In this study, we demonstrated that: (i) at least two distinct intracellular pathways were involved in TDP-43 cytoplasmic aggregation, including aggresome formation and LLPS; (ii) the two pathways were independent of each other; and (iii) in spinal motor neurons of sporadic ALS, both HDAC6-positive and HDAC6-negative aggregates were found in the same ALS patient. Therefore, to the best of our knowledge, we are the first to confirm that HDAC6-dependent aggresome formation is partially involved in skein-like TDP-43 inclusion of sporadic ALS.
Our study revealed that over 70% of the examined inherited ALS/FTLD causative 19 genes induced TDP-43 co-aggregation in cultured neuronal cells. Our screening system enabled us to assess whether any genes of interest induce cytoplasmic TDP-43 aggregation by a simple transfection-based screening. Moreover, our screening system has been proved useful for investigating the mechanism of TDP-43 aggregation. Indeed, as described above, we identified two distinct mechanisms involved in TDP-43 aggregation by using this system (i.e., aggresome formation and LLPS). Our screening system will also be useful for identifying compounds that prevent cytoplasmic TDP-43 aggregation, which may contribute to a novel therapeutic strategy for ALS/FTLD.
Although our screening system generally well recapitulated cytoplasmic TDP-43 aggregation in ALS/FTLD, our results were partially inconsistent with some previous reports on the pathology of inherited ALS [17, 18]. Notably, we did not find any evidence of TDP-43 aggregation in the neuronal cells expressing the inherited ALS/FTLD causative genes related to protein degradation (e.g., VCP, UBQLN2, SQSTM1, and TBK1), although the previous studies reported TDP-43 pathology in inherited ALS patients with these mutations . Moreover, the co-localization of TDP-43 and the inherited ALS/FTLD causative gene products are still controversial even though TDP-43 pathology is widely observed in the patients with ALS. For example, some studies have suggested the co-localization of TDP-43 with TIA-1 or FUS  but other studies do not [32, 33]. One possible interpretation of this inconsistency is an experimental limitation of our screening system based on the overexpression of the inherited ALS/FTLD causative genes with a short incubation time before evaluation. Therefore, a careful interpretation is required to compare our results with the inherited ALS/FTLD neuropathology data.
The co-aggregation of TDP-43 and RBPs was sensitive to 1,6-Hd, suggesting that LLPS drives cytoplasmic RBP-induced aggregation. Nucleotides, enriched in nuclei, prevent aggregation driven by LLPS [34, 35]. Indeed, the previous study demonstrated that an artificially designed RNA, which specifically binds to TDP-43, prevents intracellular aggregation of TDP-43 triggered by light-driven forced multimerization of TDP-43 in the cytoplasm . This mechanism could explain our observations that RBPs only aggregated with TDP-43 when they leaked from nuclei into the cytoplasm. On the other hand, the MRPs-induced co-aggregation of TDP-43 was dependent on aggresome formation. The role of aggresomes in the accumulation of misfolded proteins has been pointed out in various neurodegenerative diseases . ALS-linked PFN1 mutant proteins that aggregated in the cytoplasm were dependent on microtubules, which was similar to our observations . Intriguingly, these two pathways of TDP-43 aggregation are not complementary and are independent of each other. Inhibiting LLPS prevented RBPs-mediated TDP-43 aggregation, but did not affect MRPs-induced TDP-43 aggregation. Conversely, HDAC6 suppression and microtubular destabilization only affected MRPs-induced TDP-43 aggregation. All these observations indicate that targeting the intracellular mechanism specific to each pathway is key to preventing TDP-43 accumulation. Nonetheless, there is still a possibility that other independent pathways are also involved in cytoplasmic TDP-43 aggregation. Further investigation into the potential mechanisms of TDP-43 accumulation is required.
HDAC6 is a key molecule in aggresome formation. HDAC6 facilitates the transport of misfolded proteins by connecting microtubules and ubiquitinated proteins [27, 36]. The involvement of HDAC6 has been reported in various neurodegenerative diseases such as Huntington's disease , Parkinson's disease , and Alzheimer’s disease . In ALS, it was reported that deletion of Hdac6 ameliorates the disease progression in SOD1-ALS mice  and TDP-43 deficiency drastically reduces HDAC6 expression in cultured cells . Moreover, HDAC6 inhibition reversed axonal transports, which are defective in iPS-derived motor neurons from ALS patients with FUS mutation . These observations suggest the possibility that HDAC6 is also involved in the ALS pathology similar to other neurodegenerative diseases and that it may be a therapeutic target for ALS through restoring axonal transport. In addition to this, our results strengthen the possibility that inhibition of HDAC6 is a potential therapeutic target for ALS by providing a novel mechanism for preventing microtubule-dependent TDP-43 pathology. HDAC6 inhibition is probably effective for MRPs-linked inherited ALS and partially to sporadic ALS.
Since most of the ALS cases are sporadic, it is important to identify the mechanism responsible for TDP-43 aggregation in the sporadic ALS. Recent reports showed that cytoplasmic TDP-43 was sequestered into aggresome dependent on microtubules in fibroblasts derived from sporadic ALS patients . Consistent with the cited study, we found that TDP-43 was sequestered into aggregates of MRPs in N2a cells and approximately half of TDP-43 skein-like inclusions in sporadic ALS patients were co-localized with HDAC6, an aggresomal marker. Our results suggest that the HDAC6-dependent aggresome pathway may be involved in the formation of skein-like inclusion in ALS/FTLD. However, we also found that both HDAC6-positive and HDAC6-negative skein-like inclusions were observed in the same sporadic ALS patient, as shown in Table 2. In addition, the round and granular inclusions, which are not co-localized with HDAC6, were also found in the same sporadic ALS patient. These observations suggest that the mechanisms responsible for TDP-43 aggregation are different from each other among the remaining motor neurons in the one sporadic ALS patient and that the morphology of TDP-43 inclusions may reflect the corresponding intracellular mechanism. Further studies are needed to determine the mechanism responsible for each type of cytoplasmic TDP-43 aggregation in motor neurons.