Hand, Foot, and Mouth Disease (HFMD) is a viral illness caused by enterovirus infections. While the introduction of the enterovirus 71 (EV71) vaccine has significantly reduced the number of EV71-related cases, the continued spread of Coxsackievirus A16 (CVA16) remains a major public health threat. Previous studies have shown that human SCARB2 (hSCARB2) knock-in (KI) mice, generated using embryonic stem cell (ESC) technology, are susceptible to CVA16. However, these models have failed to reproduce the clinical pathology and neurotoxicity after CVA16 infection. Therefore, there is an urgent need for a more reliable and effective animal model to study CVA16. In this study, we successfully created a hSCARB2 KI mouse model targeting the ROSA26 locus using CRISPR/Cas9 gene editing technology. The application of CRISPR/Cas9 enabled stable and widespread expression of hSCARB2 in the model. After infection, the KI mice exhibited a clinical pathology that closely mimics human infection, with prominent limb weakness and paralysis. The virus was detectable in multiple major organs of the mice, with peak viral load observed on day 7 post-infection, gradually clearing thereafter. Further analysis revealed widespread neuronal necrosis and infiltration of inflammatory cells in the brain and spinal cord of the KI mice. Additionally, significant activation of astrocytes (GFAP-positive) and microglia (IBA1-positive) was observed in the brain, suggesting that CVA16 infection may induce limb paralysis by attacking neuronal cells. Overall, this model effectively replicates the neuropathological changes induced by CVA16 infection and provides a potential experimental platform for studying CVA16-associated pathogenesis and neurotoxicity.

Objectives: This study aimed to establish a transgenic mouse model expressing nucleus-localized human α-synuclein (α-syn) to investigate its impact on the central nervous system and behavior and the underlying mechanisms involved. Methods: A nuclear localization sequence (NLS) was added to the end of the human SNCA (hSNCA) gene. Subsequently, an empty vector and a mammalian lentiviral vector of the hSNCA-NLS were constructed. Transgenic mice were generated via microinjection, with genotyping and protein expression confirmed by PCR and western blotting. Only male mice were used in subsequent behavioral and molecular experiments. Immunofluorescence identified the colocalization of human α-syn with the cell nucleus in mouse brain tissues. Behavioral changes in transgenic mice were assessed using open field, rotarod, and O-maze tests. qPCR and Western blotting detected expression levels of genes and proteins related to inflammation, endoplasmic reticulum stress (ERS), and apoptosis. Bulk RNA sequencing was used to screen for differentially expressed genes and signaling pathways. Results: We successfully constructed a transgenic mouse model expressing human α-syn. Human α-syn was widely expressed in the heart, liver, spleen, kidneys, and brain of the mice, with distinct nuclear localization observed. Behavioral assessments demonstrated that, by 2 months of age, the mice exhibited motor dysfunction alongside astrocyte proliferation and neuroinflammation. At 6 months, the elevated expression of ERS-related genes (ATF6, PERK, and IRE1) and activation of the PERK-Beclin1-LC3II pathway indicated progressive ERS. By 9 months, apoptotic events had occurred, accompanied by significant anxiety-like behaviors. Bulk RNA sequencing further identified key differentially expressed genes, including IL-1α, TNF, PERK, BECLIN, GABA, IL-6α, P53, LC3II, NOS, and SPAG, suggesting their involvement in the observed pathological and behavioral phenotypes. Conclusions: The nuclear localization human α-syn transgenic mice were successfully established. These findings demonstrate that nucleus-localized α-syn induces early motor deficits, which are likely mediated by neuroinflammation, whereas later anxiety-like behaviors may result from ERS-induced apoptosis. This model provides a valuable tool for elucidating the role of nuclear α-syn in Parkinson’s disease and supports further mechanistic and therapeutic research.