Browsing by Subject "Extracellular Matrix Proteins"

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ApoE Receptors in Alzheimer's and CNS Function
(2017-03-24) Lane-Donovan, Courtney; Powell, Craig M.; Herz, Joachim; Eisch, Amelia J.; Huber, Kimberly M.
Alzheimer's disease (AD) is a currently incurable neurodegenerative disorder and the most common form of dementia over age 65. The predominant genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4). Other genes related to lipid metabolism and lipoprotein receptor signaling have also been identified as risk modifiers for AD. Despite nearly two decades of research, the mechanisms by which lipid transport proteins cause central nervous system (CNS) disease are not completely understood. Here, the ApoE receptor family and its ligands and their roles in CNS function and neurological disease were explored. It has been previously shown that ApoE4 renders neurons resistant to the neuromodulator and ApoE receptor ligand Reelin, which enhances synaptic plasticity and protects against amyloid β (Aβ) oligomer-induced synaptic toxicity in vitro. Here, mice with reduced Reelin expression were more sensitive to amyloid-induced synaptic suppression, and had memory and learning disabilities at very low amyloid levels. However, this effect of Reelin loss did not extend to other forms a neurological insult, since the Reelin conditional knockout mice were not more susceptible to transient middle cerebral artery occlusions. Together, these findings highlight the specific physiological importance of Reelin for protecting the brain against Aβ-induced synaptic dysfunction and memory impairment. One of the continuing debates in the AD field is whether ApoE is required for synaptic function. ApoE knockout mice have synaptic loss; however, they also have severely increased plasma lipids, which could independently contribute to CNS dysfunction. A novel mouse with normal plasma ApoE, but severely depleted brain ApoE, shares a similar synaptic phenotype as ApoE knockout mice, suggesting central ApoE is required for brain function. To determine if diet can modulate ApoE levels, wildtype, ApoE3, and ApoE4 targeted replacement mice were fed a chow, high-fat, or ketogenic (high-fat, very-low-carb) diet. Surprisingly, high-fat diet reduced hippocampal ApoE levels in ApoE3 TR mice, indicating an intersection of genetic (ApoE isoform) and lifestyle (diet) risk factors on AD pathogenesis. Taken together, these findings highlight the importance of ApoE receptors and their ligands in AD biology, and future studies will have to determine how to target these mechanisms to treat AD and improve patient outcomes.
Does Reelin Affect Recovery from a Stroke?
(2016-01-19) DeSai, Charisma; Herz, Joachium; Stowe Ann M.; Lane-Donovan, Courtney
Reelin is an extraceullular glycoprotein that modulates synaptic plasticity and increases long-term potentiation. Since Reelin has neuroprotective effects, we were interested to see if Reelin plays a role in recovery after significant neurological damage. Earlier studies with reeler mice showed that mice lacking Reelin have increased susceptibility to stroke and suffer more damage post-stroke. Since Reelin is important in neuronal migration during development, it is possible that the effects seen were due to improper brain development, instead of Reelin deficiency itself. Instead of using Reeler mice, we used the Reelin conditional knockout(cKO) mouse model. Thus, we were able to see the effects of Reelin loss while permitting normal brain development. Stroke size and post-stroke behavior were investigated after inducing transient middle cerebral artery occlusion in four-month-old Reelin cKO mice. No significant difference was seen between wild type and Reelin cKO mice in infarct size or behavior, suggesting that while Reelin does play in important function in the brain, it does not play a significant role in post-stroke recovery.
Fibulin-5 Promotes Pancreatic Tumor Growth through Inhibition of Integrin-induced ROS: Insights into Tumor-Matrix Signaling
(2016-07-18) Topalovski, Mary; Terada, Lance; Cobb, Melanie H.; Abrams, John M.; Brekken, Rolf A.
Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anti-cancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We found that Fbln5 is abundantly expressed in mouse and human pancreatic cancer compared to normal pancreas. By employing genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA), we showed that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDA therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5 mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin (FN) signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDA progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. The latter part of this thesis is focused on the underlying mechanism that leads to upregulation of Fbln5 in PDA. The deposition of ECM is a defining feature of PDA where ECM signaling can promote cancer cell survival and epithelial plasticity programs. ECM-mediated signaling is governed by expression of the ECM proteins, the presence of cell surface receptors and the expression and activity of matricellular proteins that function as extracellular adaptors to reduce ECM-cell interaction. As stated above, Fbln5 is a matricellular protein that blocks FN-integrin interaction and thus directly limits ECM-driven ROS production and supports PDA progression. Compared to normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a transforming growth factor β (TGF-β)- and PI3K-dependent manner. Pharmacologic inhibition of TGF-β receptor (TGF-βR), PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-βR and PI3K/Akt inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression while pharmacologic inhibition of TGF-β signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.
Molecular and Ocular Characterization of Novel Fibulin-3 Variants Involved in Retinal Degeneration
(2021-12-10) Woodard, Danae Rochelle; Kohler, Jennifer J.; Petroll, W. Matthew; Tu, Benjamin; Hulleman, John D.
Distinct mutations in fibulin-3 (F3), a secreted extracellular matrix glycoprotein, have been associated with various ocular diseases including Malattia Leventinese (ML) and the most common macular degenerative disease, age-related macular degeneration (AMD), which ultimately lead to vision loss. AMD is a late onset disease characterized by the progressive loss of photoreceptors and retinal pigment epithelial (RPE) cells that result in irreversible blindness. Although AMD is an etiologically complex disease due to a variety of genetic and environmental risk factors, insight into its pathogenesis can be gained by studying phenotypically similar early-onset monogenic macular diseases. One such disease is ML, a rare macular dystrophy caused by an autosomal dominant Arg345Trp (R345W) mutation in the F3 protein. Previous research has demonstrated that the R345W mutation leads to protein misfolding, inefficient secretion, and accumulation at higher intracellular steady state levels in cultured cells. However, it remained unclear whether other potentially pathogenic or clinically-identified F3 variants recently reported in the human population also share features similar to that of R345W. We hypothesized that secretion defects in one or more F3 mutants may be a shared mechanism that ultimately contributes to ocular disease. First, I characterized 15 clinically-identified F3 mutations, some of which were identified in patients with AMD, primary open-angle glaucoma (POAG), or had non-discript retinal abnormalities. I found that of the mutants tested, only a single F3 variant, L451F, presented with a significant secretion defect as well as similarities in its biochemical and molecular properties to that of R345W. Subsequently, I generated a retinal disease mouse model of the L451F mutant utilizing recombinant adeno-associated virus (rAAV) in order to robustly evaluate disease phenotypes and uncover how L451F and other F3 mutations (i.e. R345W) are involved in retinal degeneration.
Novel Function for Fibulin-5 in Controlling Integrin-Induced ROS Production: Implications on Angiogenesis and Cancer
(2010-01-12) Schluterman, Marie Kay; Brekken, Rolf A.
Tumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins are secreted into the ECM and impact signaling pathways required for pro-tumorgenic activities such as angiogenesis. Fibulin-5 (Fbln5) is a matricellular protein recently shown to regulate angiogenesis, however its effect on tumor angiogenesis and thus tumor growth is currently unknown. We report that the growth of pancreatic tumors and tumor angiogenesis was suppressed in Fbln5 null (Fbln5-/-) mice compared to wild-type (WT) littermates. Furthermore, we observed an increase in the level of reactive oxygen species (ROS) in tumors grown in Fbln5-/- animals. Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor and represented the underlying cause for the reduction in angiogenesis and tumor growth. In vitro, we identified a novel pathway by which Fbln5 controls ROS production through a mechanism dependent on _1 integrins. These results were validated in Fbln5RGE/RGE mice, which harbor a point mutation in the integrin-binding RGD motif of Fbln5 preventing its interaction with integrins. Tumor growth and angiogenesis was reduced in Fbln5RGE/RGE mice, however treatment with an antioxidant rescued angiogenesis and elevated tumor growth to WT levels. These findings introduce a novel function for Fbln5 in the regulation of integrin-induced ROS production and establish a rationale for future studies to examine whether blocking Fbln5 function could be an effective anti-tumor strategy, alone or in combination with other therapies.
A Novel Role for HAPLN1 in Skeletal Development
(2022-12-01T06:00:00.000Z) Patel, Chandreshkumar Vishnubhai; Madhuranthakam, Ananth; Wise, Carol A.; Cleaver, Ondine; Evers, Bret M.; Risbud, Makarand
Adolescent idiopathic scoliosis (AIS) is a potentially progressive spinal curvature that occurs during rapid growth and has a multi-factorial genetic etiology. Prior genetic studies in humans and animal models support differences in cartilage development in the pathogenesis of AIS. Here, we performed genome sequencing in families with inherited AIS and identified a co-segregating rare variant in the HAPLN1 gene. HAPLN1 encodes Hyaluronan and Proteoglycan Link Protein 1, also known as Cartilage Link Protein, that is reported to stabilize the extracellular matrix (ECM) by binding hyaluronic acid (HA) and proteoglycans in multiple tissues. One variant, p.C304S, was predicted to disrupt a critical disulfide bridge of the HAPLN1 C-terminal loop domain. In transient over-expression experiments, we detected wild type mouse Hapln1 protein in the cell lysate, media, and ECM, while Hapln1 p.C306S (C304S in human) was detected in cell lysate and media, but not in the ECM. A truncated Hapln1 p.C306fs*5 (ET) lacking the C-terminal loop domain was detected only in the cell lysate. By mutagenesis experiments we found that replacing cysteines in Hapln1 primary disulfide bridges disrupted secretion and retention in the ECM, whereas replacing cysteines in secondary loops disrupted ECM retention only. Thus, all disulfide loops are required for ECM retention. Mice were engineered to harbor the orthologous mutations. While Hapln1ET/ET mice died shortly after birth, exhibiting a chondrodysplastic phenotype with shortened limbs and dome-shaped head, Hapln1C306S/C306S mice were viable, born at expected Mendelian ratios, and showed no significant skeletal malformations up to one year of age. Surprisingly, we were unable to detect Hapln1 protein in tissues of Hapln1C306S/C306S mice although they were phenotypically normal. Furthermore, we found that Hapln1 negatively regulates apoptosis in growth plate hypertrophic zones. Collectively, our data suggest an essential function for Hapln1 that is potentially independent of its role in the ECM.