained Problem Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a disease affecting the blood vessels of the brain. Patients suffering from CADASIL experience migraine with aura, strokes and cognitive decline. CADASIL is caused by mutations in a specific gene, NOTCH3, and is currently incurable with no therapies in clinical use. Results We have developed an active immunization approach aimed at targeting the CADASIL-associated NOTCH3 pathology and assessed efficacy in a transgenic mouse model for CADASIL. We find that active repeated immunization with a short CADASIL-mutated NOTCH3 peptide resulted in reduced deposition of the extracellular domain of the NOTCH3 receptor around the smallest vessels in the brain (capillaries) and reduced levels of NOTCH3 extracellular domain in the blood in the CADASIL mouse model. There were no signs of kidney toxicity, inflammation, neurodegeneration or loss of vascular smooth muscle cells in the vasculature in the immunized mice. Impact Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy is a dominant disease, meaning that individuals carrying a CADASIL NOTCH3 mutation will develop the disease, and the fact that there are yet no functional therapies for CADASIL fuels efforts to explore novel therapy strategies. Our finding that active immunization reduces accumulation of the NOTCH3 extracellular domain around capillaries in the brain of a CADASIL mouse model is therefore encouraging. Importantly, the finding that immunization does not alter normal Notch signaling nor lead to kidney damage indicates that active immunotherapy may be an efficacious and tolerable therapeutic strategy for CADASIL therapy development. Introduction Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL, OMIN No. 125310) is the most common monogenic form of cerebral small vessel disease (SVD), affecting approximately 5/100,000 individuals, but is in all likelihood underdiagnosed (Rutten et al, 2016). Patients suffering from CADASIL experience migraine with aura, subcortical ischemic events, mood disturbances, apathy, and cognitive impairment (Joutel et al, 1996; Chabriat et al, 2009; Coupland et al, 2018; Joutel, 2020). CADASIL results in white matter lesions, neuronal loss, and widespread vascular pathology characterized by degenerating vascular smooth muscle cells (VSMC) and thickening of the arterial wall (fibrosis), which leads to lumen stenosis, for review see Coupland et al (2018). Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy is exclusively caused by mutations in the NOTCH3 gene (Joutel et al, 1996). The NOTCH3 transmembrane receptor undergoes proteolytic cleavages upon activation by ligands presented on juxtaposed cells, ultimately releasing the Notch intracellular domain (ICD) into the interior of the cell, while the Notch extracellular domain (ECD) is shed from the cell surface (see Fig 1A for details on Notch signaling) (Andersson et al, 2011; Siebel & Lendahl, 2017; Coupland et al, 2018). Most CADASIL mutations are missense mutations confined to the 34 epidermal growth factor (EGF)-like repeats of the NOTCH3 ECD moiety (Rutten et al, 2014), resulting in an altered number of cysteine residues in the EGF-like repeats (Fig 1B; Joutel et al, 1996). The CADASIL cysteine-altering mutations perturb the structure of NOTCH3 ECD resulting in NOTCH3 ECD multimerization and aggregation. This in turn leads to the recruitment of microvascular extracellular matrix proteins including metalloproteases and vitronectin, that form the so called granular osmiophilic dense material, GOM, a histopathological hallmark of CADASIL (Karlstrom et al, 2002; Duering et al, 2011; Monet-Lepretre et al, 2013; Capone et al, 2016; Fig 1B). NOTCH3 ECD accumulation is one of the earliest events in CADASIL pathogenesis, indicating that it may cause cellular pathology by inducing changes in the brain microvascular extracellular matrix (Joutel et al, 2001, 2010; Monet-Lepretre et al, 2013; Capone et al, 2016). Collectively, these data argue that CADASIL may be considered a protein misfolding and aggregation disease. Figure 1. Schematic representation of Notch signaling and Notch3 CADASIL mutations Schematic representation of Notch signaling. (1) Furin (S1 cleavage) cleaves the NOTCH3 precursor protein in the Golgi system, resulting in a non-covalently bound bipartite protein that is transported to the cell surface. (2) A mechanical traction force is applied to the NOTCH3 ECD when a Notch ligand binds to the EGF repeats 10–11, exposing the extracellular NRR near the cell membrane, which consists of LNR and the heterodimerization domain (in green). Subsequently, ADAM17 cleaves the C-terminal portion of the heterodimerization domain (S2-cleavage). (3) The NEXT, which is made up of a RAM domain, the ANK domains, a PEST domain, and a transmembrane domain, is cleaved by the γ-secretase (S3-cleavage) releasing the N3ICD. (4) The N3ICD binds to the CSL protein and together with the co-activator Mastermind-like (MAM) trigger downstream gene transcription in the nucleus. (5) The NOTCH3 ECD and ligand are normally endocytosed by the ligand-expressing cell and degraded in the lysosome. Schematic representation of NOTCH3 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) mutations. NOTCH3 ECD contains 34 EGF repeat domains, each of which has six cysteine residues (WT). Mutations in CADASIL change the number of cysteines to an uneven number of cysteines (Mutant). These unpaired cysteines residues result in incorrect EGF repeat folding, irregular protein folding which leads to an enhanced NOTCH3 ECD multimerization. Distribution of the cysteine-altering mutations that cause CADASIL are shown. In the CADASIL mutant NOTCH3 ECD, the endocytosis is hampered, and NOTCH ECD remains outside of the VSMC and starts to accumulate and aggregate around the vessels. ADAM17, a disintegrin and metalloproteinase domain-containing protein 17; ANK, ankyrin repeats; EGF, epidermal growth factor; HD, heterodimerization domain; LNR, Lin-Notch repeats; PEST, proline (P), glutamic acid (E), serine (S), and threonine (T) degradation domain; RAM, Rbp-associated molecule domain; TM, transmembrane domain. Download figure Download PowerPoint There are currently no therapies to abrogate or ameliorate the disease process for CADASIL patients but given that NOTCH3 accumulation is a hallmark of the disease, immunotherapy targeting NOTCH3 aggregation or aggregates may be an attractive therapeutic strategy. Immunotherapeutic approaches are gaining increasing attention as novel disease modifying therapies for protein misfolding and aggregation diseases such as certain neurodegenerative diseases (Forman et al, 2004; Shrivastava et al, 2017). Promising preclinical and clinical results have been obtained in particular for the clearance of amyloid beta (Aβ) deposits (the so called "senile plaques") in Alzheimer's disease (AD). Therapies based on both active and passive immunization can clear amyloid in the brain with great efficiency in preclinical mouse models of Aβ amyloidosis as well as in patients. Recently, a number of different monoclonal antibodies targeting Aβ aggregates have demonstrated promising clinical improvement in association with amyloid clearance in Phase II and III clinical trials with aducanumab recently being approved for treatment of AD by the Food and Drug Association (Budd Haeberlein et al, 2017; Tolar et al, 2020; Mintun et al, 2021). Although the efficacy of the compound has been questioned and challenged (Knopman et al, 2021), this passive vaccine is still of interest not only for the AD field but also encourages development of immunotherapies for other disorders where protein misfolding and aggregation are believed to play a pivotal pathological role. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy shares a number of features with AD, including that both are considered protein misfolding and aggregation diseases and that both NOTCH3 and Aβ aggregates accumulate in the extracellular milieu. It is thus conceivable that NOTCH3 aggregates are accessible to efficient antibody-mediated clearance, as has been demonstrated for Aβ immunotherapies. Thus far, passive immunization has been explored in pre-clinical CADASIL mouse models with potentially encouraging results (Machuca-Parra et al, 2017; Ghezali et al, 2018), including ameliorative effects on cerebrovascular dysfunctions such as impaired blood flow and myogenic tone, although no attenuation of NOTCH3 ECD or GOM deposition was observed (Ghezali et al, 2018). To directly target the NOTCH3 ECD aggregation, it may therefore be interesting to explore an active immunization strategy, a therapeutic strategy which has been proven safe in several AD trials with different vaccines targeting Aβ (Vandenberghe et al, 2017; Novak et al, 2019; Rosenberg & Lambracht-Washington, 2020). Active immunization may be particularly appealing as a treatment for CADASIL for a number of reasons. First, the NOTCH3 deposits are located in the vessel walls and thus readily accessible to the circulating humoral immune defense enabling efficient NOTCH3 aggregate targeting. Second, given the dominant nature and high penetrance of the NOTCH3 mutations, a large number of CADASIL patients and CADASIL mutation carriers could be identified at a young age, when an active immunization has a greater potential to mount an efficient immune response. Finally, CADASIL is a chronic life-long disease and vaccine injections restricted to a few times yearly, as opposed to monthly or even more frequently, which could be the case with passive vaccines, would be advantageous from a patient perspective. In this study, we report on the development of an active immunization therapy aimed at targeting the CADASIL-associated NOTCH3 pathology as a novel disease-modifying therapy for the treatment of CADASIL. We take advantage of a CADASIL mouse model (TgN3R182C150), which expresses a human NOTCH3 R182C receptor and which develops a progressive cerebrovascular NOTCH3 ECD and GOM deposition phenotype in arterioles (Rutten et al, 2015), and thus is a suitable preclinical model for the development of therapies targeting CADASIL-associated NOTCH3 pathology. We find that active repeated immunization starting at 3 months of age results in reduced NOTCH3 ECD deposition around capillaries and lowered levels of NOTCH3 ECD in the blood at 7 months of age, which was the end point of the experiments. No loss of VSMC in the retina, and no inflammation, neuronal or kidney damage was observed after NOTCH3 immunization, indicating that the active immunotherapy does not alter normal Notch signaling. Collectively, the data suggest that active immunization that specifically targets aggregated NOTCH3 may be an efficacious and tolerable therapeutic strategy for CADASIL therapy development. Results Production of recombinant N3 EGF1–5 antigen for active immunization To perform active immunization in the CADASIL TgN3R182C150 mouse model, we first generated a suitable antigen, that would be selective for aggregated NOTCH3 and spare monomeric NOTCH3 receptors, potentially minimizing adverse effects on NOTCH3 signaling. To this end, we produced an immunogen based on aggregates of the NOTCH3 EGF repeat (EGF1–5), the part of NOTCH3 which contains the majority of all CADASIL-causing mutations identified to date. Although using a mouse model carrying the NOTCH3 R182C mutation, we opted for another cysteine-altering mutation (NOTCH3 R133C) for generation of the aggregated antigen, since this protein previously has been shown to generate aggregates and represents a mutation in the "hot-spot" region in the NOTCH3 protein (EGF1–6), but also to determine whether an aggregation-general rather than a mutation-specific therapy could be efficacious. By mixing the NOTCH3 EGF1–5 R133C with wild-type (WT) NOTCH3 EGF1–5, we obtained excellent in vitro-aggregation, in line with previous reports (Opherk et al, 2009; Duering et al, 2011), which would increase the chances of producing an immunogenic aggregate. From stable cells lines producing NOTCH3 EGF1–5 from either WT or NOTCH3R133C, we purified poly-histidine- and c-Myc-tagged NOTCH3 EGF1–5 WT or NOTCH3R133C fragments (Duering et al, 2011) from cell culture medium by metal ion affinity chromatography capturing the poly-histidine tag (Fig 2A). This resulted in a yield of approximately 90% of the total protein content in the aggregated form (Fig 2B). We next explored the potential of the NOTCH3 EGF1–5 peptides to multimerize and produce an amorphous aggregate (Duering et al, 2011) by incubating equal amounts of WT with R133C fragments or the WT and R133C fragments separately at 37°C for 5 days. Spontaneous multimer aggregation increased with time of incubation, which is in line with previous observations (Opherk et al, 2009; Duering et al, 2011), with a prominent loss of monomeric NOTCH3 EGF1–5 when WT and R133C fragments were mixed when compared with incubating them separately (Fig 2C). Aggregates from mixed WT and R133C (WT/R133C aggregates) were therefore selected for the active immunization experiments. Figure 2. Schematic representation of NOTCH3 and NOTCH3 EGF1–5 proteins and NOTCH3 EGF1–5 antigen purification Schematic representation of NOTCH3 and NOTCH3 EGF1–5. NOTCH3 represents the full-length protein, and NOTCH3 EGF1–5 represents the NOTCH3 protein with exon 1–5 fused with a myc-His-Tag at the C-terminus used for purification of the aggregated protein. Western blot of the NOTCH3 EGF1–5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α-myc antibody. Western blot of NOTCH3 EGF1–5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF1–5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were visualized after 1–5 days incubation by western blot using α-myc antibody under non-reducing conditions. Source data are available online for this figure. Source Data for Figure 2 [emmm202216556-sup-0003-SDataFig2.zip] Download figure Download PowerPoint N3 EGF1–5 antigen evokes a robust immune response in TgN3R182C150 mice We next assessed the immunogenic potential of the aggregated NOTCH3 EGF1–5 peptides and whether they could prevent NOTCH3 ECD aggregation. Active vaccination was initiated at 3 months of age, 2 months before NOTCH3 protein accumulation is observed in the TgN3R182C150 mice (Rutten et al, 2015). Aggregated NOTCH3 EGF1–5 WT/R133C protein plus adjuvant (with PBS plus adjuvant as sham-immunization control) was used to immunize TgN3R182C150 mice at 3 months of age (Fig 3A; Kontsekova et al, 2014). A booster shot containing aggregated protein plus adjuvant, or PBS plus adjuvant as control, was administered 1 month later. Two weeks later, another booster shot containing only NOTCH3 EGF1–5 WT/R133C protein or PBS was injected, and further booster shots were administered every 2 weeks until 7 months of age, which was the end point of the analysis (Fig 3A; Kontsekova et al, 2014). Using a NOTCH3 EGF1–5 aggregate ELISA, we assessed the immune response in the serum
Referência(s)