Faculty Bibliography

BACKGROUND: Recent studies have implicated specific assembly subtypes of beta-amyloid (Abeta) peptide, specifically soluble oligomers (soAbeta) as disease-relevant structures that may underlie memory loss in Alzheimer disease. Removing existing soluble and insoluble Abeta assemblies is thought to be essential for any attempt at stabilizing brain function and slowing cognitive decline in Alzheimer disease. IV immunoglobulin (IVIg) therapies have been shown to contain naturally occurring polyclonal antibodies that recognize conformational neoepitopes of soluble or insoluble Abeta assemblies including soAbeta. These naturally occurring polyclonal antibodies have been suggested to underlie the apparent clinical benefits of IVIg. However, direct evidence linking anti-Abeta antibodies to the clinical bioactivity of IVIg has been lacking. METHODS: Five-month-old female Dutch APP E693Q mice were treated for 3 months with neat IVIg or with IVIg that had been affinity-depleted over immobilized Abeta conformers in 1 of 2 assembly states. Memory was assessed in a battery of tests followed by quantification of brain soAbeta levels using standard anti-soAbeta antibodies. RESULTS: We provide evidence that NU4-type soAbeta (NU4-soAbeta) assemblies accumulate in the brains of Dutch APP E693Q mice and are associated with defects in memory, even in the absence of insoluble Abeta plaques. Memory benefits were associated with depletion from APP E693Q mouse brain of NU4-soAbeta and A11-soAbeta but not OC-type fibrillar Abeta oligomers. CONCLUSIONS: We propose that targeting of specific soAbeta assembly subtypes may be an important consideration in the therapeutic and/or prophylactic benefit of anti-Abeta antibody drugs.

Vitiligo, characterized by progressive skin depigmentation, results from autoimmune-mediated melanocyte loss. The mechanisms underlying disease onset are poorly delineated. Triggers, including exposure to phenols such as monobenzone (MB), are thought to disrupt melanocyte homeostasis and ultimately instigate an autoimmune reaction. We have shown that MB disrupts cellular homeostasis and induces endoplasmic reticulum (ER) stress that leads to activation of the unfolded protein response (UPR). Three proteins, including PERK, each activate UPR arms that orchestrate the restoration of homeostasis. When activated, PERK phosphorylates eIF2a, a translation initiation factor, thus reducing protein synthesis and ER stress. In this study, we investigated the impact of the PERKeIF2a cascade on melanocyte viability and sensitivity to MB. Basal levels of phospho-eIF2a are higher in melanocytes compared to cutaneous fibroblasts or keratinocytes. When PERK expression is downregulated by RNAi, there is a significant reduction in melanocyte viability (88% decrease, p < 0.05 shPERK versus non-target/ shNT; n = 3). Some melanocytes (shPERKLT) can however survive despite prolonged PERK downregulation. Survival correlated with a paradoxical increase in phospho-eIF2a levels and reduced sensitivity to MB (Cleaved/c-PARP levels lower in shPERKLT cells treated with 400 muM MB compared to shNT cells). Chemical inhibition of PERK kinase activity, using GSK2606414, prevented eIF2a phosphorylation and sensitized melanocytes to MB (c-PARP observed with 250 muM MB+ GSK2606414, compared to 400 muM MB + vehicle). PERK-eIF2a axis activity contributes to melanocyte viability and determines sensitivity to MB. Pathways, such the UPR, which has also been implicated in autoimmune diabetes, may link exposure to vitiligo-inducing triggers and onset of autoimmunity. These pathways represent novel therapeutic targets to prevent vitiligo progression or improve efficacy of repigmentation protocols

INTRODUCTION: Selective serotonin re-uptake inhibitors (SSRIs) are currently the most commonly prescribed antidepressant on the U.S. market. Recent studies have linked chronic SSRI use to osteoporosis and an increased fracture risk. To date there are no studies investigating the effect of SSRIs on fracture healing. Here, we first examined the direct effect of SSRIs on osteoprogenitor cells in an in vitro setting. We aim at understanding the underlying mechanism of action of SSRI-induced impaired bone repair. METHODS: Bone marrow derived OPC were harvested using the bone marrow flush-and-scrape technique. Cells were passed through a cell strainer, then counted and plated. Once cells were attached, the media was changed and non-adherent cells were removed. Once confluent, cells were trypsinized and split for the following experiments. Cells were treated with 5, 10, 20, 50, lOOuM of fluoxetine or media as a control. Media was changed every two days. BrdU assays were performed at 3 days, RNA isolation for qPCR on day 7 and differentiation assays (alkaline phosphatase and alizarin red) at 14 days. All experiments were run in triplicates. RESULTS SECTION: Selective serotonin re-uptake inhibitors decrease osteoprogenitor cells proliferation in vitro In this study we sought to investigate if SSRIs had a direct effect on primary osteoblasts. We harvested bone marrow derived mesenchymal stem cells, using a scrape and flush technique. Cells were plated on tissue culture plastic and split for the experiments once they had reached confluence. First, we tested whether treatment with fluoxetine affected the mitotic activity of these primary cell cultures. Cells were either treated with standard osteogenic differentiation media containing beta-glycerophosphate, ascorbic acid and dexamethasone or with standard growth media. A subset of cell received media with 5, 10, 20, 50, lOOuM fluoxetine. Media was changed every two days. After three days, we assessed the proliferative activity of the cells in the fluoxetine-treated and control cells using a Bromodeoxyuridine (BrdU) incorporation assay. Treatment with fluoxetine resulted in a significant reduction of proliferative activity compared to the control cells (p=0.032)(Fig. 1). This experiment indicated that the SSRI fluoxetine inhibits the mitotic activity of primary osteoblasts. Selective serotonin re-uptake inhibitors decrease osteoprogenitor cells differentiation in vitro Next, we assessed whether differentiation was affected by fluoxetine treatment. We chose multiple approaches involving molecular and cellular assays. First, we treated cells with osteogenic differentiation media +/- fluoxetine for 7 days and then performed an alkaline phosphatase assay. The alkaline phosphatase assay is a widely accepted method to assess osteoblast activity. After 7 days in osteogenic media +/- fluoxetine, cells were analyzed using a colometric assay. Results from this experiment showed a significant reduction in alkaline phosphatase activity after fluoxetine treatment (p=0.009)(Fig. 2). We also isolated RNA from cells treated with osteogenic media +/- fluoxetine for 7 days and performed quantitative PCR. In agreement with the alkaline phosphatase assay, osteoblastic markers, such as runx2, collagen type 1, osterix, osteocalcin and ALP were down regulated in the fluoxetine treated cells (p<0.002)(Fig. 3). Finally, we stained cells treated with growth media and osteogenic differentiation media +/- fluoxetine with Alizarin red to quantify bone matrix deposition. As shown in figure 4, fluoxetine significantly reduced bone matrix deposition. Dose response experiments (data not shown) demonstrated that differentiation was inhibited with lower fluoxetine doses (lOuM), while higher doses (20uM-100uM) still resulted in decreased osteogenic differentiation, however the difference was less significant. These experiments demonstrate that fluoxetine inhibits osteoprogenitor cell (OPC)/osteoblast proliferation and impedes osteogenic differentiation. Ongoing in vivo experiments in a murine fracture model are confirming the in vitro observation. DISCUSSION: Animal research and human clinical data have unmistakably shown that chronic SSRI use leads to osteoporosis, thus putting patients at risk for fragility fractures. If in fact SSRIs have a negative effect on bone formation after fracture, then this patient cohort will be at risk for delayed union and non-union. This potentially prolonged recovery may jeopardize the positive effect of the SSRI on the depression, for which it was prescribed initially. The discovery of a mechanism of action of SSRIs during regenerative bone formation may identify a new therapeutic target for a biomimetic approach to enhance fracture healing. Results from this study will lead to future in depth studies of the serotonin signaling cascade and its effect on the key players during fracture healing. SIGNIFICANCE: Antidepressants are one of the most commonly prescribed medications in the U.S. A significant portion of fracture patients are taking SSRIs, and are therefore prone to delayed union or non-union. Results form this study and from an ongoing prospective clinical trial will be able to offer a clinical recommendation in regards to SSRI use in fracture patients

INTRODUCTION: Osteoarthritis (OA) is a degenerative disease of the entire joint.¹ In healthy joints the articulating surfaces are encased in a smooth layer of hyaline cartilage and are further protected by the surrounding synovial fluid. High molecular weight hyaluronan (HMWHA) is present at relatively high levels in the synovial fluid and cartilage matrix.'²' HMWHA provides viscoelastic protection and lubrication of the cartilage surfaces, and has also been shown to have important anti-inflammatory properties.'³-⁵' During inflammation hyaluronan (HA) can be degraded to lower molecular weight HA (LMWHA) by the increased levels of free radicals and endogenous hyaluronidases present in the inflamed joint.'⁶' Interestingly, LMWHA, which can signal through a number of different receptors (TLR-2 and 4, CD44 and RHAMM), was suggested to act as potent inflammatory mediator in the joint.'⁷' Our collaborators identified a 15-mer peptide that binds to LMWHA and reduced inflammation and fibrogenesis in excisional skin wounds.^<8' In this study we hypothesized that LMWHA generated in the OA joint is a key mediator for stimulating catabolic and inflammatory events in joint cells and that interfering with LMWHA signaling using the novel peptide inhibitor will attenuate joint inflammation. Methods: Human articular chondrocytes were isolated from articular cartilage samples obtained from patients (donor age range 48 - 67) undergoing total knee replacement surgery at NYU Hospital for Joint Diseases. Knee cartilage was harvested from regions with no macroscopically evident degeneration. The collection of tissue from patients undergoing knee replacement surgery was approved by the Institutional Regulatory Board at NYU School of Medicine. Human chondrocytes were isolated from these cartilage samples and cultured as described previously.'⁹' Cultured chondrocyte were switched to serum-free medium for 24 h and then treated with inflammatory stimuli (interleukin-lbeta (IL-lp), LMWHA; average molecular weight of lOkDa). In addition, serum-starved cells were treated with the 15-mer peptide at various concentrations. A synovial fibroblast cell line (SW982) was also used and was cultured as described previously.'¹⁰' The analysis of HA concentrations in the culture medium was done as described by us previously.'¹¹' Western blotting and real time PCR analysis was performed as described by us.^<12) Results: In the present study we used human articular chondrocytes and a synovial fibroblast cell line (SW982) to determine the involvement of LMWHA promoting inflammation in the joint cavity. We show that LMWHA stimulated catabolic markers (Cox-2, IL-6, iNOS, MMP-13) and inhibited the expression of articular cartilage markers (aggrecan, type II collagen) in human articular chondrocytes and SW982 synovial cells. LMWHA treatment stimulated the NF-KB and the MAP kinase (ERK, JNK, p38) signaling pathways in human chondrocytes similar to IL-lp treatment. Whereas the stimulation of these signaling pathways occurred within the first hour of treatment with IL-lp, the stimulation of these pathways by LMWHA occurred at later time points (6h and 24h). The novel peptide inhibitor markedly reduced the activation of these signaling pathways in LMWHA-treated chondrocytes. In addition, it markedly decreased the expression of catabolic markers and increased the expression of articular cartilage markers in LMWHA-treated chondrocytes. The peptide also inhibited the expression of catabolic markers in IL-1 p-treated human articular chondrocytes and SW982 cells and increased articular cartilage marker expression in IL-1 p-treaded chondrocytes. Treatment of human articular chondrocytes with IL-lp resulted in a marked increase of HA released into the culture medium over time. This increase in HA release correlated with an increase in catabolic markers and a reduction in anabolic markers over time, similar to the effects of the LMWHA treatment. Discussion: Our findings demonstrate that LMWHA stimulates inflammatory and catabolic events in joint cells via activation of NF-Kb and ERK, JNK and p38 signaling pathways. In addition, we demonstrate that a novel peptide that binds to LMWHA and interferes with LMWHA signaling attenuates inflammatory and catabolic events in joint cells mediated by LMWHA and IL-lp. More specifically, we have shown that the novel peptide inhibitor dramatically reduced IL-6 and Cox-2 levels in articular chondrocytes and synovial fibroblasts. Both of these catabolic markers have been previously shown to be associated with increased pain levels in patients with OA.'¹³' These findings suggest that LMWHA plays a key role in mediating inflammation in joint cells during OA pathology, and that a novel peptide inhibitor of LMWHA signaling may act as a novel compound to specifically reduce inflammation and pain in the joint and ultimately slow down cartilage degradation during OA. SIGNIFICANCE: This study identified LMWHA as a key mediator of inflammatory and catabolic events in joint cells. In addition, we determined that interfering with LMWHA signaling using a novel peptide that binds LMWHA may provide a novel therapeutic strategy to reduce inflammation in the OA joint and ultimately slow down cartilage degradation during OA