º£½ÇÆƽâ°æ

Alzheimer¡¯s disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined iPSC-derived neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding disease pathogenesis.

It is advantageous to culture the ex?vivo retina and other tissues at the air-liquid interface to allow for more efficient gas exchange. However, gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery, but typically requires submergence in liquid buffer for electrical current flow. We have developed a submergence-free electroporation technique that incorporates an agarose hydrogel disk between the positive electrode and retina. Inner retinal neurons and M¨¹ller glia are transfected with increased propensity toward M¨¹ller glia transfection after extended time in culture. We also observed an increase in BrdU incorporation in M¨¹ller glia following electrical stimulation, and variation in detection of transfected cells from expression vectors with different promoters. This method advances our ability to use ex?vivo retinal tissue for genetic studies and should be adaptable for other tissues cultured at an air-liquid interface. Subject areas: Genetic engineering, Methodology in biological sciences, Bioelectrical engineering

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study, we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures, including upregulation of inflammatory chemokines, lack of neuronal apoptosis, and axonal transmission of viruses in neuronal networks. In addition, we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies, which could lead to improved control methods.