Combination of pathological, biochemical and behavioral evaluations for peripheral neurotoxicity assessment in isoniazid-treated rats.

In drug development, assessment of non-clinical peripheral neurotoxicity is important to ensure human safety. Clarifying the pathological features and mechanisms of toxicity enables the management of safety risks in humans by estimating the degree of risk and proposing monitoring strategies. Published guidelines for peripheral neurotoxicity assessment do not provide detailed information on which endpoints should be monitored preferentially and how the results should be integrated and discussed. To identify an optimal assessment method for the characterization of peripheral neurotoxicity, we conducted pathological, biochemical (biomaterials contributing to mechanistic considerations and biomarkers), and behavioral evaluations of isoniazid-treated rats. We found a discrepancy between the days on which marked pathological changes were noted and those on which biochemical and behavioral changes were noted, suggesting the importance of combining these evaluations. Although pathological evaluation is essential for pathological characterization, the results of biochemical and behavioral assessments at the same time points as the pathological evaluation are also important for discussion. In this study, since the measurement of serum neurofilament light chain could detect changes earlier than pathological examination, it could be useful as a biomarker for peripheral neurotoxicity. Moreover, examination of semi-thin specimens and choline acetyltransferase immunostaining were useful for characterizing morphological neurotoxicity, and image analysis of semi-thin specimens enabled us to objectively show the pathological features.

Classification of proteins inducing liquid-liquid phase separation: sequential, structural and functional characterization.

Liquid-liquid phase separation (LLPS) within the cell can form biological condensates, which are increasingly recognized to play important roles in various biological processes. Most proteins involved in LLPS are known to be intrinsically disordered proteins containing intrinsically disordered regions (IDRs) with low complexity regions (LCRs). The proteins driving LLPS were selected from databases of LLPS-related proteins and then classified into three classes according to the components in the condensates. Through in silico analyses, we found that proteins in the homo class, those that induce LLPS without partner molecules, have different IDRs and LCRs compared with the reference proteome. In contrast, proteins in the other classes, those that induce LLPS with partner proteins (the hetero class) or nucleic acids (the mixed class), did not show a clear difference to the reference proteome in IDRs and LCRs. The hetero-class proteins contained structural domains to serve protein-protein interactions, and the mixed-class ones had the structural domains associated with nucleic acids. These results suggest that IDRs in the homo-class proteins have unique IDRs, which provide multivalent interaction sites required for LLPS, whereas the hetero- and mixed-class proteins can induce LLPS through the combination of the interaction among LCRs, structural domains and nucleic acids.

Karnovsky's fixative prevents artifacts appearing as vacuolation derived from tissue processing in kidneys treated with antisense oligonucleotide.

Antisense oligonucleotide (ASO) therapies have been identified as a new treatment modality for intractable diseases. In kidneys treated with ASOs, vacuoles, in addition to basophilic granules, are often observed in the proximal tubules. Some reports have described that these vacuoles are likely to be a secondary phenomenon resulting from the extraction of ASOs during tissue processing. In this study, we compared renal morphology after fixation with Karnovsky's fixative or 4% paraformaldehyde phosphate buffer (PFA) with that of 10% neutral-buffered formaldehyde solution (NBF). Female Sprague-Dawley rats, intravenously treated four times with 50 mg/kg locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) for 1 or 2 weeks, were examined. Microscopically, vacuoles and basophilic granules in the proximal tubules were observed in the kidneys fixed with NBF. Basophilic granules are indicative of the accumulation of ASOs. Moreover, some of the vacuoles also contained faint basophilic granules, suggesting that the vacuoles were relevant to the accumulation of ASOs. Although moderate vacuolation was observed in the proximal tubules, the majority of the vacuolated epithelia were negative for kidney injury molecule-1 on immunohistochemical staining. Vacuoles in the proximal tubules were not observed in samples subjected to Karnovsky's fixation, although basophilic granules were observed. In samples subjected to PFA fixation, vacuoles and basophilic granules were observed in the proximal tubules, similar to those in samples subjected to NBF fixation. Overall, our findings demonstrated the possibility of overestimation of vacuolation due to artifacts during tissue processing when using conventional NBF fixation. Karnovsky's fixative is considered a useful alternative for distinguishing artificial vacuoles from true nephrotoxicity.