Publicação
Dissecting the role of bin1 in alzheimer’s disease
| Resumo: | Bridging Integrator 1 (BIN1) is the second most significant genetic risk factor for late-onset Alzheimer’s disease. It regulates endosomal trafficking and its loss has been linked to β-amyloid (Aβ) production, endosomal dysfunction, and tau propagation in mouse neurons. However, the mechanisms by which these processes occur in human neurons are not yet fully understood. Given BIN1’s critical role at vGAT-positive inhibitory synapses, we first generated wild-type inhibitory neurons derived from human-induced pluripotent stem cells (hiPSCs) to model BIN1-driven inhibitory synaptic dysfunction. Transient expression of ASCL1-DLX2 produced a GABAergic neuronal population. These iNeurons formed synapses as early as 14 days in vitro and continued to do so throughout 45 days in vitro. Bin1 colocalized with vGAT, indicating a possible role in human inhibitory synapses. Next, we examined the BIN1-KR missense mutation by differentiating CRISPR-Cas9-edited mutant hiPSCs and isogenic unedited controls. During initial differentiation, KR iNeurons thrived, whereas control iNeurons failed to differentiate. Nonetheless, we observed notable findings: KR neurons developed inhibitory synapses, and mutant BIN1 localized to vGAT-positive synapses. Aβ42 was observed in a punctate pattern, indicating accumulation in vesicular compartments, but assessment of Aβ42 within Rab5-positive early endosomes was not possible. While this model represents an important step toward humanizing BIN1 studies, definitive conclusions about the pathogenicity of the mutation cannot yet be made. Lastly, we investigated the role of Bin1 in tau propagation via extracellular vesicles. Using pHluorin-CD63 expression, we visualized extracellular vesicle and their cargo. Bin1 knockdown did not significantly affect EV secretion or the incorporation of tau and synaptic proteins into EVs. Our developed model allows for the study of human inhibitory synapse-specific phenotypes with increased translational relevance, especially concerning BIN1 mutations linked to Alzheimer’s disease. |
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| Autores principais: | Fernandes,Ana Rafaela |
| Assunto: | Late-onset Alzheimer’s disease BIN1 iPSC-derived neurons extracellular vesicles synapse |
| Ano: | 2025 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Bridging Integrator 1 (BIN1) is the second most significant genetic risk factor for late-onset Alzheimer’s disease. It regulates endosomal trafficking and its loss has been linked to β-amyloid (Aβ) production, endosomal dysfunction, and tau propagation in mouse neurons. However, the mechanisms by which these processes occur in human neurons are not yet fully understood. Given BIN1’s critical role at vGAT-positive inhibitory synapses, we first generated wild-type inhibitory neurons derived from human-induced pluripotent stem cells (hiPSCs) to model BIN1-driven inhibitory synaptic dysfunction. Transient expression of ASCL1-DLX2 produced a GABAergic neuronal population. These iNeurons formed synapses as early as 14 days in vitro and continued to do so throughout 45 days in vitro. Bin1 colocalized with vGAT, indicating a possible role in human inhibitory synapses. Next, we examined the BIN1-KR missense mutation by differentiating CRISPR-Cas9-edited mutant hiPSCs and isogenic unedited controls. During initial differentiation, KR iNeurons thrived, whereas control iNeurons failed to differentiate. Nonetheless, we observed notable findings: KR neurons developed inhibitory synapses, and mutant BIN1 localized to vGAT-positive synapses. Aβ42 was observed in a punctate pattern, indicating accumulation in vesicular compartments, but assessment of Aβ42 within Rab5-positive early endosomes was not possible. While this model represents an important step toward humanizing BIN1 studies, definitive conclusions about the pathogenicity of the mutation cannot yet be made. Lastly, we investigated the role of Bin1 in tau propagation via extracellular vesicles. Using pHluorin-CD63 expression, we visualized extracellular vesicle and their cargo. Bin1 knockdown did not significantly affect EV secretion or the incorporation of tau and synaptic proteins into EVs. Our developed model allows for the study of human inhibitory synapse-specific phenotypes with increased translational relevance, especially concerning BIN1 mutations linked to Alzheimer’s disease. |
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