Activation of a synapse weakening pathway by human Val66 but not Met66 pro-brain-derived neurotrophic factor (proBDNF).

This study describes a fundamental functional difference between the two main polymorphisms of the pro-form of brain-derived neurotrophic factor (proBDNF), providing an explanation as to why these forms have such different age-related neurological outcomes. Healthy young carriers of the Met66 form (present in ∼30% Caucasians) have reduced hippocampal volume and impaired hippocampal-dependent memory function, yet the same polymorphic population shows enhanced cognitive recovery after traumatic brain injury, delayed cognitive dysfunction during aging, and lower risk of late-onset Alzheimer's disease (AD) compared to those with the more common Val66 polymorphism. To examine the differences between the protein polymorphisms in structure, kinetics of binding to proBDNF receptors and in vitro function, we generated purified cleavage-resistant human variants. Intriguingly, we found no statistical differences in those characteristics. As anticipated, exogenous application of proBDNF Val66 to rat hippocampal slices dysregulated synaptic plasticity, inhibiting long-term potentiation (LTP) and facilitating long-term depression (LTD). We subsequently observed that this occurred via the glycogen synthase kinase 3ß (GSK3ß) activation pathway. However, surprisingly, we found that Met66 had no such effects on either LTP or LTD. These novel findings suggest that, unlike Val66, the Met66 variant does not facilitate synapse weakening signaling, perhaps accounting for its protective effects with aging.

Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.

The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein-ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a ß-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF ( 1WWW .pdb), and the (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

Sequences located within the N-terminus of the PD-linked LRRK2 lead to increased aggregation and attenuation of 6-hydroxydopamine-induced cell death.

Clinical symptoms of Parkinson's disease (PD) arise from the loss of substantia nigra neurons resulting in bradykinesia, rigidity, and tremor. Intracellular protein aggregates are a pathological hallmark of PD, but whether aggregates contribute to disease progression or represent a protective mechanism remains unknown. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to PD in both familial cases and idiopathic cases and aggregates of the LRRK2 protein are present in postmortem PD brain samples. To determine whether LRRK2 contains a region of protein responsible for self-aggregation, two independent, bioinformatic algorithms were used to identify an N-terminal amino acid sequence as being aggregation-prone. Cells subsequently transfected with a construct containing this domain were found to have significantly increased protein aggregation compared to wild type protein or a construct containing only the last half of the molecule. Finally, in support of the hypothesis that aggregates represent a self-protection strategy, aggregated N-terminal LRRK2 constructs significantly attenuated cell death induced by the PD-mimetic, 6-hydroxydopamine (6-OHDA).

Responses of human endothelial cells to pathogenic and non-pathogenic Leptospira species.

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.

Leptospira interrogans binds to human cell surface receptors including proteoglycans.

Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.

Dexamethasone promotes calcium pyrophosphate dihydrate crystal formation by articular chondrocytes.

OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joints and correlate with a poor prognosis. Intraarticular corticosteroids, such as dexamethasone (Dxm), are commonly used therapies for osteoarthritis with or without CPPD deposition. Dxm has variable effects in mineralization models. We investigated the effects of Dxm on CPPD crystal formation in a well established tissue culture model. METHODS: Porcine articular chondrocytes were incubated with ATP to generate CPPD crystals. Chondrocytes incubated with or without ATP were exposed to 1-100 nM Dxm in the presence of 45Ca. Mineralization was measured by 45Ca uptake in the cell layer. We also investigated the effect of Dxm on mineralization-regulating enzymes such as alkaline phosphatase, nucleoside triphosphate pyrophosphohydrolase (NTPPPH), and transglutaminase. RESULTS: Dxm significantly increased ATP-induced mineralization by articular chondrocytes. While alkaline phosphatase and NTPPPH activities were unchanged by Dxm, transglutaminase activity increased in a dose-responsive manner. Levels of Factor XIIIA mRNA and protein were increased by Dxm, while type II Tgase protein was unchanged. Transglutaminase inhibitors suppressed Dxminduced increases in CPPD crystal formation. CONCLUSION: These findings suggest a potential for Dxm to contribute to pathologic mineralization in cartilage and reinforce a central role for the transglutaminase enzymes in CPPD crystal formation.

Feasibility of a tetracycline-binding method for detecting synovial fluid basic calcium phosphate crystals.

OBJECTIVE: Basic calcium phosphate (BCP) crystals are common components of osteoarthritis (OA) synovial fluid. Progress in understanding the role of these bioactive particles in clinical OA has been hampered by difficulties in their identification. Tetracyclines stain calcium phosphate mineral in bone. The aim of this study was to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in synovial fluid. METHODS: A drop of oxytetracycline was mixed with a drop of fluid containing synthetic or native BCP, calcium pyrophosphate dihydrate (CPPD), or monosodium urate (MSU) crystals and placed on a microscope slide. Stained and unstained crystals were examined by light microscopy, with and without a portable broad-spectrum ultraviolet (UV) pen light. A small set of characterized synovial fluid samples were compared by staining with alizarin red S and oxytetracycline. Synthetic BCP crystals in synovial fluid were quantified fluorimetrically using oxytetracycline. RESULTS: After oxytetracycline staining, synthetic and native BCP crystals appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or MSU crystals or other particulates. Oxytetracycline staining had fewer false-positive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid. CONCLUSION: With further validation, oxytetracycline staining may prove to be a useful adjunct or alternative to currently available methods for identifying BCP crystals in synovial fluid.

Promotion of articular cartilage matrix vesicle mineralization by type I collagen.

OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur in up to 60% of osteoarthritic joints and predict an increased severity of arthritis. Articular cartilage vesicles (ACVs) generate CPPD crystals in the presence of ATP and BCP crystals with added beta-glycerophosphate. While ACVs are present in normal articular cartilage, they mineralize primarily in cartilage from osteoarthritic joints. The aim of this study was to explore the hypothesis that ACV mineralization is regulated by components of the surrounding extracellular matrix. METHODS: Porcine ACVs were embedded in agarose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as (45)Ca accumulation stimulated by ATP or beta-glycerophosphate and reflects both nucleation and growth. Synthetic CPPD and BCP crystals were embedded in similar gels to isolate the effect of matrix components on crystal growth. RESULTS: After establishing baseline responsiveness of ACVs to ATP and beta-glycerophosphate in agarose gels, we examined the ability of ATP and beta-glycerophosphate to stimulate mineral formation in gels containing various matrix components. Type II collagen suppressed the ability of ATP to stimulate mineralization, while a combination of type II plus type I collagen increased the effect of ATP and beta-glycerophosphate on mineralization. Type I collagen affected ACV mineralization in a dose-responsive manner. Neither type of collagen significantly affected crystal growth or levels of mineralization-regulating enzymes. Proteoglycans suppressed mineral formation by ACVs in gels containing both type I and type II collagen. CONCLUSION: Cartilage matrix changes that occur with osteoarthritis, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization.

Human ProNGF: biological effects and binding profiles at TrkA, P75NTR and sortilin.

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75(NTR) and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer's disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild-type and cleavage-resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M-proNGF and NGF bound to p75(NTR) with similar affinities, whilst M-proNGF had a lower affinity than NGF for TrkA. M-proNGF behaved neurotrophically, albeit less effectively than NGF. M-proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M-proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M-proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M-proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75(NTR) : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.

Bilateral femoral supracondylar stress fractures in a cross country runner.

Several high-risk factors lead to stress fractures. They include excessive training in athletes leading to overuse injuries, nutritional deficiencies, and endocrine disorders. While stress fractures are common, bilateral stress fractures are rarely seen. Few cases have been reported of bilateral femoral stress fractures in young athletes. This article presents a case of a 14-year-old cross country runner with a bilateral femoral supracondylar stress fracture. He presented with bilateral supracondylar stress fractures from running. The patient followed a strict vegan diet, but his parents stated that, to their knowledge, he was getting adequate protein and calcium. Treatment consisted of decreased activity to pain-free levels with acetaminophen for pain. Low-impact conditioning such as swimming and bicycling was allowed. Hamstring and quadricep stretching was suggested. Nutritional consultation was obtained to ensure appropriate nutrition on a vegan diet. At 1-month follow-up, he was pain free and allowed to proceed with a gradual return to running activities. In this case, the onset of a new workout routine was intolerable for this patient's low bone density, causing insufficiency fractures. Appropriate vegan diets were not associated with stress fracture in our literature review. He may have had an inadequate diet prior to this injury. As in this case, full recovery can be made after this rest period, and the patient may return to his or her original activity safely. In young athletes, diet and nutrition must be kept in mind.

Calcific tendonitis : a model.

Calcific tendonitis is a common clinical condition associated with high rates of tendon rupture, prolonged symptoms, and poor response to therapy. Little is known about the pathogenesis of calcifications in tendons and consequently few effective therapies are available. We hypothesized that tendon calcification, like pathologic calcification in other sites, was generated by extracellular organelles known as matrix vesicles and that isolated matrix vesicles would constitute the basis for a useful model of this process. Tendon matrix vesicles were isolated from adult porcine patellar tendons using enzymatic digestion and differential centrifugation. Vesicle morphology was examined with electron microscopy. Levels of calcium, phosphate, pyrophosphate, ATP, and mineralization-associated enzymes were measured and compared with articular cartilage vesicles from porcine articular cartilage. Vesicles were embedded in agarose gels with or without type I collagen or dermatan sulfate and incubated in calcifying salt solution trace labeled with (45)calcium. (45)Calcium in the vesicle fraction was measured after 5-7 days. The type of mineral formed was determined by micro-x-ray diffraction. Matrix vesicles isolated from adult porcine tendon were similar morphologically to those obtained from articular cartilage. They contained mineralization-related enzymes and formed hydroxyapatite mineral in vitro. Mineralization was suppressed by levamisole and modulated by extracellular matrix components. Matrix vesicles isolated from tendons mineralize in vitro. This model may aid in the study of the pathogenesis of calcific tendonitis as well as serve as a means to identify effective therapies for this common disorder.

TrkAd5: A novel therapeutic agent for treatment of inflammatory pain and asthma.

Elevated levels of nerve growth factor have been linked to the onset and persistence of many pain-related disorders and asthma. Described here are the design, expression, refolding, and purification of a monomeric (nonstrand-swapped) form of the binding domain of the nerve growth factor receptor, designated TrkAd5. We have shown that TrkAd5 produced recombinantly binds nerve growth factor with picomolar affinity. TrkAd5 has been characterized using a variety of biophysical and biochemical assays and is shown here to be stable in both plasma and urine. The palliative effects of TrkAd5 are demonstrated in animal models of inflammatory pain and allergic asthma. We conclude that TrkAd5 will prove effective in ameliorating both acute and chronic conditions where nerve growth factor acts as a mediator and suggest a role for its application in vivo as a novel therapeutic.