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Chemotactic responses of growing neurites to precisely controlled gradients of nerve growth factor.

ABSTRACT: Chemotaxis plays a key role in many biological systems. In particular in the context of the developing nervous system, growing neurites can respond in vitro to shallow gradients of chemotropic molecules such as nerve growth factor (NGF). However, in such studies the gradient parameters are often not well controlled. Here we present a dataset of ~3500 images of early postnatal rat dorsal root ganglion (DRG) explants growing in 40 different precisely controlled combinations of absolute concentration and gradient steepness of NGF. Each image has been segmented into neurite and explant-body regions. We provide computer code for exploration and quantification of the data, including a Fourier analysis of the outer contour of neurite growth, which allows quantities such as outgrowth and guidance as a function of concentration and gradient steepness to be easily extracted. This is the most comprehensive quantitative dataset of chemotactic responses yet available for any biological system, which we hope will be useful for exploring the biological mechanisms governing chemotaxis.

SUBMITTER: Bicknell BA

PROVIDER: S-EPMC6122170 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Chemotactic responses of growing neurites to precisely controlled gradients of nerve growth factor.

Bicknell Brendan A BA Pujic Zac Z Feldner Julia J Vetter Irina I Goodhill Geoffrey J GJ

Scientific data 20180904

Chemotaxis plays a key role in many biological systems. In particular in the context of the developing nervous system, growing neurites can respond in vitro to shallow gradients of chemotropic molecules such as nerve growth factor (NGF). However, in such studies the gradient parameters are often not well controlled. Here we present a dataset of ~3500 images of early postnatal rat dorsal root ganglion (DRG) explants growing in 40 different precisely controlled combinations of absolute concentrati ...[more]

PMID: 30179228

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Project description:ImportanceAlzheimer disease (AD) is the most common neurodegenerative disorder and lacks effective disease-modifying therapies. In 2001, we initiated a clinical trial of nerve growth factor (NGF) gene therapy in AD, the first effort at gene delivery in an adult neurodegenerative disorder. This program aimed to determine whether a nervous system growth factor prevents or reduces cholinergic neuronal degeneration in patients with AD. We present postmortem findings in 10 patients with survival times ranging from 1 to 10 years after treatment.ObjectiveTo determine whether degenerating neurons in AD retain an ability to respond to a nervous system growth factor delivered after disease onset.Design, setting, and participantsPatients in this anatomicopathological study were enrolled in clinical trials from March 2001 to October 2012 at the University of California, San Diego, Medical Center in La Jolla. Ten patients with early AD underwent NGF gene therapy using ex vivo or in vivo gene transfer. The brains of all 8 patients in the first phase 1 ex vivo trial and of 2 patients in a subsequent phase 1 in vivo trial were examined.Main outcomes and measuresBrains were immunolabeled to evaluate in vivo gene expression, cholinergic neuronal responses to NGF, and activation of NGF-related cell signaling. In 2 patients, NGF protein levels were measured by enzyme-linked immunosorbent assay.ResultsAmong 10 patients, degenerating neurons in the AD brain responded to NGF. All patients exhibited a trophic response to NGF in the form of axonal sprouting toward the NGF source. Comparing treated and nontreated sides of the brain in 3 patients who underwent unilateral gene transfer, cholinergic neuronal hypertrophy occurred on the NGF-treated side (P < .05). Activation of cellular signaling and functional markers was present in 2 patients who underwent adeno-associated viral vectors (serotype 2)-mediated NGF gene transfer. Neurons exhibiting tau pathology and neurons free of tau expressed NGF, indicating that degenerating cells can be infected with therapeutic genes, with resultant activation of cell signaling. No adverse pathological effects related to NGF were observed.Conclusions and relevanceThese findings indicate that neurons of the degenerating brain retain the ability to respond to growth factors with axonal sprouting, cell hypertrophy, and activation of functional markers. Sprouting induced by NGF persists for 10 years after gene transfer. Growth factor therapy appears safe over extended periods and merits continued testing as a means of treating neurodegenerative disorders.

Project description:BackgroundNerve growth factor (NGF) contributes to pain in knee osteoarthritis (KOA) patients. Transforming growth factor-beta (TGF-β) stimulates NGF expression in chondrocytes from KOA patients. However, the correlation between synovial TGF-β and NGF levels has not been sufficiently studied in human KOA patients. Further, the mechanism governing NGF regulation by TGF-β in synovial cells is unclear.MethodsDuring total knee arthroplasty, we extracted the synovial tissue (SYT) of 107 subjects with unilateral Kellgren/Lawrence grade 3-4 KOA confirmed by radiography. We examined the distribution of TGF-β and NGF using immunohistochemistry, and analyzed the relationship between NGF and TGFB mRNA levels. Cultured synovial cells extracted from SYT were exposed to culture medium (control), human recombinant TGF-β (rhTGF-β), rhTGF-β + ALK5 inhibitor SB505124, rhTGF-β + transforming growth factor activating kinase 1 (TAK1) inhibitor (5Z)-7-oxozeaenol, or rhTGF-β + p38 inhibitor SB203580 for 30 min, 6 h and 24 h. NGF mRNA expressed by the cultured cells and NGF protein levels in the cell supernatant were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Phosphorylation of p38 was evaluated by western blotting.ResultsNGF mRNA levels were positively correlated with those of TGFB. Cells expressing TGF-β and NGF protein were observed in the lining layer of SYT. TGF-β stimulated increased NGF mRNA expression and NGF protein production. The ALK5 inhibitor completely suppressed the TGF-β-mediated increase in NGF expression and NGF production in synovial cells. ALK5, TAK1 and p38 inhibitors inhibited the TGF-β-induced phosphorylation of p38, and TAK1 and p38 inhibitors partially inhibited the TGF-β-mediated increase in NGF expression and NGF production in synovial cells.ConclusionTGF-β regulates NGF production via the TGF-β/ALK5 signaling pathway in osteoarthritic synovium. This effect may partially occur through inhibition of the TAK1/p38 pathway in the SYT of KOA patients.

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Chemotactic responses of growing neurites to precisely controlled gradients of nerve growth factor.

Publications

Chemotactic responses of growing neurites to precisely controlled gradients of nerve growth factor.

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