# Quantitative Immunofluorescence Mapping of HSP70's Neuroprotective Effects in FUS-ALS Mouse Models
## 4 Discussion
A large body of evidence had been already demonstrated for HSP40-mediated HSP70 activity in ALS [40]. [PERSON] et al. also showed double transgenic mice (Hsp40 and SODI) to have a higher degree of motor neuron survival [41]. Further, [PERSON] and colleagues reported Hsp40 expression to reduce signs of damage in TDP-43-associated ALS in vitro and in patients [17], and the consistent effect was achieved for HSP70 [42]. For another key player of ALS, C9 orf72 protein, a presence of Hsp40 enhanced clearance of aberrant C9 orf72 [43]. However, history of studying HSP70 interactions in ALS is not long in regard to FUS-associated pathology, and covers molecular studies and research of direct FUS-HSP70 intercommunication. For molecular studies, it was found that pathological stress granules with FUS are rather stable [13], although HSP70 and HSP40 were supposed to disintegrate such aberrant agglomerates [44]. Thus, [PERSON] and colleagues experimentally discovered an increased mobility of mutant FUS aggregates having HSP40- and therefore HSP70-dependent manner [45]. For direct HSP70-FUS interaction, there were attempts to regard the administration of HSP70 inducers in fusopathies. In 2020, [PERSON] and colleagues showed aberrant _FUS\({}^{R525L}\)_ expression to regress in vitro after treatment with armoclomol, which provided an enhanced HSP70 binding to its upregulated, HSF-1 [46]. Nevertheless, excellent series of studies from the same team revealed almost no positive changes in motor neurons with mutant _FUS\({}^{R521G}\)_ isoform after HSP70 induction by armoclomol [47, 48]. Therefore, cells with different types of mutant FUS may experience a different degree of involvement into HSP70-driven changes. This ambivalence requires further understanding, as shown by our study, where we used a _FUS\({}^{[1-359]}\) Hemi_ version of aberrant FUS protein. Another approach is to test absolutely other molecular chaperones for their potential to resist to aberrant FUS. For example, HSP104 functioning requires no HSP70 engagement [49]. It was also found that HSP104-related machinery is highly efficient for disaggregating misfolded FUS but is only appropriate as external chemical because HSP104 is typical for yeast and not synthesized by metazoans [50]. Discussing our data, we should outline the difference of two HSPA1A isoforms tested. A theoretical thinking allows to consider extracellular HSPA1A usage to be worse. Exactly,HSP70/HSPA1A is a ligand for TLR [33]. TLR-mediated HSPA1A overexpression outside the cells can lead to generalized seizures as shown in HSP70-transgenic mice [51]. Interestingly, we observed no damaging effects of a direct HSP70 treatment to nervous tissue in our earlier study, so the phenomenon described by [PERSON] and colleagues is not universal [52]. HSP70 takes part in many cellular processes during neuroinflammation and neuronal damage [53]. Thus, HSP70 obviously has side effects as its overexpression results not only in FUS modification but in changing a number of other cellular pathways it participates in. Even in ALS, HSP70 may act via different mechanisms including, e.g., histone deacetylase management [47]. This implies possible effects of excessive HSP70 on FUS models by different mechanisms. Therefore, we cannot claim a direct effect of HSP70 to FUS mislocalization and/or aggregation, but a beneficial role of HSP70 presence together with aberrant FUS expression is observed. One may also expect a potential of this presence to be studied not only in ALS but in other neurodegenerative diseases with a proven pathogenesis of fusopathies, such as frontotemporal dementia and Alzheimer's disease. Further, HSP70 is well-known for its tumorigenic potential [31]. Although this effect may even become a benefit in neurodegeneration, as there were reports about cell proliferation stimulated by HSP70 in hippocampal region [54], we cannot deny possible risks. The anti-apoptotic activity of HSP70 may also result in cardiac hypertrophy and not only in tumorigenesis [55].