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Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity cause ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH 4 + ). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal-resident protein that preserves organelle homeostasis by controlling ammonium and chloride levels. SLC12A9 knockout (KO) cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 KO cells, and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 function is central for the handling of lysosomal ammonium and chloride, an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors., Competing Interests: Declaration of interests M.C.B. declares outside interest in DEM Biopharma. A.K. is on the scientific advisory board of PatchBio, SerImmune, AINovo, TensorBio, and OpenTargets; was a consultant with Illumina until January 2023; and owns shares in DeepGenomics, Immunai, and Freenome. Y.K. is a cofounder of Esya and Macrologic., (Copyright © 2024. Published by Elsevier Inc.)

Endocytosis is the fundamental uptake process through which cells internalize extracellular materials and species. Neurodegenerative diseases (NDs) are characterized by a progressive accumulation of intrinsically disordered protein species, leading to neuronal death. Misfolding in many proteins leads to various NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and other disorders. Despite the significance of disordered protein species in neurodegeneration, their spread between cells and the cellular uptake of extracellular species is not entirely understood. This review discusses the major internalization mechanisms of the different conformer species of these proteins and their endocytic mechanisms. We briefly introduce the broad types of endocytic mechanisms found in cells and then summarize what is known about the endocytosis of monomeric, oligomeric and aggregated conformations of tau, Aβ, α-Syn, Huntingtin, Prions, SOD1, TDP-43 and other proteins associated with neurodegeneration. We also highlight the key players involved in internalizing these disordered proteins and the several techniques and approaches to identify their endocytic mechanisms. Finally, we discuss the obstacles involved in studying the endocytosis of these protein species and the need to develop better techniques to elucidate the uptake mechanisms of a particular disordered protein species., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

One of the biggest challenges limiting the biological applications of fluorescent carbon-based nanoparticles is their capacity to emit in the red region of the spectrum and simultaneously maintaining the smaller size. These two parameters always go in inverse proportion, thus lagging their applications in biological imaging. Endocytic pathways play important roles in regulating major cellular functions such as cellular differentiation. The Spatio-temporal dynamics of endocytic pathways adopted by various ligands (including nanoparticles) over longer durations in cellular differentiation remain unstudied. Here we have used red-emitting fluorescent carbon nanoparticles to study the endocytic pathways in neuronal cells at different stages of differentiation. These small-sized, bright, red-emitting carbon nanoparticles (CNPs) can be internalized by live cells and imaged for extended periods, thus capturing the Spatio-temporal dynamics of endocytic pathways in model SH-SY5Y derived neuroblastoma neurons. We find that these nanoparticles are preferably taken up via clathrin-mediated endocytosis and follow the classical recycling pathways at all the stages of neuronal differentiation. These nanoparticles hold immense potential for their size, composition, surface and fluorescence tunability, thus maximizing their applications in spatio-temporally tracking multiple cellular pathways in cells and tissues simultaneously.

Alpha-synuclein (α-Syn), an intrinsically disordered protein (IDP), is associated with neurodegenerative disorders, including Parkinson's disease (PD or other α-synucleinopathies. Recent investigations propose the transmission of α-Syn protein fibrils, in a prion-like manner, by entering proximal cells to seed further fibrillization in PD. Despite the recent advances, the mechanisms by which extracellular protein aggregates internalize into the cells remain poorly understood. Using a simple cell-based model of human neuroblastoma-derived differentiated neurons, we present the cellular internalization of α-Syn PFF to check cellular uptake and recycling kinetics along with the standard endocytic markers Transferrin (Tf) marking clathrin-mediated endocytosis (CME) and Galectin3 (Gal3) marking clathrin-independent endocytosis (CIE). Specific inhibition of endocytic pathways using chemical inhibitors reveals no significant involvement of CME, CIE and caveolae-mediated endocytosis (CvME). A substantial reduction in cellular uptake was observed after perturbation of actin polymerization and treatment with macropinosomes inhibitor. Our results show that α-Syn PFF mainly internalizes into the SH-SY5Y cells and differentiated neurons via the macropinocytosis pathway. The elucidation of the molecular and cellular mechanism involved in the α-Syn PFF internalization will help improve the understanding of α-synucleinopathies including PD, and further design specific inhibitors for the same., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Designing programmable biomaterials that could act as extracellular matrices and permit functionalization is a current need for tissue engineering advancement. DNA based hydrogels are gaining significant attention owing to their self-assembling properties, biocompatibility, chemical robustness and low batch to batch variability. The real potential of DNA hydrogels in the biomedical domain remains to be explored. In this work, a DNA hydrogel was coated on a glass surface and coupled to a synthetic IKVAV peptide by a chemical crosslinker. We observe enhanced neuronal differentiation, prolonged neurite length, dynamic movement of microtubules and cytoskeleton, and altered endocytic mechanisms in neuroblastoma-based stem cells for the peptide modified DNA hydrogel compared to the unmodified DNA hydrogel and controls. We anticipate that a peptide-modified DNA hydrogel could emerge as a promising scaffold coating material to develop nerve tissue conduits in the future for application in neuroscience and neuroregeneration.

The direct write printing method has gained popularity in synthesizing scaffolds for tissue engineering. To achieve an excellent printability of scaffolds, a thorough evaluation of rheological properties is required. We report the synthesis, characterization, rheology, and direct-write printing of chitosan - graphene oxide (CH - GO) nanocomposite hydrogels at a varying concentration of GO in 3 and 4 wt% CH polymeric gels. Rheological characterization of CH - GO hydrogels shows that an addition of only 0.5 wt% of GO leads to a substantial increase in storage modulus (G'), viscosity, and yield stress of 3 and 4 wt% of CH hydrogels. A three-interval thixotropy test (3ITT) shows that 3 wt% CH with 0.5 wt% GO hydrogel has 94% recovery of G' after 7 sequential stress cycles and is the best candidate for direct-write printing. Neuronal cell culture on 3 wt% CH with 0.5 wt% hydrogels reveals that GO promotes the differentiation of SH-SY5Y cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We demonstrate a benign and straightforward method to modify the chitosan (CH) by carbamoylation. The free amines on CH are converted into carbamyl functionalities by reacting with potassium cyanate (KCNO). One wt% CH solution, when reacted with KCNO ⩾ 0.1 M, leads to the sol-gel transition of CH through the hydrogen bonding to form carbamoylated chitosan (CCH) hydrogel. Gelation time of CCH decreases with an increase in the KCNO concentration and an interconnected porous network is formed as observed under SEM. Rheological studies show that while one wt% CH solution is a viscous liquid, the CCH hydrogel with 0.5 M KCNO has a storage modulus (G') of 10 4 Pa. The CCH hydrogel is proved to be non-cytotoxic and promotes the attachment and growth of the small lung cancer model A549, and the neuroblastoma SH-SY5Y cell lines. CCH hydrogel also promotes the differentiation of SH-SY5Y cells into neuronal cells, as supported by immunostaining and thus demonstrating its utility as a versatile scaffold for three-dimensional cell-culture systems., (© 2021 IOP Publishing Ltd.)

A series of triazole-based compounds was synthesized using a click chemistry approach and evaluated for the inhibition of α-synuclein (α-syn) fibrillogenesis and its disaggregation. Compounds Tr3, Tr7, Tr12, Tr15, and Tr16 exhibited good effect in inhibiting α-syn fibrillogenesis confirmed by Thioflavin-T assay and fluorescence microscopy and α-syn disaggregation confirmed by fluorescence microscopy. Molecular docking was used to understand the plausible mechanism of the test compounds for inhibiting the α-syn fibrillogenesis and to verify the in vitro results. Compounds Tr3, Tr7, Tr12, Tr15 and Tr16 showed good binding interactions with the essential amino acid residues of α-syn. The compounds which were found to be good inhibitors or disaggregators had no toxic effects on the SH-SY5Y cell line. These compounds have the potential to be developed as therapeutic interventions against synucleinopathies including Parkinson's disease and Lewy body dementia.

Aggregation of α-synuclein (α-syn) is one of the central hypotheses for Parkinson's disease (PD), therefore, its inhibition and disaggregation is an optimistic approach for the treatment of PD. Here, we report design, synthesis and in-vitro efficacy studies of a series of diphenyl triazine hybrids as potential inhibitors of α-syn fibrillogenesis. From the docking studies, we concluded that compounds A1, A2, A4, A8 and A9 display promising binding affinity with the essential residues of α-syn with binding energy values: -6.0, -7.0, -6.3, -6.6 and -6.7 kcal/mol respectively. The target compounds were synthesized using multistep organic synthesis reactions. Compounds A1, A2 A4, A8 and A9 showed a significant lowering of the α-syn fibril formation during Thioflavin-T assay and fluorescence microscopy. In addition, these compounds A1, A2, A4, A8 and A9 also proved to be good disaggregators in the pre-aggregated form of α-syn. Most of the compounds exhibited no cytotoxicity in mouse embryonic fibroblast (MEF) and human adenocarcinomic alveolar basal epithelial cells (A549) except A2. Overall, diphenyl triazine-based compounds can be further investigated for the treatment of synucleinopathies and for Lewy body dementia in which α-syn is predominantly observed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)