Aging-Accelerated Differential Production And Aggregation Of STAT3 Protein In Neuronal Cells And Neural Stem Cells In The Male Mouse Spinal Cord

Background Spinal cord aging undergoes significant structural and functional changes associated with neuronal cell loss,inflammation,and proteasome inhibition.The cellular composition of the spinal cord affected by senescence is diverse and regionally specific.The signaling sensor and activator of signal transducer and activator of transcription 3(STAT3)is a major activator of immunosuppressive cells that counteracts the chronic low-grade inflammation associated with the aging process.Neural stem cells(NSC)contribute to learning,memory,maintenance of homeostasis,energy metabolism and many other important processes in vivo.STAT3 plays an important role in the maintenance of NSCs and their differentiation,and NSCs have a specific response to aging and may have potential anti-aging effects.Abnormal protein folding leads to the accumulation of proteins within the cell,causing the cell to release toxic substances,which can cause damage to neurons.Dysregulated protein homeostasis is one of the hallmarks of aging,which is caused by protein accumulation due to ubiquitin(Ub)-proteasome system(UPS)and autophagy dysfunction.In addition to folding into their natural native state according to their own amino acid sequence,proteins can also form non-naturally folded intermediates in a low free energy manner,which may lead to the formation of protein aggregates.It has been well investigated that Ub-48 lysine(lysine 48,K48)and Ub-63 lysine(lysine 63,K63)linked polyubiquitin chains drive proteasomal degradation and regulate different cellular signaling events,respectively.Ub-K48 polyubiquitin chain is the most abundant link in the cell and is considered to be the main signal for proteasomalmediated degradation,and Ub-K63 polyubiquitin chain is also abundant in the cell and may provide an alternative function for proteasomal-mediated degradation,but ubiquitination of K63 chains does not induce proteasome-dependent degradation,but rather serves as a molecular platform for protein/protein interactions.Several studies have shown that aging leads to the accumulation of abnormal protein aggregates and dysregulation of proteostasis.Therefore understanding the molecular mechanisms of spinal cord aging is crucial for scientists to discover new rejuvenation therapies.Objectives In this study,we examined STAT3,Tuj1,Ub-K48,Ub-K63 and BAF45 D in spinal cord neural stem cells and neuronal cells during spinal cord senescence to elucidate the changes in STAT3 expression and aggregation and its association with ubiquitin linkage in spinal cord senescence,and to identify potential pathogenic factors and mechanisms associated with spinal cord senescence and the role of neural stem cells in spinal cord senescence This will provide a new experimental basis for the discovery of potential pathogenic factors and mechanisms associated with spinal cord aging and the role of neural stem cells in spinal cord aging.Methods Two-month-old,13-month-old and 21-month-old C57BL/6J male mice were selected as the experimental subjects for this study.Six 2-month-old mice,eight 13-month-old mice,and eight 21-month-old mice were randomly divided into two groups for immunoprotein blotting and immunohistochemistry and immunofluorescence experiments,respectively.Mice used for immunoprotein blotting experiments were only perfused with saline for heart perfusion,and then spinal cord proteins were extracted to detect the expression of STAT3 and Tuj1,and GAPDH was used as an internal reference.Mice used for immunohistochemistry and immunofluorescence experiments were first perfused with saline for cardiac perfusion,then paraformaldehyde was fixed,and the tissue was dehydrated and transparent and then sectioned with paraffin wax embedding.The expression of P16,STAT3 and Tuj1 in spinal cord sections was detected using the SP method,and PBS was used as a negative control.Immunofluorescence double-labeling was used to observe the expression,localization and co-localization of STAT3 and Ub-K48 and Ub-K63.Results1.The expression of P16 protein in the spinal cord of 13-month-old and 21-monthold male mice is significantly more than that of 2-month-old mice,and P16 protein is concentrated in the anterior horn and central canal regions of the spinal cord.2.In the spinal cord of male mice,STAT3 expression increases significantly with aging,while Tuj1 expression decreases significantly with aging,indicating an age-dependent increase in STAT3 and decrease in Tuj1.3.Immunofluorescence double-labeling results shows that Ub-K48 and Ub-K63 ubiquitin aggregates are seen in the spinal cords of aging male mice,and colocalized with STAT3 protein.4.Ub-K48 and Ub-K63 linkage formation of STAT3-ubiquitin aggregates is significantly increased in the aging spinal cord,but not in the neural stem cells of the central canal of the spinal cord.It indicates that STAT3 and its regionspecific aggregation and ubiquitin binding increase during spinal cord aging.5.Immunofluorescence double-labeling showes co-localization of BAF45 D with STAT3,a phenomenon suggesting that BAF45 D may be involved in the aggregate formation process.Conclusion In spinal cord tissue,aging causes an increase in STAT3 protein and the production of aggregates formed by STAT3 with ubiquitin K48 and K63,and the above phenomenon is more pronounced in neural stem cells compared with spinal cord neuronal cells,suggesting that neural stem cells may have a better tolerance to aging compared with mature neuronal cells.