Intracellular Vesicle Transport Research Articles

Comprehensive Analyses of the Effects of the Small-Molecule Inhibitor of the UHM Domain in the Splicing Factor U2AF1 in Leukemia Cells.

Mutations in RNA splicing factor genes including SF3B1, U2AF1, SRSF2, and ZRSR2 have been reported to contribute to development of myeloid neoplasms including myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Chemical tools targeting cells carrying these mutant genes remain limited and underdeveloped. Among the four proteins, mutant U2AF1 (U2AF1mut) acquires an altered 3' splice site selection preference and co-operates with the wild-type U2AF1 (U2AF1wt) to change various gene isoform patterns to support MDS cells survival and proliferation. U2AF1 mutations in MDS cells are always heterozygous and the cell viability is reduced when exposed to additional insult affecting U2AF1wt function. To investigate if the pharmacological inhibition of U2AF1wt function can provoke drug-induced vulnerability of cells harboring U2AF1 mut , we conducted a fragment-based library screening campaign to discover compounds targeting the U2AF homology domain (UHM) in U2AF1 that is required for the formation of the U2AF1/U2AF2 complex to define the 3' splice site. The most promising hit (SF1-8) selectively inhibited growth of leukemia cell lines overexpressingU2AF1 mut and human primary MDS cells carrying U2AF1 mut . RNA-seq analysis of K562-U2AF1mut following treatment with SF1-8 further revealed alteration of isoform patterns for a set of proteins that impair or rescue pathways associated with endocytosis, intracellular vesicle transport, and secretion. Our data suggested that further optimization of SF1-8 is warranted to obtain chemical probes that can be used to evaluate the therapeutic concept of inducing lethality to U2AF1 mut cells by inhibiting the U2AF1wt protein.

Before Translating Extracellular Vesicles into Personalized Diagnostics and Therapeutics: What We Could Do.

Extracellular vesicle (EV) research is rapidly advancing from fundamental science to translational applications in EV-based personalized therapeutics and diagnostics. Yet, fundamental questions persist regarding EV biology and mechanisms, particularly concerning the heterogeneous interactions between EVs and cells. While we have made strides in understanding virus delivery and intracellular vesicle transport, our comprehension of EV trafficking remains limited. EVs are believed to mediate intercellular communication through cargo transfer, but uncertainties persist regarding the occurrence and quantification of EV-cargo delivery within acceptor cells. This ambiguity is crucial to address, given the significant translational impact of EVs on therapeutics and diagnostics. This perspective article does not seek to provide exhaustive recommendations and guidance on EV-related studies, as these are well-articulated in position papers and statements by the International Society for Extracellular Vesicles (ISEV), including the 'Minimum Information for Studies of Extracellular Vesicles' (MISEV) 2014, MISEV2018, and the recent MISEV2023. Instead, recognizing the multilayered heterogeneity of EVs as both a challenge and an opportunity, this perspective emphasizes novel approaches to facilitate our understanding of diverse EV biology, address uncertainties, and leverage this knowledge to advance EV-based personalized diagnostics and therapeutics. Specifically, this perspective synthesizes current insights, identifies opportunities, and highlights exciting technological advancements in ultrasensitive single EV or "digital" profiling developed within the author's multidisciplinary group. These newly developed technologies address technical gaps in dissecting the molecular contents of EV subsets, contributing to the evolution of EVs as next-generation liquid biopsies for diagnostics and providing better quality control for EV-based therapeutics.

Mannogalactoglucan from mushrooms protects pancreatic islets via restoring UPR and promotes insulin secretion in T1DM mice

Type 1 diabetes mellitus (T1DM) lacks insulin secretion due to autoimmune deficiency of pancreatic β-cells. Protecting pancreatic islets and enhancing insulin secretion has been therapeutic approaches. Mannogalactoglucan is the main type of polysaccharide from natural mushroom, which has potential medicinal prospects. Nevertheless, the antidiabetic property of mannogalactoglucan in T1DM has not been fully elucidated. In this study, we obtained the neutral fraction of alkali-soluble Armillaria mellea polysaccharide (AAMP-N) with the structure of mannogalactoglucan from the fruiting body of A. mellea and investigated the potential therapeutic value of AAMP-N in T1DM. We demonstrated that AAMP-N lowered blood glucose and improved diabetes symptoms in T1DM mice. AAMP-N activated unfolded protein response (UPR) signaling pathway to maintain ER protein folding homeostasis and promote insulin secretion in vivo. Besides that, AAMP-N promoted insulin synthesis via upregulating the expression of transcription factors, increased Ca2+ signals to stimulate intracellular insulin secretory vesicle transport via activating calcium/calmodulin-dependent kinase II (CamkII) and cAMP/PKA signals, and enhanced insulin secretory vesicle fusion with the plasma membrane via vesicle-associated membrane protein 2 (VAMP2). Collectively, these studies demonstrated that the therapeutic potential of AAMP-N on pancreatic islets function, indicating that mannogalactoglucan could be natural nutraceutical used for the treatment of T1DM.

Immune-phenotyping and transcriptomic profiling of blood monocytes from patients with breast cancer under neoadjuvant chemotherapy

Introduction. Chemotherapy is a common treatment for breast cancer. Chemotherapeutic drugs effect blood monocytes, which are major contributors to cancer pathogenesis. However, to date, pro-tumor or anti-tumor programming by chemotherapy of monocytes is controversial.Aim. To characterize changes in phenotypic and transcriptomic profiles of monocytes of breast cancer patients before and after chemotherapeutic treatment.Materials and methods. In a cohort of 50 breast cancer patients, monocyte populations were identified based on their expression of CD14, CD16, CD163, and HLA-DR evaluated by flow cytometry before and after neoadjuvant chemotherapy. Bulk RNA sequencing was adopted to explore the transcriptomic profile of CD14+ monocytes before and after treatment. After treatment, we observed an increase in the activity of signaling pathways related to lipid metabolism and intracellular transport of vesicles from the endoplasmic reticulum, against the background of a decreased response to exposure to interferon γ and interferon α, and foreign molecules (exogenous nucleic acids, viruses and bacteria).Results. In breast cancer patients, neoadjuvant chemotherapy decreased in CD14+16+HLA-DR+ monocytes. Under cytostatic treatment, increased gene expression of MGLL, NR4A2, UCK1, YOD1, ABCA2, PAPSS2, ATP10 (log2FoldChange ≥0.8; false discovery rate (FDR) ≤0.01) and decreased gene expression of KPNA2, ERCC4, JAGN1, RUBCNL, SMYD4, B3GALT4 (log2FoldChange ≥0.8; FDR ≤0.01) were observed in monocytes of patients. Using discriminant analysis, the relative numbers of CD14+16–, CD14+16+, CD14-16+, CD14+16-HLA-DR+, CD14+16+HLA-DR+ and CD14–16+HLA-DR+ monocytes in the blood were found to be valuable in predicting response to neoadjuvant chemotherapy.Conclusion. Thus, association of blood monocytes with chemotherapeutic treatment in breast cancer was revealed.

The exocyst complex and intracellular vesicles mediate soluble protein trafficking to the primary cilium

The efficient transport of proteins into the primary cilium is a crucial step for many signaling pathways. Dysfunction of this process can lead to the disruption of signaling cascades or cilium assembly, resulting in developmental disorders and cancer. Previous studies on the protein delivery to the cilium were mostly focused on the membrane-embedded receptors. In contrast, how soluble proteins are delivered into the cilium is poorly understood. In our work, we identify the exocyst complex as a key player in the ciliary trafficking of soluble Gli transcription factors. In line with the known function of the exocyst in intracellular vesicle transport, we demonstrate that soluble proteins, including Gli2/3 and Lkb1, can use the endosome recycling machinery for their delivery to the primary cilium. Finally, we identify GTPases: Rab14, Rab18, Rab23, and Arf4 that are involved in vesicle-mediated Gli protein ciliary trafficking. Our data pave the way for a better understanding of ciliary transport and uncover transport mechanisms inside the cell.

Release of extracellular vesicles triggered by low-intensity pulsed ultrasound: immediate and delayed reactions.

Previous studies have shown that ultrasound may stimulate the release of extracellular vesicles, improving the efficiency of tumor detection. However, it is unclear whether ultrasonic stimulation affects the distribution of extracellular vesicles, and the duration of such stimulation release has not been extensively studied. In this study, we stimulated cells with low-intensity pulsed ultrasound and used liposomes containing black hole quenchers to simulate natural extracellular vesicles, confirming that ultrasound has a destructive effect on vesicles and thus affects particle size distribution. Furthermore, we used proteomics technology to examine the protein expression profile of small vesicles and discovered that the expression of proteins involved in exosome biogenesis was down-regulated. We then looked into the regulation of the actin cytoskeleton and endocytosis pathways, which are required for intracellular vesicle transport, and discovered that ultrasound might induce F-actin depolymerization. The intracellular transport of the cation-independent mannose-6-phosphate receptor (CI-MPR) in the trans-Golgi network (TGN) and the amount of Rab7a protein were proportional to the culture time after LIPUS treatment.

Deficiency of Pdcd10 causes urothelium hypertrophy and vesicle trafficking defects in ureter.

The scaffolding protein programmed cell death protein 10 (Pdcd10) has been demonstrated to play a critical role in renal epithelial cell homeostasis and function by maintaining appropriate water reabsorption in collecting ducts. Both ureter and kidney collecting duct systems are derived from the ureter bud during development. Here, we report that cadherin-16 (Cdh16)-cre drives gene recombination with high specificity in the ureter, but not the bladder, urothelium. The consequences of Pdcd10 deletion on the stratified ureter urothelium were investigated using an integrated approach including messenger RNA (mRNA) expression analysis, immunocytochemistry, and high-resolution confocal and electron microscopy. Loss of Pdcd10 in the ureter urothelium resulted in increased expression of uroplakins (Upks) and keratins (Krts), as well as hypertrophy of the ureter urothelium with an associated increase in the number of proliferation marker protein Ki-67 (Ki67)-expressing cells specifically within the basal urothelium layer. Ultrastructural analysis documented significant modification of the intracellular membrane system, including intracellular vesicle genesis and transport along the basal- to umbrella-cell-layer axis. Additionally, Pdcd10 loss resulted in swelling of Golgi compartments, disruption of mitochondrial cristae structure, and increased lysosomal fusion. Lack of Pdcd10 also resulted in decreased fusiform vesicle formation in umbrella cells, increased secretion of exosome vesicles, and alteration in microvillar structure on apical membranes. Our findings indicate that Pdcd10 expression and its influence on homeostasis is associated with modulation of endomembrane trafficking and organelle biogenesis in the ureter urothelium.

Interleukin‑22 alleviates arginine‑induced pancreatic acinar cell injury via the regulation of intracellular vesicle transport system: Evidence from proteomic analysis.

Acute pancreatitis (AP) is a severe inflammatory condition characterized by the activation of pancreatic enzymes within acinar cells, leading to tissue damage and inflammation. Interleukin (IL)-22 is a potential therapeutic agent for AP owing to its anti-inflammatory properties and ability to promote tissue repair. The present study evaluated the differentially expressed proteins in arginine-induced pancreatic acinar cell injury following treatment with IL-22, and the possible mechanisms involved in IL-22-mediated alleviation of AP. AR42J cells were stimulated using L-arginine to establish an acinar cell injury model in vitro and the damaged cells were subsequently treated with IL-22. The characteristics of the model and the potential therapeutic effects of IL-22 were examined by CCK-8 assay, flow cytometry, TUNEL assay, transmission electron microscopy and ELISA. Differentially expressed proteins in cells induced by arginine and treated with IL-22 were assessed using liquid chromatography-mass spectrometry. The identified proteins were further subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to elucidate their functional roles. The present study demonstrated that arginine-stimulated cells showed significant pathological changes resembling those in AP, which were alleviated after IL-22 treatment. Proteomic analysis then demonstrated that in IL-22-treated cells, proteins related to the formation and fusion of autophagosomes with lysosomes were significantly downregulated, whereas endocytosis related proteins were enriched in the upregulated proteins. After IL-22 treatment, western blotting demonstrated reduced expression of autophagy-associated proteins. In conclusion, by inhibiting the formation and fusion of autophagosomes with lysosomes, IL-22 may have mitigated premature trypsinogen activation, subsequently minimizing acinar cell injury induced by L-arginine. This was accompanied by concurrent upregulation of endocytosis, which serves a pivotal role in sustaining regular cellular material transport and signal propagation. This research underscored the potential of IL-22 in mitigating arginine-induced AR42J injury, which could be valuable in refining treatment strategies for AP.

Galectin-9 interacts with Vamp-3 to regulate cytokine secretion in dendritic cells

Intracellular vesicle transport is essential for cellular homeostasis and is partially mediated by SNARE proteins. Endosomal trafficking to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of immune responses. Despite its critical importance, the specific molecular components that regulate DC cytokine secretion are poorly characterised. Galectin-9, a ß-galactoside-binding protein, has emerged as a novel cellular modulator although its exact intracellular roles in regulating (immune) cell homeostasis and vesicle transport are virtually unknown. We investigated galectin-9 function in primary human DCs and report that galectin-9 is essential for intracellular cytokine trafficking to the cell surface. Galectin-9-depleted DCs accumulate cytokine-containing vesicles in the Golgi complex that eventually undergo lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using immunoprecipitation-mass-spectrometry and identified galectin-9 was required for rerouting Vamp-3-containing endosomes upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle transport and sheds new light into the multiple roles galectins play in governing cell function.Graphical abstract

The Zika virus infection remodels the expression of the synaptotagmin-9 secretory protein.

The exact mechanisms involved in flaviviruses virions' release and the specific secretion of viral proteins, such as the Non Structural protein-1 (NS1), are still unclear. While these processes might involve vesicular transport to the cell membrane, NS1 from some flaviviruses was shown to participate in viral assembly and release. Here, we assessed the effect of the Zika virus (ZIKV) NS1 expression on the cellular proteome to identify trafficking-related targets that may be altered in the presence of the viral protein. We detected an increase in the synaptotagmin-9 (SYT9) secretory protein, which participates in the intracellular transport of protein-laden vesicles. We confirmed the effect of NS1 on SYT9 levels by transfection models while also detecting a significant subcellular redistribution of SYT9. We found that ZIKV prM-Env proteins, required for the viral particle release, also increased SYT9 levels and changed its localization. Finally, we demonstrated that ZIKV cellular infection raises SYT9 levels and promotes changes in its subcellular localization, together with a co-distribution with both Env and NS1. Altogether, the data suggest SYT9's implication in the vesicular transport of viral proteins or virions during ZIKV infection, showing for the first time the association of synaptotagmins with the flavivirus' life cycle.

Inceptor facilitates acrosomal vesicle formation in spermatids and is required for male fertility.

Spermatogenesis is a crucial biological process that enables the production of functional sperm, allowing for successful reproduction. Proper germ cell differentiation and maturation require tight regulation of hormonal signals, cellular signaling pathways, and cell biological processes. The acrosome is a lysosome-related organelle at the anterior of the sperm head that contains enzymes and receptors essential for egg-sperm recognition and fusion. Even though several factors crucial for acrosome biogenesis have been discovered, the precise molecular mechanism of pro-acrosomal vesicle formation and fusion is not yet known. In this study, we investigated the role of the insulin inhibitory receptor (inceptor) in acrosome formation. Inceptor is a single-pass transmembrane protein with similarities to mannose-6-phosphate receptors (M6PR). Inceptor knockout male mice are infertile due to malformations in the acrosome and defects in the nuclear shape of spermatozoa. We show that inceptor is expressed in early spermatids and mainly localizes to vesicles between the Golgi apparatus and acrosome. Here we show that inceptor is an essential factor in the intracellular transport of trans-Golgi network-derived vesicles which deliver acrosomal cargo in maturing spermatids. The absence of inceptor results in vesicle-fusion defects, acrosomal malformation, and male infertility. These findings support our hypothesis of inceptor as a universal lysosomal or lysosome-related organelle sorting receptor expressed in several secretory tissues.

Rab2 and Rab6 are Implicated in Acrosome Formation during Spermatogenesis in Eriocheir sinensis: Based on Sperm Proteome.

Rab proteins are GTP-dependent small proteins that function as regulators of intracellular vesicle transport, fusion, and localization. However, few studies have investigated their function in Decapoda reproduction. The Eriocheir sinensis sperm has no tail and the nuclei are uncondensed. With the acrosome forming the majority of the sperm mass, it provides an ideal model for studying acrosome formation. We firstly analyzed the sperm proteome using LC-MS/MS. To study the functions of Rab2 and Rab6, related to the Golgi apparatus, in the acrosome formation during spermatogenesis, the genes of Rab2 and Rab6 were cloned based on the testis transcriptome of E.sinensis and poly-clonal antibodies were prepared. The presence of 2 Rab proteins was confirmed in the testis and sperm by western blot. We further observed the characteristics of target 2 Rab proteins using immunofluorescence (IF). A total of 1247 proteins including 7 Rab proteins, Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18 were identified in the sperm proteome. The IF results showed that Rab2 co-localizes with GM130, a cis-Golgi matrix protein, in the spermatagonia and spermatocytes. In the early spermatids, Rab2 and Rab6 participate in the formation of pre-acrosomal vesicles. In maturing spermatids, both Rab2 and Rab6 settle on the acrosomal membrane but present different characteristics wrapping the pre-acrosome. In the mature sperm, Rab2 localizes in the perinuclear theca surrounding the nuclei cup, while Rab6 remains on the acrosomal membrane. Our research found 7 Rab proteins based on the analysis of the sperm proteome in E.sinensis, and confirmed the involvement of Rab2 and Rab6 in acrosome formation. These findings provide a foundation for studying the functions of Rab proteins during spermatogenesis in Decapoda animals.

Membrane inward/outward budding and transition pathway induced by the asymmetric solutions

The study of membrane morphology transitions is crucial for understanding various biological processes, such as cell division, and intracellular vesicle transport. In this work, we aim to clarify the molecular mechanism underlying the asymmetric solution-induced membrane shape transition in lipid bilayers. Using giant unilamellar vesicles (GUVs) as model membranes, we investigated the interaction of fully hydrated lipid membranes with two distinct sugar solutions—glucose and sucrose. We applied advanced imaging techniques and theoretical analyses to study membrane deformation and its transition pathways. Our experimental observations revealed that asymmetric sugar solutions induce spontaneous curvature in the phospholipid bilayer, resulting in the formation of outward(/inward) budding vesicles over a wide range of parameters. The direction of the curvature can be reversed by exchanging the internal and external solutions, indicating that the sugar-lipid interactions generate a significant positive(/negative) spontaneous curvature. Theoretically, we attempted to determine the parameter range of stable membrane shapes (i.e., phase diagram) using two dimensionless physical parameters—volume-to-area ratio and normalized spontaneous curvature. Obtaining a phase diagram helps identify the transition pathways of GUVs membrane shapes (experimentally, we captured two possible outward budding vesicles and one inward budding vesicle transition pathway). Our findings can be extended to other types of lipid membranes and provide new insights into the deformation of cell-like micron-sized vesicles, such as exosomes and endosomes, which are increasingly used as biomarkers and drug delivery systems. Moreover, lipid vesicles containing anticancer drugs exhibit fewer side effects than non-liposomal anticancer agents and can effectively target solid tumors.

End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network.

The organization of the mitochondrial network is relevant for the metabolic fate of T cells and their ability to respond to TCR stimulation. This arrangement depends on cytoskeleton dynamics in response to TCR and CD28 activation, which allows the polarization of the mitochondria through their change in shape, and their movement along the microtubules towards the immune synapse. This work focus on the role of End-binding protein 1 (EB1), a protein that regulates tubulin polymerization and has been previously identified as a regulator of intracellular transport of CD3-enriched vesicles. EB1-interferred cells showed defective intracellular organization and metabolic strength in activated T cells, pointing to a relevant connection of the cytoskeleton and metabolism in response to TCR stimulation, which leads to increased AICD. By unifying the organization of the tubulin cytoskeleton and mitochondria during CD4+ T cell activation, this work highlights the importance of this connection for critical cell asymmetry together with metabolic functions such as glycolysis, mitochondria respiration, and cell viability.

A universal strategy of facilitating intracellular delivery of nanomedicines based on tuning ARF6 GTPase to its GTP-bound form

Intracellular delivery crossing the endomembrane barrier is the “last mile to target” for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we establish a universal strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. ARF6, one member of ARF subfamily in small GTPases, is verified to regulate intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. ARF6 biased to GTP-bound state causes the increased endocytosis and reduced exocytosis of eleven types of nanoparticles. This universal effect is derived from the formation of a hybrid type of endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Due to the mild microenvironment in hybrid endosomes, the internalized protein and nanoparticles are steadily delivered to the cytoplasm, avoiding the intensive degradation in lysosomes. Based on these findings, we identify QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, significantly enhances the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, these findings reveal that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.

Elucidation of the Cellular Interactome of African Swine Fever Virus Fusion Proteins and Identification of Potential Therapeutic Targets.

African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins, which potentially mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for those ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Rab geranyl geranylation emerged as a significant hit, and also Rab proteins, which are crucial regulators of the endocytic pathway and interactors of both p34 and E199L. Rab proteins co-ordinate a tight regulation of the endocytic pathway that is necessary for ASFV infection. Moreover, several interactors were proteins involved in the molecular exchange at ER membrane contacts. These ASFV fusion proteins shared interacting partners, suggesting potential common functions. Membrane trafficking and lipid metabolism were important categories, as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages.

Expression of tau and phosphorylated tau in the brain of normal and hemiparkinsonian rhesus macaques.

Tau is a neuronal protein involved in microtubule stabilization and intracellular vesicle transport in axons. In neurodegenerative disorders termed "tauopathies," like Alzheimer's and Parkinson's disease, tau becomes hyperphosphorylated and forms intracellular inclusions. Rhesus macaques are widely used for studying ageing processes and modeling neurodegenerative disorders, yet little is known about endogenous tau expression in their brains. In this study, immunohistochemical methods were used to map and characterize total tau, 3R- and 4R-tau isoforms, and phosphorylated tau (pThr231-tau and pSer202/Thr205-tau/AT8) expression bilaterally in 16 brain regions of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced hemiparkinsonian adult rhesus macaques. Tau-immunoreactivity (-ir), including both 3R and 4R isoforms, was observed throughout the brain, with varying regional intensities. The anterior cingulate cortex, entorhinal cortex, and hippocampus displayed the most robust tau-ir, while the subthalamic nucleus and white matter regions had minimal expression. Tau was present in neurons of gray matter regions; it was preferentially observed in fibers of the globus pallidus and substantia nigra and in cell bodies of the thalamus and subthalamic nucleus. In white matter regions, tau was abundantly present in oligodendrocytes. Additionally, neuronal pThr231-tau-ir was abundant in all brain regions, but not AT8-ir. Differences in regional and intracellular protein expression were not detected between control subjects and both brain hemispheres of MPTP-treated animals. Specifically, tau-ir in the substantia nigra of all subjects colocalized with GABAergic neurons. Overall, this report provides an in-depth characterization of tau expression in the rhesus macaque brain to facilitate future investigations for understanding and modeling tau pathology in this species.

Palmitate-mediated disruption of the endoplasmic reticulum decreases intracellular vesicle motility

Essential cellular processes such as metabolism, protein synthesis, and autophagy require the intracellular transport of membrane-bound vesicles. The importance of the cytoskeleton and associated molecular motors for transport is well documented. Recent research has suggested that the endoplasmic reticulum (ER) may also play a role in vesicle transport through a tethering of vesicles to the ER. We use single-particle tracking fluorescence microscopy and a Bayesian change-point algorithm to characterize vesicle motility in response to the disruption of the ER, actin, and microtubules. This high-throughput change-point algorithm allows us to efficiently analyze thousands of trajectory segments. We find that palmitate-mediated disruption of the ER leads to a significant decrease in vesicle motility. A comparison with the disruption of actin and microtubules shows that disruption of the ER has a significant impact on vesicle motility, greater than the disruption of actin. Vesicle motility was dependent on cellular region, with greater motility in the cell periphery than the perinuclear region, possibly due to regional differences in actin and the ER. Overall, these results suggest that the ER is an important factor in vesicle transport.

Hesperetin Ameliorates Inhibition of Neuronal and Oligodendroglial Cell Differentiation Phenotypes Induced by Knockdown of Rab2b, an Autism Spectrum Disorder-Associated Gene Product.

Autism spectrum disorder (ASD) is a central nervous system (CNS) neurodevelopmental disorder that includes autism, pervasive developmental disorder, and Asperger's syndrome. ASD is characterized by repetitive behaviors and social communication deficits. ASD is thought to be a multifactorial disorder with a range of genetic and environmental factors/candidates. Among such factors is the rab2b gene, although it remains unclear how Rab2b itself is related to the CNS neuronal and glial developmental disorganization observed in ASD patients. Rab2 subfamily members regulate intracellular vesicle transport between the endoplasmic reticulum and the Golgi body. To the best of our knowledge, we are the first to report that Rab2b positively regulates neuronal and glial cell morphological differentiation. Knockdown of Rab2b inhibited morphological changes in N1E-115 cells, which are often used as the neuronal cell differentiation model. These changes were accomplished with decreased expression levels of marker proteins in neuronal cells. Similar results were obtained for FBD-102b cells, which are used as the model of oligodendroglial cell morphological differentiation. In contrast, knockdown of Rab2a, which is another Rab2 family member not known to be associated with ASD, affected only oligodendroglial and not neuronal morphological changes. In contrast, treatment with hesperetin, a citrus flavonoid with various cellular protective effects, in cells recovered the defective morphological changes induced by Rab2b knockdown. These results suggest that knockdown of Rab2b inhibits differentiation in neuronal and glial cells and may be associated with pathological cellular phenotypes in ASD and that hesperetin can recover their phenotypes at the in vitro level at least.

The function of Golgi apparatus in LRRK2-associated Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease associated with the intracellular organelles. Leucine-rich repeat kinase 2 (LRRK2) is a large multi-structural domain protein, and mutation in LRRK2 is associated with PD. LRRK2 regulates intracellular vesicle transport and function of organelles, including Golgi and lysosome. LRRK2 phosphorylates a group of Rab GTPases, including Rab29, Rab8, and Rab10. Rab29 acts in a common pathway with LRRK2. Rab29 has been shown to recruit LRRK2 to the Golgi complex (GC) to stimulate LRRK2 activity and alter the Golgi apparatus (GA). Interaction between LRRK2 and Vacuolar protein sorting protein 52 (VPS52), a subunit of the Golgi-associated retrograde protein (GARP) complex, mediates the function of intracellular soma trans-Golgi network (TGN) transport. VPS52 also interacts with Rab29. Knockdown of VPS52 leads to the loss of LRRK2/Rab29 transported to the TGN. Rab29, LRRK2, and VPS52 work together to regulate functions of the GA, which is associated with PD. We highlight recent advances in the roles of LRRK2, Rabs, VPS52, and other molecules, such as Cyclin-dependent kinase 5 (CDK5) and protein kinase C (PKC) in the GA, and discuss their possible association with the pathological mechanisms of PD.