GPC3-Unc5 receptor complex structure and role in cell migration.

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.

SMOC-1 interacts with both BMP and glypican to regulate BMP signaling in C. elegans.

Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium-binding (EC) domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. In this study, we used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. We showed that CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. We provide both in vitro biochemical and in vivo functional evidence demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. We further showed that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. Our work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling.

PIK3CA variants selectively initiate brain hyperactivity during gliomagenesis.

Glioblastoma is a universally lethal form of brain cancer that exhibits an array of pathophysiological phenotypes, many of which are mediated by interactions with the neuronal microenvironment1,2. Recent studies have shown that increases in neuronal activity have an important role in the proliferation and progression of glioblastoma3,4. Whether there is reciprocal crosstalk between glioblastoma and neurons remains poorly defined, as the mechanisms that underlie how these tumours remodel the neuronal milieu towards increased activity are unknown. Here, using a native mouse model of glioblastoma, we develop a high-throughput in vivo screening platform and discover several driver variants of PIK3CA. We show that tumours driven by these variants have divergent molecular properties that manifest in selective initiation of brain hyperexcitability and remodelling of the synaptic constituency. Furthermore, secreted members of the glypican (GPC) family are selectively expressed in these tumours, and GPC3 drives gliomagenesis and hyperexcitability. Together, our studies illustrate the importance of functionally interrogating diverse tumour phenotypes driven by individual, yet related, variants and reveal how glioblastoma alters the neuronal microenvironment.

Glypicans shield the Wnt lipid moiety to enable signalling at a distance.

A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1-3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4-6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.

Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

Astrocyte-secreted glypican-4 drives APOE4-dependent tau hyperphosphorylation.

Tau protein aggregates are a major driver of neurodegeneration and behavioral impairments in tauopathies, including in Alzheimer's disease (AD). Apolipoprotein E4 (APOE4), the highest genetic risk factor for late-onset AD, has been shown to exacerbate tau hyperphosphorylation in mouse models. However, the exact mechanisms through which APOE4 induces tau hyperphosphorylation remains unknown. Here, we report that the astrocyte-secreted protein glypican-4 (GPC-4), which we identify as a binding partner of APOE4, drives tau hyperphosphorylation. We discovered that first, GPC-4 preferentially interacts with APOE4 in comparison to APOE2, considered to be a protective allele to AD, and second, that postmortem APOE4-carrying AD brains highly express GPC-4 in neurotoxic astrocytes. Furthermore, the astrocyte-secreted GPC-4 induced both tau accumulation and propagation in vitro. CRISPR/dCas9-mediated activation of GPC-4 in a tauopathy mouse model robustly induced tau hyperphosphorylation. In the absence of GPC4, APOE4-induced tau hyperphosphorylation was largely diminished using in vitro tau fluorescence resonance energy transfer-biosensor cells, in human-induced pluripotent stem cell-derived astrocytes and in an in vivo mouse model. We further show that APOE4-mediated surface trafficking of APOE receptor low-density lipoprotein receptor-related protein 1 through GPC-4 can be a gateway to tau spreading. Collectively, these data support that APOE4-induced tau hyperphosphorylation is directly mediated by GPC-4.

GPC2 promotes prostate cancer progression via MDK-mediated activation of PI3K/AKT signaling pathway.

Prostate cancer is a major medical problem for men worldwide. Advanced prostate cancer is currently incurable. Recently, much attention was paid to the role of GPC2 in the field of oncology. Nevertheless, there have been no investigations of GPC2 and its regulatory mechanism in prostate cancer. Here, we revealed a novel action of GPC2 and a tumor promoting mechanism in prostate cancer. GPC2 was upregulated in prostate cancer tissues and cell lines. Higher expression of GPC2 was correlated with higher Gleason score, lymphatic metastasis, and worse overall survival in prostate cancer patients. Decreased expression of GPC2 inhibited cell proliferation, migration, and invasion in prostate cancer, whereas GPC2 overexpression promoted these properties. Mechanistically, GPC2 promoted the activation of PI3K/AKT signaling pathway through MDK. The rescue assay results in prostate cancer cells demonstrated that overexpression of MDK could attenuate GPC2 knockdown induced inactivation of PI3K/AKT signaling and partly reverse GPC2 knockdown induced inhibition of cell proliferation, migration, and invasion. In all, our study identified GPC2 as an oncogene in prostate cancer. GPC2 promoted prostate cancer cell proliferation, migration, and invasion via MDK-mediated activation of PI3K/AKT signaling pathway. GPC2 might be a promising prognosis predictor and potential therapeutic target in prostate cancer.

Regulation of Wnt distribution and function by Drosophila glypicans.

The extracellular distribution of secreted Wnt proteins is crucial for their ability to induce a response in target cells at short and long ranges to ensure proper development. Wnt proteins are evolutionarily conserved ligands that are lipid-modified, and their hydrophobic nature interferes with their solubility in the hydrophilic extracellular environment. This raises the question of how Wnt proteins spread extracellularly despite their lipid modifications, which are essential for both their secretion and function. Seminal studies on Drosophila Wingless (Wg), a prototypical Wnt, have discovered multiple mechanisms by which Wnt proteins spread. A central theme emerges from these studies: the Wnt lipid moiety is shielded from the aqueous environment, allowing the ligands to spread and remain viable for signaling. Wnt distribution in vivo is primarily facilitated by glypicans, which are cell-surface heparan sulfate proteoglycans, and recent studies have further provided mechanistic insight into how glypicans facilitate Wnt distribution. In this Review, we discuss the many diverse mechanisms of Wnt distribution, with a particular focus on glypican-mediated mechanisms.

Hedgehog pathway modulation by glypican 3-conjugated heparan sulfate.

Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure. Here, we investigate how glypican 3 (GPC3), which is mutated in Simpson-Golabi-Behmel tissue overgrowth syndrome, regulates Hedgehog signaling. We find that GPC3 is necessary for the Hedgehog response, surprisingly controlling a downstream signal transduction step. Purified GPC3 ectodomain rescues signaling when artificially recruited to the surface of GPC3-deficient cells but has dominant-negative activity when unattached. Strikingly, the purified stalk, modified with heparan sulfate but not chondroitin sulfate, is necessary and sufficient for activity. Our results demonstrate a novel function for GPC3-associated heparan sulfate and provide a framework for the functional dissection of glycosaminoglycans by in vivo biochemical complementation. This article has an associated First Person interview with the first author of the paper.

Steric Hindrance-Mediated Enzymatic Reaction Enable Homogeneous Dual Fluorescence Indicators Aptasensing of Hepatocellular Carcinoma CTCs.

Circulating tumor cells (CTCs) serve as important biomarkers in the liquid biopsy of hepatocellular carcinoma (HCC). Herein, a homogeneous dual fluorescence indicators aptasensing strategy is described for CTCs in HCC, with the core assistance of a steric hindrance-mediated enzymatic reaction. CTCs in the sample could specifically bind to a 5'-biotin-modified glypican-3 (GPC3) aptamer and remove the steric hindrance formed by the biotin-streptavidin system. This influences the efficiency of the terminal deoxynucleotidyl transferase enzymatic reaction. Then, methylene blue (MB) was introduced to react with the main product poly cytosine (polyC) chain, and trivalent cerium ion (Ce3+) was added to react with the byproduct pyrophosphate to form fluorescent pyrophosphate cerium coordination polymeric nanoparticles. Finally, the CTCs were quantified by dual fluorescence indicators analysis. Under optimized conditions, the linear range was 5 to 104 cells/mL, and the limits of detection reached 2 cells/mL. Then, 40 clinical samples (15 healthy and 25 HCC patients) were analyzed. The receiver operating characteristic curve analysis revealed an area under the curve of 0.96, a sensitivity of 92%, and a specificity of 100%. Therefore, this study established a sensitive and accurate CTCs sensing system for clinical HCC patients, promoting early tumor diagnosis.

Glypican 4 regulates planar cell polarity of endoderm cells by controlling the localization of Cadherin 2.

Noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling has been implicated in endoderm morphogenesis. However, the underlying cellular and molecular mechanisms of this process are unclear. We found that, during convergence and extension (C&E) in zebrafish, gut endodermal cells are polarized mediolaterally, with GFP-Vangl2 enriched at the anterior edges. Endoderm cell polarity is lost and intercalation is impaired in the absence of glypican 4 (gpc4), a heparan-sulfate proteoglycan that promotes Wnt/PCP signaling, suggesting that this signaling is required for endodermal cell polarity. Live imaging revealed that endoderm C&E is accomplished by polarized cell protrusions and junction remodeling, which are impaired in gpc4-deficient endodermal cells. Furthermore, in the absence of gpc4, Cadherin 2 expression on the endodermal cell surface is increased as a result of impaired Rab5c-mediated endocytosis, which partially accounts for the endodermal defects in these mutants. These findings indicate that Gpc4 regulates endodermal planar cell polarity during endoderm C&E by influencing the localization of Cadherin 2. Thus, our study uncovers a new mechanism by which Gpc4 regulates planar cell polarity and reveals the role of Wnt/PCP signaling in endoderm morphogenesis.

Soluble vascular endothelial glycocalyx proteoglycans as potential therapeutic targets in inflammatory diseases.

Reducing the activity of cytokines and leukocyte extravasation is an emerging therapeutic strategy to limit tissue-damaging inflammatory responses and restore immune homeostasis in inflammatory diseases. Proteoglycans embedded in the vascular endothelial glycocalyx, which regulate the activity of cytokines to restrict the inflammatory response in physiological conditions, are proteolytically cleaved in inflammatory diseases. Here we critically review the potential of proteolytically shed, soluble vascular endothelial glycocalyx proteoglycans to modulate pathological inflammatory responses. Soluble forms of the proteoglycans syndecan-1, syndecan-3 and biglycan exert beneficial anti-inflammatory effects by the removal of chemokines, suppression of proinflammatory cytokine expression and leukocyte migration, and induction of autophagy of proinflammatory M1 macrophages. By contrast, soluble versikine and decorin enhance proinflammatory responses by increasing inflammatory cytokine synthesis and leukocyte migration. Endogenous syndecan-2 and mimecan exert proinflammatory effects, syndecan-4 and perlecan mediate beneficial anti-inflammatory effects and glypican regulates Hh and Wnt signaling pathways involved in systemic inflammatory responses. Taken together, targeting the vascular endothelial glycocalyx-derived, soluble syndecan-1, syndecan-2, syndecan-3, syndecan-4, biglycan, versikine, mimecan, perlecan, glypican and decorin might be a potential therapeutic strategy to suppress overstimulated cytokine and leukocyte responses in inflammatory diseases.

Oncofetal protein glypican-3 is a biomarker and critical regulator of function for neuroendocrine cells in prostate cancer.

Neuroendocrine (NE) cells comprise ~1% of epithelial cells in benign prostate and prostatic adenocarcinoma (PCa). However, they become enriched in hormonally treated and castration-resistant PCa (CRPC). In addition, close to 20% of hormonally treated tumors recur as small cell NE carcinoma (SCNC), composed entirely of NE cells, which may be the result of clonal expansion or lineage plasticity. Since NE cells do not express androgen receptors (ARs), they are resistant to hormonal therapy and contribute to therapy failure. Here, we describe the identification of glypican-3 (GPC3) as an oncofetal cell surface protein specific to NE cells in prostate cancer. Functional studies revealed that GPC3 is critical to the viability of NE tumor cells and tumors displaying NE differentiation and that it regulates calcium homeostasis and signaling. Since our results demonstrate that GPC3 is specifically expressed by NE cells, patients with confirmed SCNC may qualify for GPC3-targeted therapy which has been developed in the context of liver cancer and displays minimal toxicity due to its tumor-specific expression. © 2023 The Pathological Society of Great Britain and Ireland.

Synthesis and preclinical evaluation of a novel probe [18F]AlF-NOTA-IPB-GPC3P for PET imaging of GPC3 positive tumor.

Glypican-3 (GPC3) is markedly overexpressed in hepatocellular carcinoma (HCC) and not expressed in normal liver tissues. In this study, a novel peptide PET imaging agent ([18F]AlF-NOTA-IPB-GPC3P) was developed to target GPC3 expressed in tumors. The overall radiochemical yield of [18F]AlF-NOTA-IPB-GPC3P was 10-15 %, and its lipophilicity, expressed as the logD value at a pH of 7.4, was -1.18 ± 0.06 (n = 3). Compared to the previously reported tracer [18F]AlF-GP2633, [18F]AlF-NOTA-IPB-GPC3P exhibited higher cellular uptake (15.13 vs 5.96) and internalized rate (80.63 % vs 35.93 %) in Huh7 cells at 120 min. Micro-PET/CT and biodistribution studies further demonstrated that [18F]AlF-NOTA-IPB-GPC3P exhibited significantly increased tumor uptake and prolonged tumor residence in Huh7 tumors compared to [18F]AlF-GP2633 (4.66 ± 0.22 % ID/g vs 0.72 ± 0.09 % ID/g at 60 min, p < 0.001; 5.05 ± 0.23 % ID/g vs 0.35 ± 0.08 % ID/g at 120 min, p < 0.001, respectively). Furthermore, the tumor-to-organ ratios of [18F]AlF-NOTA-IPB-GPC3P surpassed those of [18F]AlF-GP2633. Our results support the utilization of [18F]AlF-NOTA-IPB-GPC3P as a PET imaging agent targeting the GPC3 receptor for tumor detection.

GPC3-targeted CAR-T cells expressing GLUT1 or AGK exhibit enhanced antitumor activity against hepatocellular carcinoma.

Chimeric antigen receptor-expressing T (CAR-T) cells induce robust antitumor responses in patients with hematologic malignancies. However, CAR-T cells exhibit only limited efficacy against solid tumors such as hepatocellular carcinoma (HCC), partially due to their limited expansion and persistence. CD8+ T cells, as key components of the adaptive immune response, play a central role in antitumor immunity. Aerobic glycolysis is the main metabolic feature of activated CD8+ T cells. In the tumor microenvironment, however, the uptake of large amounts of glucose by tumor cells and other immunosuppressive cells can impair the activation of T cells. Only when tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment have a glycolytic advantage might the effector function of T cells be activated. Glucose transporter type 1 (GLUT1) and acylglycerol kinase (AGK) can boost glycolytic metabolism and activate the effector function of CD8+ T cells, respectively. In this study, we generated GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK for the treatment of HCC. GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK specifically and effectively lysed GPC3-positive tumor cells in vitro in an antigen-dependent manner. Furthermore, GLUT1 or AGK overexpression protected CAR-T cells from apoptosis during repeated exposures to tumor cells. Compared with second-generation CAR-T cells, GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK exhibited greater CD8+ T-cell persistence in vivo and better antitumor effects in HCC allograft mouse models. Finally, we revealed that GLUT1 or AGK maintained anti-apoptosis ability in CD8+ T cells via activation of the PI3K/Akt pathway. This finding might identify a therapeutic strategy for advanced HCC.

20(S)-Ginsenoside Rh2 inhibits hepatocellular carcinoma by suppressing angiogenesis and the GPC3-mediated Wnt/ß­catenin signaling pathway.

20(S)-Ginsenoside Rh2 has significant anti-tumor effects in various types of cancers, including human hepatocellular carcinoma (HCC). However, its molecular targets and mechanisms of action remain largely unknown. Here, we aim to elucidate the potential mechanisms by which Rh2 suppresses HCC growth. We first demonstrate the role of Rh2 in inhibiting angiogenesis. We observe that Rh2 effectively suppresses cell proliferation and induces apoptosis in HUVECs. Furthermore, Rh2 significantly inhibits HepG2-stimulated HUVEC proliferation, migration and tube formation, accompanied by the downregulation of VEGF and MMP-2 expressions. We also reveal that Rh2 inhibits HCC growth through the downregulation of glypican-3-mediated activation of the Wnt/ß-catenin pathway. We observe a dose-dependent inhibition of proliferation and induction of apoptosis in HepG2 cells upon Rh2 treatment, which is mediated by the inhibition of glypican-3/Wnt/ß-catenin signaling. Moreover, downregulation of glypican-3 expression enhances the effects of Rh2 on the glypican-3/Wnt/ß-catenin signaling pathway, resulting in greater suppression of tumor growth in HepG2 cells. Collectively, our findings shed light on the molecular mechanisms through which Rh2 modulates HCC growth, which involve the regulation of angiogenesis and the glypican-3/Wnt/ß-catenin pathway. These insights may pave the way for the development of novel therapeutic strategies targeting these pathways for the treatment of HCC.

Alpha-fetoprotein producing endometrioid carcinoma arising in an adenomyoma of the uterus.

We report a case of alpha-fetoprotein-producing endometrioid carcinoma (AFP-EC) that originated within an adenomyoma of the uterine corpus. A 76-year-old Japanese woman was incidentally discovered to have a uterine tumor along with multiple lung nodules. Upon surgical removal of the uterus, it was revealed that the tumor was situated within the adenomyoma. The tumor exhibited microfollicular structures and solid growth patterns, with hyaline globules, clear cell glands, and primitive tumor cells. Immunohistochemical analysis indicated the presence of germ cell markers, including AFP, SALL4, and glypican3, leading to final diagnosis of AFP-EC. Histopathologically, AFP-ECs exhibit characteristics similar to those of AFP-producing neoplasms in other organs. Furthermore, a nomenclature issue arises when distinguishing AFP-ECs from yolk sac tumors of the endometrium in older patients due to their shared features. The concept of retrodifferentiation or neometaplasia suggests that "endometrioid carcinoma with yolk sac tumor differentiation" or "endometrioid carcinoma with a primitive phenotype" may serve as more fitting terms for the diverse spectrum of AFP-producing neoplasms in the endometrium. In conclusion, this case underscores the diagnostic challenges posed by AFP-ECs arising from adenomyomas and emphasizes the need for refining the nomenclature and classification of AFP-producing neoplasms within the endometrium.

Proteomic Investigation of Differential Interactomes of Glypican 1 and a Putative Disease-Modifying Variant of Ataxia.

In a currently 13-year-old girl of consanguineous Turkish parents, who developed unsteady gait and polyneuropathy at the ages of 3 and 6 years, respectively, we performed whole genome sequencing and identified a biallelic missense variant c.424C>T, p.R142W in glypican 1 (GPC1) as a putative disease-associated variant. Up to date, GPC1 has not been associated with a neuromuscular disorder, and we hypothesized that this variant, predicted as deleterious, may be causative for the disease. Using mass spectrometry-based proteomics, we investigated the interactome of GPC1 WT and the missense variant. We identified 198 proteins interacting with GPC1, of which 16 were altered for the missense variant. This included CANX as well as vacuolar ATPase (V-ATPase) and the mammalian target of rapamycin complex 1 (mTORC1) complex members, whose dysregulation could have a potential impact on disease severity in the patient. Importantly, these proteins are novel interaction partners of GPC1. At 10.5 years, the patient developed dilated cardiomyopathy and kyphoscoliosis, and Friedreich's ataxia (FRDA) was suspected. Given the unusually severe phenotype in a patient with FRDA carrying only 104 biallelic GAA repeat expansions in FXN, we currently speculate that disturbed GPC1 function may have exacerbated the disease phenotype. LC-MS/MS data are accessible in the ProteomeXchange Consortium (PXD040023).

Glypican-4 regulated actin cytoskeletal reorganization in glucocorticoid treated trabecular meshwork cells and involvement of Wnt/PCP signaling.

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.

Interplay between APP and glypican-1 processing and α-synuclein aggregation in undifferentiated and differentiated human neural progenitor cells.

In Parkinson's disease, there is an accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. In neural cells, cytokine-induced SYN aggregation is modulated by heparan sulfate (HS) derived from glypican-1 (GPC1) by amyloid precursor protein (APP) and nitric oxide (NO)-dependent cleavage. We have explored possible interplay between APP, GPC1, and SYN in undifferentiated and differentiated neural progenitor cells (NPCs) by modulating APP and GPC1 processing. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies recognizing APP degradation products, HS released from GPC1, and SYN aggregates (filamentous SYN [SYNfil]). Suppression of HS release from GPC1 by inhibition of ß-secretase or by NO deprivation resulted in no or slight increase in SYNfil aggregation. Stimulation of HS release by ascorbate did not further increase SYNfil staining. Interleukin-6 (IL-6) induced increased APP and GPC1 processing and SYNfil formation, which was reduced when ß-secretase was inhibited and when HS release was impeded by NO deprivation. Ascorbate restored APP and GPC1 processing but did not affect SYNfil formation. Ascorbate-dependent differentiation of NPC resulted in the expression of tyrosine hydroxylase (TH) which colocalized with SYNfil. Suppression of APP processing by inhibition of ß-secretase greatly disturbed the differentiation process. IL-6 induced coclustering of APP-degradation products, TH, HS, and SYNfil, which could be reversed by stimulation of HS release from GPC1 by excess ascorbate. We suggest that continuous release of HS from GPC1 moderates SYN aggregation and supports differentiation of NPC to dopaminergic neurons.