Winnok H. De Vos

Long considered to fluctuate between pro‐ and anti‐inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18‐seeded P301L) and an accelerated aging model (Senescence‐Accelerated Mouse‐Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18‐seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease‐associated microglia. At the cell population‐level, we observed a marked upregulation of the calprotectin‐encoding genes S100a8 and S100a9 . In 9‐month‐old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease‐associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8‐positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8‐positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.

Photoporation is an up-and-coming technology for the gentle and efficient transfection of cells. Inherent to the application of photoporation is the optimization of several process parameters, such as laser fluence and sensitizing particle concentration, which is typically done one factor at a time (OFAT). However, this approach is tedious and runs the risk of missing a global optimum. Therefore, in this study, we explored whether response surface methodology (RSM) would allow for more efficient optimization of the photoporation procedure. As a case study, FITC-dextran molecules of 500 kDa were delivered to RAW264.7 mouse macrophage-like cells, making use of polydopamine nanoparticles (PDNPs) as photoporation sensitizers. Parameters that were varied to obtain an optimal delivery yield were PDNP size, PDNP concentration and laser fluence. Two established RSM designs were compared: the central composite design and the Box-Behnken design. Model fitting was followed by statistical assessment, validation, and response surface analysis. Both designs successfully identified a delivery yield optimum five- to eight-fold more efficiently than when using OFAT methodology while revealing a strong dependence on PDNP size within the design space. In conclusion, RSM proves to be a valuable approach to efficiently optimize photoporation conditions for a particular cell type.