# Quantitative Immunofluorescence Mapping of HSP70's Neuroprotective Effects in FUS-ALS Mouse Models
## 4 Discussion  
In our study, we bred out the mice carrying aberrant form of the human FUS gene with the mice overexpressing human HSPA1A, both intra- and extracellular. We revealed animal survival to be even shorter in double FUS [1-359]/HSP70 (out) transgenic mice than in FUS [1-359] animals. However, increased terms of survival were characteristic for FUS [1-359]/HSP70 (in) transgenic mice, thus pretending HSPA1A overexpression with intracellular accumulation is more beneficial. It has been reported that not only survival but body mass also decreases in animals with FUS-related ALS pathology [28], although no body mass decrease occurs in HSP70 overexpression [29]. As ALS is principally a motor neurons degeneration, the primary zone of our interest in histological study included the motor cortex. We preferred NeuN over other possible neuronal molecular markers as it is the most universal one for mature cerebral neurons [30]. Figure 5: Immunofluorescent study. Iba1 and SOX9 expression in the primary motor cortex of double FUS [1-359] /HSP70 transgenic mice compared to single FUS [1-359] transgenic animals and control groups (\(n=6\) for each group). (**A**,**B**) Microphotographs of cerebral sections, d = 5 μm, ob. 10. Scale bar size: 20 μm. Shades of red color mark the primary antibody ((**A**)—Iba1, (**B**)—SOX9) used, blue color marks DAPI counterstainedaining. (**C**,**D**) Boxplots of statistical data: names of study groups are given at X axis, parameters measured—at Y axis. *—statistical difference is significant at \(p<0.001\) (\(<\)0.0001 for both S100\(\&\) SOX9). We observed a sharp decline in NeuN-positive cells for FUS-transgenic animals compared to all other groups. In this study, we reveal for the first time a neuroprotective impact of both intracellular and extracellular HSPA1A overexpressed together with FUS [1-359] in motor neurons of the primary motor cortex. For the NeuN/DAPI ratio, we did not mention a significant difference between FUS [1-359] mice and double transgenes. This may be explained as an evidence of cell damage reduction in HSP70-affected animals not only in neuronal population. If total number of mature neurons increases, and no considerable increase in ratio to all cells occurs, this suggests that the total number of cells also increases, so total cellularity increases. However, there may also be a different interpretation. For instance, increased cellularity is specific for tumorigenic phenomena, whereas HSP70 is a well-known anti-apoptotic and thereby pro-oncogenic agent [31]. Despite the lack of significant difference between two double transgene groups for the NeuN/DAPI ratio, we can easily notice a larger peak in Tg FUS + HSP70 (out), i.e., with extracellular secretion (Figure 3C). This is probably due to a lower inhibiting impact of extracellular HSP70 on apoptotic enzymatic cascades inside the cytosol in cells other than NeuN-positive ones. Other markers elucidate the HSP70-related potential of glial cell populations in the cerebral cortex. For astrocytes, we used three markers. GFAP is specific for cytoskeleton of astrocytes, so its content increases in GFAP activation during defensive reactions [32], e.g., in neuroinflammation, neurodegeneration, and traumas. However, all FUS-overexpressing groups have normal number of GFAP-positive cells. In contrast, single Tg HSP70 (out) mice have larger GFAP expression than even single FUS [1-359] transgenic mice. We explain this by the fact that not all the neurodegenerative processes lead to the same level of considerable changes outside the cells, and aberrant FUS stays in cytoplasm with no aggregation in extracellular matrix. At the same time, extracellular HSP70 binds Toll-like receptors (TLR) on astroglial cells [33], thereby invoking innate immune reactions and therefore astrocytic non-specific activation. Further, GFAP is more characteristic for white matter and not for the cortex. Meanwhile, an increase in S100\(\beta\)-positive cells, which are usually grey matter astrocytes [32], is observed for all FUS-containing groups.