Alleviation of neurotoxicity induced by polystyrene nanoplastics by increased exocytosis from neurons.

Nanoplastics (NPs) are potentially toxic and pose a health risk as they can induce an inflammatory response and oxidative stress at cellular and organismal levels. Humans can be exposed to NPs through various routes, including ingestion, inhalation, and skin contact. Notably, uptake into the body via inhalation could result in brain accumulation, which may occur directly across the blood-brain barrier or via other routes. NPs that accumulate in the brain may be endocytosed into neurons, inducing neurotoxicity. Recently, we demonstrated that exposure to polystyrene (PS)-NPs reduces the viability of neurons. We have also reported that inhibiting the retrograde transport of PS-NPs by histone deacetylase 6 (HDAC6) prevents their intracellular accumulation and promotes their export in mouse embryonic fibroblasts. However, whether HDAC6 inhibition can improve neuronal viability by increasing exocytosis of PS-NPs from neurons remains unknown. In this study, mice were intranasally administered fluorescent PS-NPs (PS-YG), which accumulated in the brain and showed potential neurotoxic effects. In cultured neurons, the HDAC6 inhibitor ACY-1215 reduced the fluorescence signal detected from PS-YG, suggesting that the removal of PS-YG from neurons was promoted. Therefore, these results suggest that blocking the retrograde transport of PS-NPs using an HDAC6 inhibitor can alleviate the neurotoxic effects of PS-NPs that enter the brain.

Reduced secretion of LCN2 (lipocalin 2) from reactive astrocytes through autophagic and proteasomal regulation alleviates inflammatory stress and neuronal damage.

LCN2/neutrophil gelatinase-associated lipocalin/24p3 (lipocalin 2) is a secretory protein that acts as a mammalian bacteriostatic molecule. Under neuroinflammatory stress conditions, LCN2 is produced and secreted by activated microglia and reactive astrocytes, resulting in neuronal apoptosis. However, it remains largely unknown whether inflammatory stress and neuronal loss can be minimized by modulating LCN2 production and secretion. Here, we first demonstrated that LCN2 was secreted from reactive astrocytes, which were stimulated by treatment with lipopolysaccharide (LPS) as an inflammatory stressor. Notably, we found two effective conditions that led to the reduction of induced LCN2 levels in reactive astrocytes: proteasome inhibition and macroautophagic/autophagic flux activation. Mechanistically, proteasome inhibition suppresses NFKB/NF-κB activation through NFKBIA/IκBα stabilization in primary astrocytes, even under inflammatory stress conditions, resulting in the downregulation of Lcn2 expression. In contrast, autophagic flux activation via MTOR inhibition reduced the intracellular levels of LCN2 through its pre-secretory degradation. In addition, we demonstrated that the N-terminal signal peptide of LCN2 is critical for its secretion and degradation, suggesting that these two pathways may be mechanistically coupled. Finally, we observed that LPS-induced and secreted LCN2 levels were reduced in the astrocyte-cultured medium under the above-mentioned conditions, resulting in increased neuronal viability, even under inflammatory stress.Abbreviations: ACM, astrocyte-conditioned medium; ALP, autophagy-lysosome pathway; BAF, bafilomycin A1; BTZ, bortezomib; CHX, cycloheximide; CNS, central nervous system; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; JAK, Janus kinase; KD, knockdown; LCN2, lipocalin 2; LPS, lipopolysaccharide; MACS, magnetic-activated cell sorting; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin kinase; NFKB/NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105; NFKBIA/IκBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha; OVEX, overexpression; SLC22A17, solute carrier family 22 member 17; SP, signal peptide; SQSTM1, sequestosome 1; STAT3, signal transducer and activator of transcription 3; TNF/TNF-α, tumor necrosis factor; TUBA, tubulin, alpha; TUBB3/ß3-TUB, tubulin, beta 3 class III; UB, ubiquitin; UPS, ubiquitin-proteasome system.

Neurotoxic potential of polystyrene nanoplastics in primary cells originating from mouse brain.

Polystyrene (PS) and chemically modified compounds in the PS family have long been used in commercial and industrial fields. However, it is poorly understood whether nanoscale-PS microplastic or PS nanoplastic exposure leads to perturbations in fundamental cellular functions, such as proliferation, differentiation, and apoptosis. Herein, we cultured three types of primary cells, including mouse embryonic fibroblasts (MEFs), mixed neuronal cells isolated from embryonic cortex, and cortical astrocytes, and investigated the effects of their exposure to PS nanoplastics with a 100 nm diameter. Although PS nanoplastic exposure did not affect the viability of MEFs or astrocytes, it significantly reduced the viability of mixed neuronal cells. Consistent with the observed effect on cellular viability, levels of the apoptosis marker, cleaved caspase-3, were elevated exclusively in mixed neuronal cells. To investigate whether cells uptake PS nanoplastics into the cytoplasm, we exposed MEFs and neurons to fluorescent PS latex beads and monitored fluorescence over time. We found that PS nanoplastics were deposited and accumulated in the cytoplasm in a concentration-dependent manner. Although astrocytes were not apoptotic upon exposure to PS nanoplastics, they underwent reactive astrocytosis, with increased levels of lipocalin-2 and proinflammatory cytokines. Therefore, our findings suggested that the vulnerability of cells to the deposition and accumulation of PS nanoplastics in the cytoplasm was dependent on cell type. Furthermore, based on our data from primary cells originating from mouse brains, we suggest that reactive astrocytosis may contribute to the neuronal apoptosis seen in defective neurons with PS nanoplastics accumulated in the cell body.

Simultaneous Disruption of Both Polyubiquitin Genes Affects Proteasome Function and Decreases Cellular Proliferation.

The ubiquitin (Ub) proteasome system is important for maintaining protein homeostasis and has various roles in cell signaling, proliferation, and cell cycle regulation. In mammals, Ub is encoded by two monoubiquitin and two polyubiquitin genes. Although reduced levels of Ub due to the disruption of one polyubiquitin gene are known to decrease cell proliferation, the effect of disrupting both polyubiquitin genes remains elusive. Polyubiquitin gene Ubc knockout mice are embryonically lethal and polyubiquitin gene Ubb knockout mice are infertile. Thus, it is difficult to study the effects of double knockouts (DKOs). In the present study, the CRISPR/Cas9 system was used to simultaneously knockout both polyubiquitin genes, UBB and UBC, in HEK293T and HeLa cells. In DKO cells, growth decreased significantly compared to the control cells. We observed reduced proteasome function and reduced levels of free Ub in DKO cells. However, the levels of purified proteasome were not different between control and DKO cells, although the mRNA levels of proteasomal subunits were significantly increased in latter. We propose that the reduction of Ub levels, by disruption of both polyubiquitin genes, resulted in an altered proteasomal status, leading to the reduced proteasome activity, and decreased cellular proliferation.

Cytoprotective role of ubiquitin against toxicity induced by polyglutamine-expanded aggregates.

Ubiquitin (Ub) homeostasis is important for cellular function and survival, especially under stress conditions. Recently, we have demonstrated that Ubc-/- (Ub-deficient) mouse embryonic fibroblasts (MEFs) exhibited reduced viability under oxidative stress induced by arsenite, which was not due to dysregulation of the antioxidant response pathway, but rather due to the potential toxicity caused by the misfolded protein aggregates. However, it is still not clear whether Ub deficiency is directly related to the accumulation of toxic protein aggregates, as arsenite itself triggers protein aggregation and renders cells into aberrant conditions such as reduced proteasome function and inhibition of autophagic flux. Therefore, under arsenite treatment, the outcome could be derived from the combination of multiple defective pathways. Furthermore, it has also been suggested that ubiquitination status of misfolded proteins may not be important for the formation of inclusion bodies composed of misfolded protein aggregates. We therefore wondered whether Ub deficiency is sufficient to trigger the accumulation of toxic protein aggregates inside the cells. In this study, we ectopically expressed polyQ-expanded aggregates (Q103) in MEFs and observed inclusion body formation at the juxtanuclear region, which was independent of cellular Ub levels. In contrast to arsenite treatment, polyQ expression did not affect proteasome function. However, we observed an increased accumulation of Q103 aggregates in Ubc-/- MEFs, which was due to impaired autophagic clearance. Finally, we demonstrated that the increased accumulation of Q103 aggregates under Ub deficiency dramatically reduced the viability of cells. Therefore, our results suggest that the maintenance of proper levels of cellular Ub is important to protect cells against the toxicity induced by the accumulation of protein aggregates.