# Quantitative Immunofluorescence Mapping of HSP70's Neuroprotective Effects in FUS-ALS Mouse Models
## 4 Discussion
Thus, these data show a slight upregulation of marker expression for grey matter astrocytes (S100\(\beta\)) and almost no changes in white matter astrocytes marker (GFAP). The original study of FUS [1-359] transfection model in mice showed an increase in GFAP content but the analysis was performed with specimens of the spinal cord and brainstem there [20], whereas we observed the primary motor cortex. Third marker studied for astrocytes, SOX9, was included as reported in previous studies being upregulated in rodents with ALS modeled, but for SOD1 and not FUS models [34]. We, however, did not observe any difference involving transgenic FUS mice for this marker. Curiously, FUS [1-359]/HSP70 (in) animals got a strong rise of SOX9 expression in the primary motor cortex compared to controls, and this phenomenon is not typical for other study groups. This fact deserves a supplementary investigation due to its probable role in explaining molecular mechanisms of intracellular interaction between HSPA1A and FUS in stress granules directly inside astrocytes or in neuron-astrocyte intercommunication. To estimate differences in microglial cell populations between the groups, we studied the number of Iba1-positive cells, as protein Iba1 is evenly distributed in the cytoplasm and processes of microglial cells [35]. Iba1 was also shown to increase sharply in single FUS [1-359] transgenic animals before [36]. FUS [1-359]/HSP70 (in) group attracts the most attention, being significantly different in sample distribution from both controls and FUS [1-359] animals. Moreover, single HSP70 (in) mice have a similar pattern of difference from controls and FUS [1-359] animals. These data for Iba1 are consistent with those for SOX9 marker and are probably explained by a more efficient phagocytosis of damaged cellular structures after chaperone-launched signaling in neuron-astrocyte intercommunication. As the double transgenic animals, FUS [1-359]/HSP70 (in), had also no difference from FUS [1-359] animals in the NeuN/DAPI ratio, this probable phagocytosis, if exists, does not greatly affect the total cell number. As this phenomenon is not quiet typical, we consider it to be an independent finding, and the degree of its relation to HSP70 upregulation should be tested further. Together, all our data mentioned require a more profound studying of neuronal and astroglial proteome in FUS [1-359]/HSP70 (in) animals to understand a molecular interplay between astrocytes and microglial cells. Our study showed some evident signs of neuroprotection according to immunofluorescence marker assessment, whereas NeuN level is principal, as we estimated it in the primary motor cortex, thereby showing neuronal survival actually at the cortical level of motor activity regulation. Neuroprotection studies may, however, include a plenty of additional approaches as we show further in perspectives. In assessment of histological preparations, we preferred immunofluorescence (IF) assay over standard immunohistochemical staining (IHC) even though a risk for degrading of fluorochrome signal with course of time is obvious. However, for such experimental studies as we did, a diverse nature of markers estimated requires to allow multiplexing of slides if needed what is only achievable for IF and not IHC. For example, NeuN estimation solely without the NeuN/DAPI ratio is not logical and even speculating as the level of NeuN expression may depend on duration of fixation [30], thus these factors are two be counted at the same slides together. In our study, we investigated the only protein aberration of ALS in combination with HSP70 overexpression. However, ALS pathology comprises a multifaceted RNA-protein participation [37] with RNA-dependent and RNA-independent steps of protein aggregation [38]. Further, there were reports about nuclear aggregation of FUS with even no mislocalization to cytoplasm [39]. Thus, different points of view are accessible to regard the hypothesis of interaction between molecular chaperones and aberrant RNA and proteins in ALS.