Mechanism Of Endocytosis Research Articles

Heterologous Expression and Optimization of Fermentation Conditions for Recombinant Ikarugamycin Production.

Ikarugamycin is a member of the natural product family of the polycyclic tetramate macrolactams (PoTeMs). The compound exhibits a diverse range of biological activities, including antimicrobial, antiprotozoal, anti-leukemic, and anti-inflammatory properties. In addition, it interferes with several crucial cellular functions, such as oxidized low-density lipoprotein uptake in macrophages, Nef-induced CD4 cell surface downregulation, and mechanisms of endocytosis. It is, therefore, used as a tool compound to study diverse biological processes. However, ikarugamycin commercial prices are very high, with up to 1300 € per 1 mg, thus limiting its application. We, therefore, set out to develop a high-yielding recombinant production platform of ikarugamycin by screening different expression vectors, recombinant host strains, and cultivation conditions. Overall, this has led to overproduction levels of more than 100 mg/L, which, together with a straightforward purification protocol, establishes biotechnological access to affordable ikarugamycin enabling its increased use in biomedical research in the future.

Massive endocytosis mechanisms are involved in uptake of HIV-1 particles by monocyte-derived dendritic cells

Massive endocytosis mechanisms are involved in uptake of HIV-1 particles by monocyte-derived dendritic cells

Transmission of Peripheral-blood α-Synuclein Fibrils Exacerbates Synucleinopathy and Neurodegeneration in Parkinson's Disease by Endothelial Lag3 Endocytosis.

Background: Parkinson's disease (PD) is an age-related neurodegenerative disorder. The pathological feature of PD is abnormal alpha-synuclein (α-syn) formation and transmission. Recent evidence demonstrates that α-syn preformed fibrils (α-syn PFF) can be detected in the serum of PD patients. The peripheral-blood α-syn PFF can cross the blood-brain barrier and aggravate neuronal damage, but the mechanism remains to be elucidated. Methods: We constructed the PD mouse models of different severity: the mild pathology (A53T ONLY) and the severe pathology (A53T+Brain FIB); this was followed by α-syn PFFs intravenous injection. Then, we used endothelium-specific Lag3 knockout mice (Lag3-ECs-CKO) to decrease the blood α-syn PFFs spreading. Results: We observed that intravenous transmission of α-syn PFFs significantly aggravated motor deficits, dopaminergic neuron loss, neuroinflammation and pathologic α-syn deposition in A53T ONLY, but not in A53T+Brain FIB. Blocking endothelial Lag3 endocytosis by Lag3-ECs-CKO decreased the blood α-syn PFFs spreading and improved the symptoms and pathogenesis of PD mice. Conclusions: Our findings reveal the role of peripheral-blood α-syn PFFs transmission in the mild pathology or early-stage PD and the mechanism of endothelial Lag3 endocytosis in the pathology of α-syn transmission. Targeting endothelial Lag3 to prevent α-syn from spreading from the blood to the brain may be a disease-modifying therapy in early-stage PD.

Shape Effect of Polymer-Based Multilayer Microcapsules on Cellular Internalization.

The intracellular fate of drug carriers had received extensive attention, which was profoundly influenced by the shapes of carriers. However, it has not been fully addressed due to the lack of effective strategies to prepare carriers with different shapes and the interference of other parameters (such as stiffness and chemistry of the shaped particle and the different cell lines). In this work, polymer-based microcapsules with different shapes (spherical, peanut, dumbbell, and cubic) but the same surface chemistry were engineered through the alternative deposition of polyethylenimine (PEI) and polyethylene glycol (PEG) onto the sacrificial CaCO3 templates with different well-defined shapes. Various techniques (SEM, CLSM, AFM, FTIR, and XPS) were utilized to determine the shapes and chemical composition of these microcapsules. The effect of microcapsule shape on cellular internalization kinetics and the endocytosis mechanism was thoroughly studied, and dumbbell and cubic microcapsules showed greater internalization rates and amounts than spherical and peanut microcapsules. These microcapsules were internalized through micropinocytosis, and the shapes had no obvious effect on the endocytosis mechanism. This work provides a wealth of information about the relationship between the shape of microcapsules and their performance in cellular internalization, which will be of great help in the development of related drug carriers.

Structural insights into Semiliki forest virus receptor binding modes indicate novel mechanism of virus endocytosis.

The Very Low-Density Lipoprotein Receptor (VLDLR) is an entry receptor for the prototypic alphavirus Semliki Forest Virus (SFV). However, the precise mechanisms underlying the entry of SFV into cells mediated by VLDLR remain unclear. In this study, we found that of the eight class A (LA) repeats of the VLDLR, only LA2, LA3, and LA5 specifically bind to the native SFV virion while synergistically promoting SFV cell attachment and entry. Furthermore, the multiple cryo-electron microscopy structures of VLDLR-SFV complexes and mutagenesis studies have demonstrated that under physiological conditions, VLDLR primarily binds to E1-DIII of site-1, site-2, and site-1' at the twofold symmetry axes of SFV virion through LA2, LA3, and LA5, respectively. These findings unveil a novel mechanism for viral entry mediated by receptors, suggesting that conformational transitions in VLDLR induced by multivalent binding of LAs facilitate cellular internalization of SFV, with significant implications for the design of antiviral therapeutics.

An Auger electron-loaded theranostic biosensor triggered by the ACE2-mediated virus/host endocytosis

Accurate diagnosis and effective antiviral strategies are critical to combat acute infection and to avoid damage to the host. Due to their restricted radiation range and energy, Auger electron emitters have shown potential as a RNA-destructing radionuclide therapy in oncology and infection. Focusing on the process of angiotensin-converting enzyme 2 (ACE2)-mediated endocytosis, Technetium-99m-labeled DX600 (99mTc-DX600) was synthesized as an Auger electron vector to specifically bind to surface-expressed ACE2 proteins on 293T-hACE2 cells (293T cells stably expressing human ACE2), and Technetium-99m-loaded microvesicles (99mTc-MVs) served as an antiviral tracer and effector in pseudovirus infection. The whole-body ACE2 expression evaluation was non-invasive, meanwhile, the enhanced green fluorescent protein expression of pseudoviruses was substantially inhibited as a result of the 99mTc-DX600 loading of microvesicles, though the mitochondrial and DNA stabilities of the host cells were not affected. Furthermore, the in vivo distribution of 99mTc-DX600 in humanized ACE2 mice was demonstrated to be both ACE2-specific and long-lasting, and an antiviral effect was fully exhibited with two cycles of intravenous injection at a dosage of 37 MBq. Taking advantage of the ACE2-mediated interaction and natural trigger mechanism of virus-induced endocytosis, 99mTc-MV represents a theranostic biosensor of Auger electrons that can expose viral RNA to lethal amounts of radiation, with the host cells receiving no detrimental radiation.

Elucidating the uptake and trafficking of nanostructured lipid carriers as delivery systems for miRNA

Cationic nanostructured lipid carriers (cNLCs) represent promising non-viral carriers for nucleic acids, such as miRNAs, forming stable self-assembled miRNA complexes due to electrostatic interactions. Prepared by high-pressure homogenization, cNLC formulations, both with and without Nile Red dye demonstrated stable particle sizes in the range of 100–120 nm and positive surface charges (>30 mV), which are necessary for effective cellular uptake. The miRNA complexes formed at mass ratios of 1:2.5 and 1:5 showed similar stability and size, with positive zeta potentials, as well as high cell viability (> 80 %) in 3T3-L1 and MCF-7 cell lines. The cellular uptake studies of miRNA:cNLC complexes in both cell lines revealed that uptake was time- and concentration-dependent, with rapid initial uptake in 30 min and a zig-zag pattern over 24 h. To elucidate the endocytosis mechanism of miRNA:cNLC complexes, 3T3-L1 and MCF-7 cells were incubated with different inhibitors (chlorpromazine, 5-[N-ethyl-N-isopropyl] amiloride, dynasore, nystatin, or sodium azide with 2-deoxy-d-glucose). Results showed significant inhibition of uptake at low temperatures and with ATP depletion, suggesting endocytosis, particularly macropinocytosis, as the main uptake mechanism in 3T3-L1 cells. In MCF-7 cells, the uptake was less inhibited by the substances, indicating the need for more specific methods to fully decipher the endocytic mechanisms involved. Confocal laser scanning microscopy images revealed that the complexes are internalized in vesicles, and are primarily localized in the juxtanuclear region, suggesting trafficking through the endolysosomal system. Colocalization study with LysoTracker™ Green DND-26 showed significant colocalization of miRNA:cNLC complexes with lysosomes in 3T3-L1 cells, indicating trafficking through the endolysosomal system. In MCF-7 cells, colocalization was lower, suggesting macropinocytosis as the primary uptake mechanism. Additional studies showed partial colocalization between labeled NLCs and miRNA, indicating that about 50 % of miRNA is released from NLCs within 30 min post-transfection.

Visualization of endogenous G proteins on endosomes and other organelles

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20–30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

Visualization of endogenous G proteins on endosomes and other organelles.

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

Smart Mesoporous Silica Nanoparticles Loading Curcumin Inhibit Liver Cancer.

Curcumin (CUR) as one of the natural edible pigments is approved by the World Health Organization due to its nontoxic and anticancer effect. However, the utility of CUR is restricted due to its low oral bioavailability. Nanoparticle drug delivery systems like mesoporous silica nanoparticles (MSNs) have been extensively used due to their high specific surface area, high loading rate, and ease of modification. This study developed lactobionic acid (LA)-modified carboxymethyl chitosan (CMCS)-coated MSNs to deliver CUR specifically targeting hepatocellular carcinoma. Among these nanoparticles, LA targets liver cancer cells. CMCS utilizes pH-responsive release of CUR. The LA-CMCS-MSN@CUR (MSN@CUR) were evaluated using several methods, including Fourier transform infrared spectroscopy, transmission electron microscopy, and zeta potential measurements. Liver cellular uptake of MSN@CUR depends on a specific LA receptor-mediated endocytosis mechanism. Additionally, MSN@CUR performed with a better antitumor effect than Cur in H22 orthotopic transplantation of liver cancer and H22 solid tumor mouse models. Treatment with MSN@CUR significantly reduced the protein of VEGF, p-PI3K, and AKT, increased the protein of caspases 3 and 8, ultimately inhibited tumor migration, and promoted apoptosis. This study provides a new path for delivery of natural active ingredients with excellent bioavailability in the antitumor field.

Virus-mimicking nanodrug active crossing of the blood-brain barrier via transcytosis against central nervous system leukemia

The poor central nervous system leukemia (CNSL) clinical efficacy of conventional doses of chemotherapy is mainly attributed to the limited permeability of chemotherapy agents caused by the blood-brain barrier (BBB). Effectively enhancing the accumulation of drugs across the BBB in the central nervous system is one of the key challenges in improving patient compliance and clinical efficacy of CNSL. Here, we find that the VP1 protein, the functional module of the John Cunningham (JC) virus, can safely penetrate the BBB through a sialic acid receptor-mediated transcytosis mechanism. Based on this, we develop a JC virus-mimicking nanodrug delivery platform based on VP1 protein-conjugated self-assembled nanoparticles (MFHV), which can active target and cross the BBB via a receptor-mediated transcytosis for safe and effective low-dose chemotherapy against CNSL after systemic administration. The results demonstrate that such a platform can penetrate the BBB through the dual mechanism of clathrin-mediated endocytosis and micropinocytosis pathway. When further synergistic with ferroptosis and histamine metabolism, the long-term survivors of low-dose MTX are significantly enhanced by 83.3 % and 56.7 % in two CNSL mice models. Collectively, this study takes a new perspective on natural living materials and molecule targeting of the BBB to present a promising strategy for low-dose chemotherapy against CNSL with safety and efficacy, which might provide a clinically translatable option for the prevention and treatment of CNSL.

Tissue-Specific Regulation of Vesicular Trafficking Mediated by Rab-GEF Complex MON1/CCZ1 From Solanum chilense Increases Salt Stress Tolerance in Arabidopsis thaliana.

Salt stress constrains the development and growth of plants. To tolerate it, mechanisms of endocytosis and vacuolar compartmentalization of Na+ are induced. In this work, the genes that encode a putative activator of vesicular trafficking called MON1/CCZ1 from Solanum chilense, SchMON1 and SchCCZ1, were co-expressed in roots of Arabidopsis thaliana to determine whether the increase in prevacuolar vesicular trafficking also increases the Na+ compartmentalization capacity and tolerance. Initially, we demonstrated that both SchMON1 and SchCCZ1 genes rescued the dwarf phenotype of both A. thaliana mon1-1 and ccz1a/b mutants associated with the loss of function, and both proteins colocalized with their functional targets, RabF and RabG, in endosomes. Transgenic A. thaliana plants co-expressing these genes improved salt stress tolerance compared to wild type plants, with SchMON1 contributing the most. At the sub-cellular level, co-expression of SchMON1/SchCCZ1 reduced ROS levels and increased endocytic activity, and number of acidic structures associated with autophagosomes. Notably, greater Na+ accumulation in vacuoles of cortex and endodermis was evidenced in the SchMON1 genotype. Molecular analysis of gene expression in each genotype supported these results. Altogether, our analysis shows that root activation of prevacuolar vesicular trafficking mediated by MON1/CCZ1 emerges as a promising physiological molecular mechanism to increase tolerance to salt stress in crops of economic interest.

The pathogenic responses elicited during exposure of human intestinal cell line with Giardia duodenalis excretory-secretory products and the potential attributed endocytosis mechanism.

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism.

Clathrin-Mediated Endocytosis of Multiple Nanoparticles Tends to Be Less Cooperative: A Computational Study.

The internalization of nanoparticles is of great significance for their biological applications. Clathrin-mediated endocytosis (CME) is one of the main endocytic pathways. However, there is still a lack of a fundamental understanding regarding the internalization of multiple nanoparticles via CME. Therefore, in this study, we conducted computational investigations to uncover detailed molecular mechanisms and kinetic pathways for differently shaped nanoparticles in the presence of clathrin. Particular focus is given to understanding the CME of multiple-nanoparticle systems. We found that unlike receptor-mediated endocytosis, multiple nanoparticles did not get cooperatively wrapped by the membrane but tended to undergo independent endocytosis in the presence of clathrin. To further investigate the endocytosis mechanism, we studied the effects of clathrins, nanoparticle shape, nanoparticle size, nanoparticle arrangement, and membrane surface tension. The self-assembly of clathrin prefers independent endocytosis for multiple nanoparticles. Besides, the cooperative behavior is weak with increasing nanoparticle-shape anisotropy. However, when the membrane tension is reduced, the endocytosis pathway for multiple nanoparticles is cooperative endocytosis. Moreover, we found that the self-assembly of clathrins reduces the critical size of nanoparticles to undergo cooperative wrapping by the cell membrane. Our results provide valuable insights into the molecular mechanisms of multiple nanoparticles through CME and offer useful guidance for the design of nanoparticles as drug/gene delivery carriers.

Potential Advantages of Idarubicin-Loaded Trastuzumab-Coated Liposomes for Combating Head and Neck Squamous Cancer Cells.

Head and neck squamous cell carcinoma (HNSCC) with a high mortality rate is among the most common types of cancer in the world. Human epidermal growth factor receptor 2 (HER2) is expressed higher than normal level in the most HNSCC tumors, making them resistant to chemotherapy and radiotherapy. Therefore, HER2 has been introduced as a suitable target for anticancer drugs. The aim of this study is to examine the efficacy of a treatment protocol involving targeted delivery of idarubicin encapsulated in trastuzumab-decorated liposomes to HNSCC cells. In the current experimental study, efficacies of idarubicin, prepared liposomal idarubicin, and constructed immunoliposomal idarubicin (trastuzumab-decorated) were investigated in killing HN5 cells, a HER2- overexpressing HNSCC-originating cell line. Liposomal content of idarubicin and trastuzumab were qualified by UVVisible spectroscopy and preparations were characterized for shape and size by atomic force microscopy (AFM) and dynamic light scattering (DLS). To clarify role of the missing parts of the available crystal structure (PDB ID: 1n8z) within trastuzumab-HER2 interactions, we used a 40 ns molecular dynamic simulation approach. Based on the obtained results, liposomal idarubicin showed higher toxicity of the encapsulated drug on HN5 cells compared to the traditional free drug formulations. The immunoliposomal form of idarubicin was more effective than the liposomal formulation, in killing of HN5 cells. In addition, simulation of interactions between trastuzumab and HER2 revealed that the missing parts (in the crystal structure) of HER2 have critical interaction with trastuzumab, through salt-bridges and hydrogen bonds. It seems that the prepared immunoliposomes could attach more efficiently to HER2 overexpressing cells, which consequently leads to increasing cellular uptake of idarubicin through a receptor-mediated endocytosis mechanism. Moreover, simulation of the interaction between HER2 and trastuzumab suggested considerable possibilities for increasing trastuzumab affinity to HER2.

Three-Dimensional Lymphatics-on-a-Chip Reveals Distinct, Size-Dependent Nanoparticle Transport Mechanisms in Lymphatic Drug Delivery.

Although nanoparticle-based lymphatic drug delivery systems promise better treatment of cancer, infectious disease, and immune disease, their clinical translations are limited by low delivery efficiencies and unclear transport mechanisms. Here, we employed a three-dimensional (3D) lymphatics-on-a-chip featuring an engineered lymphatic vessel (LV) capable of draining interstitial fluids including nanoparticles. We tested lymphatic drainage of different sizes (30, 50, and 70 nm) of PLGA-b-PEG nanoparticles (NPs) using the lymphatics-on-a-chip device. In this study, we discovered that smaller NPs (30 and 50 nm) transported faster than larger NPs (70 nm) through the interstitial space, as expected, but the smaller NPs were captured by lymphatic endothelial cells (LECs) and accumulated within their cytosol, delaying NP transport into the lymphatic lumen, which was not observed in larger NPs. To examine the mechanisms of size-dependent NP transports, we employed four inhibitors, dynasore, nystatin, amiloride, and adrenomedullin, to selectively block dynamin-, caveolin-, macropinocytosis-mediated endocytosis-, and cell junction-mediated paracellular transport. Inhibiting dynamin using dynasore enhanced the transport of smaller NPs (30 and 50 nm) into the lymphatic lumen, minimizing cytosolic accumulation, but showed no effect on larger NP transport. Interestingly, the inhibition of caveolin by nystatin decreased the lymphatic transport of larger NPs without affecting the smaller NP transport, indicating distinct endocytosis mechanisms used by different sizes of NPs. Macropinocytosis inhibition by amiloride did not change the drainage of all sizes of NPs; however, paracellular transport inhibition by adrenomedullin blocked the lymphatic transport of NPs of all sizes. We further revealed that smaller NPs were captured in the Rab7-positive late-stage lymphatic endosomes to delay their lymphatic drainage, which was reversed by dynamin inhibition, suggesting that Rab7 is a potential target to enhance the lymphatic delivery of smaller NPs. Together, our 3D lymphatics-on-a-chip model unveils size-dependent NP transport mechanisms in lymphatic drug delivery.

SV2A controls the surface nanoclustering and endocytic recruitment of Syt1 during synaptic vesicle recycling.

Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a 'pre-assembly' mechanism for endocytosis that ensures high-fidelity retrieval of SV cargo. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1K326A/K328A) and SV2A knockdown. SV2A co-clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin-1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs.

A new mechanism of self-lipid endocytosis by endothelial cells mediated by the receptor Mincle

A new mechanism of self-lipid endocytosis by endothelial cells mediated by the receptor Mincle

Visualization of endogenous G proteins on endosomes and other organelles.

Classical G protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

CD2AP is Co-Expressed with Tropomyosin-Related Kinase A and Ras-Related Protein Rab-5A in Cholinergic Neurons of the Murine Basal Forebrain.

Basal forebrain cholinergic neurons project to the hippocampus and cortex, are critical for learning and memory, and are central to the pathogenesis of Alzheimer's disease (AD). GWAS have consistently shown that genomic variants at the CD2AP gene locus are associated with significant increased risk of AD. GWAS studies have also shown that genetic variants in endocytosis genes, including RAB5A , significantly increase susceptibility to AD. Previous work in our lab has shown that CD2AP functions as a docking-scaffold/adaptor protein as a coordinator of nerve growth factor (NGF) and trophic signaling in neurons. We have also demonstrated that CD2AP positively regulates Rab5-mediated mechanisms of endocytosis in primary sensory neurons. The purpose of this study was to perform an in vivo characterization of CD2AP expression in cholinergic neurons of the brain regions most relevant to AD pathogenesis and to investigate the colocalization of CD2AP and Rab5 in cholinergic neurons of the murine basal forebrain. Brain tissue was perfused, harvested from ChAT BAC -eGFP transgenic mice (N=4 male, N=4 female; aged 10 mo), where cholinergic neurons (co-) express green fluorescence protein (GFP) in central and peripheral neurons that express choline acetyltransferase (ChAT). Frozen tissue sections were used to assess the specificity of the reporter in mouse brain along with localization of both CD2AP and Rab5 (co-) expression using immunofluorescence (IF) analysis of ChAT-GFP+ neurons and primary antibodies against ChAT, CD2AP and Rab5. Image J software was used to develop and optimize a colocalization assay for CD2AP and Rab5 puncta. Experiments were repeated in a follow-up cohort of aged-adult mice (N=2 male, N=2 female; aged 18 mo). IF expression of CD2AP was quantified in the basal forebrain, diagonal band of Broca (vDB), and striatal regions and compared to results from the cortical regions of the adult mouse brain. Colocalization of CD2AP was observed in the cell bodies of ChAT-GFP+ neurons of the striatum, vDB and basal forebrain regions, where CD2AP expression intensity as well as the number of cell bodies with positive signal increased incrementally. Colocalization analyses revealed near-complete overlap of CD2AP and Rab5 expression in ChAT-GFP+ cholinergic neurons of the basal forebrain region. We conclude that cholinergic neurons express CD2AP in healthy adult and aged-adult mouse brains. These data provide the first evidence of quantifiable CD2AP protein expression of cholinergic neurons specific to the diagonal band of Broca (vDB) and basal forebrain. Together with previous research from our lab, these data support a role for CD2AP in the pathogenesis of AD through orchestration of endocytosis and retrograde signaling. Ongoing studies are underway to verify these findings in a novel AD mouse model that incorporates the humanized variant of CD2AP , created by MODEL-AD, where we aim to further investigate how CD2AP variants may affect mechanistic components of Rab5 endocytosis as well as subsequent survival of cholinergic neurons in the context of known amyloid beta and Tau pathologies.