Periodic Reporting for period 1 - TSPAN14-AD (Functional analysis of TSPAN14 as a genetic risk factor for Alzheimer’s disease)
Reporting period: 2023-05-01 to 2025-04-30
Alzheimer’s disease (AD) is the most prevalent form of dementia and a leading cause of disability and death in older adults. In Europe alone, it currently affects more than 14 million people — a number projected to rise significantly as the population ages — making AD not only a pressing medical challenge, but also a growing social and economic crisis. Despite major advances in research, there is still no effective treatment to prevent, halt, or reverse the course of the disease. One of the defining biological features of AD is the accumulation of toxic amyloid-beta plaques in the brain, which are thought to interfere with communication between brain cells and trigger damaging inflammation. While reducing amyloid-beta levels has been a central goal of therapy development, it remains unclear whether this alone is sufficient to improve symptoms or slow disease progression.
Recent advances in human genetics have brought new insights into the molecular underpinnings of AD. Large-scale genome-wide association studies (GWAS) have identified novel genetic risk factors, many of which influence the function of microglia—the brain’s resident immune cells responsible for clearing toxic proteins such as amyloid-beta and other cellular waste, thereby maintaining neural health. Among these novel genetic risk factors is TSPAN14, a gene whose increased expression has been associated with a higher risk of developing AD. Interestingly, certain naturally occurring forms of the gene, which lead to lower production of the TSPAN14 protein and therefore reduce its activity, appear to protect against the disease. However, the biological mechanisms behind these genetic links remain largely unexplored.
The TSPAN14-AD project, "Functional analysis of TSPAN14 as a genetic risk factor for Alzheimer's disease", was designed to address this gap in our knowledge. The aim of the project is to gain a better understanding of the physiological role of TSPAN14 in the brain and how it may contribute to the development of AD. Specifically, the project focuses on TSPAN14’s role in microglia and their activity. It investigates how TSPAN14 influences ADAM10, a protease involved in cleaving multiple transmembrane proteins in the brain, including the AD-related proteins APP and TREM2. Emerging evidence suggests that TSPAN14 directs ADAM10’s activity in a cell-type-specific manner — for example, by potentially altering TREM2 cleavage in microglia. Since TREM2 plays a critical role in microglial function and immune response, its improper regulation may impair the brain’s ability to clear toxic proteins, thereby accelerating disease progression.
The central hypothesis of the TSPAN14-AD project is that TSPAN14 contributes to AD by altering ADAM10-mediated cleavage of TREM2, ultimately disrupting microglial function and the brain’s ability to remove amyloid-beta plaques. The project follows three key objectives:
1) To confirm GWAS findings that AD-protective TSPAN14 isoforms formed through alternative splicing mechanisms result in reduced TSPAN14 expression
2) To determine whether reduced TSPAN14 expression affects the surface expression of ADAM10 in microglia
3) To assess whether reduced TSPAN14 expression decreases TREM2 cleavage thereby enhancing the phagocytic function of microglia
By dissecting how TSPAN14 contributes to AD at the molecular level, this research aims to uncover new potential targets for treatment that go beyond traditional plaque-based approaches. The findings could pave the way for more precise, cell-type-specific interventions aimed at improving microglial function and slowing or preventing the onset of AD. This project contributes to the broader EU efforts to address major health challenges by advancing neurodegenerative disease research. It also highlights the growing importance of integrating genetics, cellular neuroscience, and immune system research, helping shape future strategies for AD prevention and treatment.
Recent advances in human genetics have brought new insights into the molecular underpinnings of AD. Large-scale genome-wide association studies (GWAS) have identified novel genetic risk factors, many of which influence the function of microglia—the brain’s resident immune cells responsible for clearing toxic proteins such as amyloid-beta and other cellular waste, thereby maintaining neural health. Among these novel genetic risk factors is TSPAN14, a gene whose increased expression has been associated with a higher risk of developing AD. Interestingly, certain naturally occurring forms of the gene, which lead to lower production of the TSPAN14 protein and therefore reduce its activity, appear to protect against the disease. However, the biological mechanisms behind these genetic links remain largely unexplored.
The TSPAN14-AD project, "Functional analysis of TSPAN14 as a genetic risk factor for Alzheimer's disease", was designed to address this gap in our knowledge. The aim of the project is to gain a better understanding of the physiological role of TSPAN14 in the brain and how it may contribute to the development of AD. Specifically, the project focuses on TSPAN14’s role in microglia and their activity. It investigates how TSPAN14 influences ADAM10, a protease involved in cleaving multiple transmembrane proteins in the brain, including the AD-related proteins APP and TREM2. Emerging evidence suggests that TSPAN14 directs ADAM10’s activity in a cell-type-specific manner — for example, by potentially altering TREM2 cleavage in microglia. Since TREM2 plays a critical role in microglial function and immune response, its improper regulation may impair the brain’s ability to clear toxic proteins, thereby accelerating disease progression.
The central hypothesis of the TSPAN14-AD project is that TSPAN14 contributes to AD by altering ADAM10-mediated cleavage of TREM2, ultimately disrupting microglial function and the brain’s ability to remove amyloid-beta plaques. The project follows three key objectives:
1) To confirm GWAS findings that AD-protective TSPAN14 isoforms formed through alternative splicing mechanisms result in reduced TSPAN14 expression
2) To determine whether reduced TSPAN14 expression affects the surface expression of ADAM10 in microglia
3) To assess whether reduced TSPAN14 expression decreases TREM2 cleavage thereby enhancing the phagocytic function of microglia
By dissecting how TSPAN14 contributes to AD at the molecular level, this research aims to uncover new potential targets for treatment that go beyond traditional plaque-based approaches. The findings could pave the way for more precise, cell-type-specific interventions aimed at improving microglial function and slowing or preventing the onset of AD. This project contributes to the broader EU efforts to address major health challenges by advancing neurodegenerative disease research. It also highlights the growing importance of integrating genetics, cellular neuroscience, and immune system research, helping shape future strategies for AD prevention and treatment.
Research on TSPAN14 research has been limited by a lack of specific tools, prompting us to develop a novel antibody against TSPAN14. This antibody reliably detects endogenous TSPAN14 via Western blot and immunoprecipitation but is currently unsuitable for immunofluorescence in brain tissue; development of additional antibodies for this purpose is ongoing. We also generated TSPAN14 knockout (KO) cell lines using CRISPR/Cas9 gene editing in human induced pluripotent stem cells (iPSCs) and mammalian cell lines, including mouse BV2 microglia and human HEK293T cells. The newly developed TSPAN14 antibody validated the absence of TSPAN14 protein in these KO lines, providing critical tools for downstream functional studies.
Compelling evidence from a recent GWAS study indicates that a SNP in TSPAN14, which induces alternative/cryptic splicing of TSPAN14, is linked to reduced AD risk. This splicing reduces the production of full-length TSPAN14 protein, a hypothesis supported by cDNA sequencing of patient-derived lymphoblastoid cell lines (LCLs) and brain tissues (frontal cortex and hippocampus) from AD patients and controls. Using our TSPAN14 antibody, we confirmed that the protective allele correlates with reduced full-length TSPAN14 protein expression, achieving a key milestone in work package 1 (WP1).
WP2 focused on TSPAN14’s regulation of ADAM10 in Alzheimer’s disease-relevant cells, primarily microglia, which exhibit high TSPAN14 expression relative to other brain cell types. We confirmed the interaction between TSPAN14 and ADAM10 using co-immunoprecipitation assays in mammalian cells and are currently working on demonstrating this in iPSC-derived microglia. In TSPAN14 KO iPSC microglia, Western blot analysis showed no alterations in ADAM10 maturation, as determined by the ratio of pro:mature forms of ADAM10. To evaluate mature ADAM10 at the cell surface, we used cell surface biotinylation with streptavidin pulldown and flow cytometry-based cell surface staining. Additionally, untargeted surface proteomics via SUSPECS revealed alterations in ADAM10 and other surface proteins in TSPAN14 KO iPSC microglia.
WP3 aimed to assess whether loss of TSPAN14 reduces ADAM10-mediated TREM2 cleavage, thus impacting microglial phagocytosis. Using TSPAN14 KO iPSC microglia, we measured soluble TREM2 (sTREM2) in conditioned media via ELISA and confirmed findings with untargeted secretome profiling (hiSPECS). Increased total TREM2 —which was measured by Western blot and proteomics— alongside decreased sTREM2, indicated reduced ADAM10-mediated cleavage. We similarly examined ADAM10-mediated cleavage of APP into soluble APP (sAPP) in TSPAN14 KO iPSC microglia, providing insight into TSPAN14’s broader role in AD pathology. Microglial activation changes were probed via proteomics of disease-associated microglia (DAM) markers. Additionally, collaborative work allowed the analysis of sTREM2 and sAPP in 130+ human CSF samples genotyped for the TSPAN14 SNP rs6586028, exploring genotype-dependent effects on protein processing.
Compelling evidence from a recent GWAS study indicates that a SNP in TSPAN14, which induces alternative/cryptic splicing of TSPAN14, is linked to reduced AD risk. This splicing reduces the production of full-length TSPAN14 protein, a hypothesis supported by cDNA sequencing of patient-derived lymphoblastoid cell lines (LCLs) and brain tissues (frontal cortex and hippocampus) from AD patients and controls. Using our TSPAN14 antibody, we confirmed that the protective allele correlates with reduced full-length TSPAN14 protein expression, achieving a key milestone in work package 1 (WP1).
WP2 focused on TSPAN14’s regulation of ADAM10 in Alzheimer’s disease-relevant cells, primarily microglia, which exhibit high TSPAN14 expression relative to other brain cell types. We confirmed the interaction between TSPAN14 and ADAM10 using co-immunoprecipitation assays in mammalian cells and are currently working on demonstrating this in iPSC-derived microglia. In TSPAN14 KO iPSC microglia, Western blot analysis showed no alterations in ADAM10 maturation, as determined by the ratio of pro:mature forms of ADAM10. To evaluate mature ADAM10 at the cell surface, we used cell surface biotinylation with streptavidin pulldown and flow cytometry-based cell surface staining. Additionally, untargeted surface proteomics via SUSPECS revealed alterations in ADAM10 and other surface proteins in TSPAN14 KO iPSC microglia.
WP3 aimed to assess whether loss of TSPAN14 reduces ADAM10-mediated TREM2 cleavage, thus impacting microglial phagocytosis. Using TSPAN14 KO iPSC microglia, we measured soluble TREM2 (sTREM2) in conditioned media via ELISA and confirmed findings with untargeted secretome profiling (hiSPECS). Increased total TREM2 —which was measured by Western blot and proteomics— alongside decreased sTREM2, indicated reduced ADAM10-mediated cleavage. We similarly examined ADAM10-mediated cleavage of APP into soluble APP (sAPP) in TSPAN14 KO iPSC microglia, providing insight into TSPAN14’s broader role in AD pathology. Microglial activation changes were probed via proteomics of disease-associated microglia (DAM) markers. Additionally, collaborative work allowed the analysis of sTREM2 and sAPP in 130+ human CSF samples genotyped for the TSPAN14 SNP rs6586028, exploring genotype-dependent effects on protein processing.
The TSPAN14-AD project led to the development and application of advanced cellular models and tools, and it has delivered early but promising results in Alzheimer’s disease-related research. Although the data remains preliminary and is not yet published, initial findings indicate potential to advance understanding of disease mechanisms and therapeutic response, going beyond current methodologies.
Overview of Preliminary Results
1) Development of Enhanced Cellular Models for Alzheimer’s disease:
Novel in vitro cellular models for the study of the newly-identified AD risk gene, TSPAN14, have been successfully developed and thoroughly characterized. These models incorporate TSPAN14 KO human iPSC lines, which can be used to generate AD-relevant cell types such as human iPSC microglia, cortical neurons, and even co-cultures or 3D brain models.
2) Potential Identification of Alzheimer’s Disease-Contributing Mechanisms:
Using exploratory omics analyses (e.g. secretomics, surfaceomics, lysate proteomics) and confirmation by orthogonal methods (ELISA, cell surface staining, Western blot), the TSPAN14-AD project has helped elucidate the role of TSPAN14 in microglia as well as its potential to regulate ADAM10-mediated TREM2 proteolysis and thus microglia function.
Indicative Impacts (Pending Validation)
1) Scientific Advancement:
The TSPAN14 KO human iPSC models offer new tools for studying TSPAN14-mediated Alzheimer’s disease relevant mechanisms.
2) Translational Potential:
If validated, findings may support the development of TSPAN14 as a therapeutic target relevant to early intervention in AD.
Key Needs to Ensure Uptake and Success
To build on these initial findings and achieve lasting impact, the following key needs are identified:
1) Further Research and Validation:
Extensive validation of TSPAN14-mediated ADAM10 substrate selectivity in human microglia cells is needed.
2) Peer-Reviewed Publication:
Dissemination of findings through high-impact journals will be essential to gain visibility and credibility in the AD research community.
Overview of Preliminary Results
1) Development of Enhanced Cellular Models for Alzheimer’s disease:
Novel in vitro cellular models for the study of the newly-identified AD risk gene, TSPAN14, have been successfully developed and thoroughly characterized. These models incorporate TSPAN14 KO human iPSC lines, which can be used to generate AD-relevant cell types such as human iPSC microglia, cortical neurons, and even co-cultures or 3D brain models.
2) Potential Identification of Alzheimer’s Disease-Contributing Mechanisms:
Using exploratory omics analyses (e.g. secretomics, surfaceomics, lysate proteomics) and confirmation by orthogonal methods (ELISA, cell surface staining, Western blot), the TSPAN14-AD project has helped elucidate the role of TSPAN14 in microglia as well as its potential to regulate ADAM10-mediated TREM2 proteolysis and thus microglia function.
Indicative Impacts (Pending Validation)
1) Scientific Advancement:
The TSPAN14 KO human iPSC models offer new tools for studying TSPAN14-mediated Alzheimer’s disease relevant mechanisms.
2) Translational Potential:
If validated, findings may support the development of TSPAN14 as a therapeutic target relevant to early intervention in AD.
Key Needs to Ensure Uptake and Success
To build on these initial findings and achieve lasting impact, the following key needs are identified:
1) Further Research and Validation:
Extensive validation of TSPAN14-mediated ADAM10 substrate selectivity in human microglia cells is needed.
2) Peer-Reviewed Publication:
Dissemination of findings through high-impact journals will be essential to gain visibility and credibility in the AD research community.