Release of nerve growth factor by human glial cells in culture

Norrgren, Gunilla; Ebendal, Ted; Belew, Makonnen; Jacobson, Carl-Olof; Porath, Jerker

doi:10.1016/0014-4827(80)90039-7

Cited by 60 publications

(16 citation statements)

References 44 publications

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“…After 10–12 D.I.V., Western blotting analysis on total protein extracts were performed by probing with antibodies against specific markers of cholinergic (TrkA high-affinity NGF receptor, p75NTR low-affinity NGF receptor, TrkB BDNF receptor, muscarinic acetylcholine receptor M1, choline acetyltransferase ChAT) glutamatergic (N-methyl-D-aspartate glutamate receptor 1 NR1, vesicular glutamate transporter 1 vGLUT-1) and GABAergic (vesicular γ-AminoButyric transporter vGAT) phenotypes to compare the extent of each neuronal-type population among three different culture conditions (2% B27, 2% B27+NGF, 0.2% B27+NGF). As shown in Figure 1 by biochemical characterization and relative densitometric quantification, septal primary neurons grown for 10–12 D.I.V in classic media supplemented with 2% B27 and in the absence of NGF (2% B27experimental group) showed a sizeable but detectable expression of three different well-confirmed cholinergic-specific markers such as M1, ChAT and TrkA (Dawbarn et al, 1988; Holtzman et al, 1992; Sobreviela et al, 1994), in line with previous findings reporting that differentiation and initial survival of central NGF-responsive neurons in the early postnatal period can occur even in the absence of NGF (Crowley et al, 1994; Ruberti et al, 2000) and that glial astrocytes are able to synthesize NGF and secrete it in culture (Lindsay, 1979; Norrgren et al, 1980; Furukawa et al, 1986). The addition of exogenous NGF (100 ng/ml) for the same period of time (10–12 D.I.V.)…”

Section: Resultssupporting

confidence: 75%

Impaired NGF/TrkA Signaling Causes Early AD-Linked Presynaptic Dysfunction in Cholinergic Primary Neurons

Latina

Caioli

Zona

et al. 2017

Front. Cell. Neurosci.

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Alterations in NGF/TrkA signaling have been suggested to underlie the selective degeneration of the cholinergic basal forebrain neurons occurring in vivo in AD (Counts and Mufson, 2005; Mufson et al., 2008; Niewiadomska et al., 2011) and significant reduction of cognitive decline along with an improvement of cholinergic hypofunction have been found in phase I clinical trial in humans affected from mild AD following therapeutic NGF gene therapy (Tuszynski et al., 2005, 2015). Here, we show that the chronic (10–12 D.I.V.) in vitro treatment with NGF (100 ng/ml) under conditions of low supplementation (0.2%) with the culturing serum-substitute B27 selectively enriches the basal forebrain cholinergic neurons (+36.36%) at the expense of other non-cholinergic, mainly GABAergic (−38.45%) and glutamatergic (−56.25%), populations. By taking advantage of this newly-developed septo-hippocampal neuronal cultures, our biochemical and electrophysiological investigations demonstrate that the early failure in excitatory neurotransmission following NGF withdrawal is paralleled by concomitant and progressive loss in selected presynaptic and vesicles trafficking proteins including synapsin I, SNAP-25 and α-synuclein. This rapid presynaptic dysfunction: (i) precedes the commitment to cell death and is reversible in a time-dependent manner, being suppressed by de novo external administration of NGF within 6 hr from its initial withdrawal; (ii) is specific because it is not accompanied by contextual changes in expression levels of non-synaptic proteins from other subcellular compartments; (ii) is not secondary to axonal degeneration because it is insensible to pharmacological treatment with known microtubule-stabilizing drug such paclitaxel; (iv) involves TrkA-dependent mechanisms because the effects of NGF reapplication are blocked by acute exposure to specific and cell-permeable inhibitor of its high-affinity receptor. Taken together, this study may have important clinical implications in the field of AD neurodegeneration because it: (i) provides new insights on the earliest molecular mechanisms underlying the loss of synaptic/trafficking proteins and, then, of synapes integrity which occurs in vulnerable basal forebrain population at preclinical stages of neuropathology; (ii) offers prime presynaptic-based molecular target to extend the therapeutic time-window of NGF action in the strategy of improving its neuroprotective in vivo intervention in affected patients.

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Section: Resultssupporting

confidence: 75%

Impaired NGF/TrkA Signaling Causes Early AD-Linked Presynaptic Dysfunction in Cholinergic Primary Neurons

Latina

Caioli

Zona

et al. 2017

Front. Cell. Neurosci.

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“…In accordance with this, culture medium containing 10% of fetal calf serum and subject to concentration 70-fold by pressure dialysis, was shown to lack biological NGF activity and to give no signal in the NGF EIA (Soderstrom et al, 1990). Norrgren et al (1980) were also unable to detect any NGF activity in fetal calf serum. They tested this in non-concentrated and concentrated culture medium supplemented with 10% of FCS or in concentrated MEM (with or without FCS) run on Sephadex columns.…”

Section: Discussionsupporting

confidence: 51%

Two‐site enzyme immunoassay for βNGF applied to human patient sera

Lorigados

Söderström

Ebendal

1992

J of Neuroscience Research

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Nerve growth factor (NGF) supports sympathetic and sensory neurons in the peripheral nervous system and serves functions in the development and maintenance of cholinergic neurons in the basal forebrain. NGF distribution can be studied with the use of a sensitive two-site enzyme immunoassay (EIA). The monoclonal antibody 27/21 to mouse NGF was recently shown to effectively block the activity of both recombinant human NGF and native mouse NGF, and a two-site EIA using monoclonal antibody 27/21 was optimized. We have now applied this assay to examine NGF levels in normal human serum and serum from Parkinson, Alzheimer, and Huntington patients. To further test the specificity of conjugate binding, dilutions of the human sera were preincubated with an excess of monoclonal NGF antibody 27/21 in solution. With this strategy it was possible to completely block the signal obtained using the two-site EIA. Furthermore, we show that recombinant BDNF and NT-3 do not cross-react with monoclonal antibody 27/21 under our conditions. We found low levels of specific NGF immunoreactivity in normal human sera (0.4 +/- 0.1 ng/ml). Significantly lower levels of NGF were found in sera from patients with Parkinson's and Huntington's disease whereas sera from Alzheimer patients showed only slight reductions in the NGF level. Two patients who had received intracerebral NGF infusions (one with Parkinson's and other with Alzheimer's disease) showed significantly elevated serum levels of NGF during the period of infusion. Due to an inhibitory activity in human serum, it was impossible to demonstrate the low levels of NGF activity in the human serum samples using explanted embryonic sympathetic ganglia, even after concentration by pressure dialysis. Thus, the serum levels are below the limit to evoke a response in NGF-sensitive neurons and thus to expect any physiological effect. Nevertheless, the levels measured may be used as indicators in clinical conditions such as Parkinson's and Huntington's disease.

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“…1989bj. It is worth mentioning that wc also found a good correlation (data not shown) between the biological activity and the recovery by EIA for native human NGF present in concentrated conditioned medium from human glial cells (Norrgren et al, 1980). By EIA, we now detected 0.3-0.5 ng human NGF per ml of concentrated conditioned medium.…”

Section: Discussionmentioning

confidence: 77%

Recombinant human β‐nerve growth factor (NGF): Biological activity and properties in an enzyme immunoassay

Söderström

Hallböök

Ibáñez

et al. 1990

J of Neuroscience Research

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Nerve growth factor (NGF) supports sympathetic and sensory neurons in the peripheral nervous system and also functions in the development and maintenance of cholinergic neurons in the basal forebrain. NGF distribution can be studied in the brain of the rat and mouse with the use of a sensitive two-site enzyme immunoassay (EIA) for mouse NGF. It would be of interest to measure the NGF protein also in the human brain, especially against the background that the cholinergic neurons are severely deteriorated in senile dementia of the Alzheimer type. The limited immunological cross-reactivity between NGFs from different species has previously hampered attempts to determine levels of the human NGF. We have now examined the biological activity and immunological properties of human recombinant NGF protein in medium conditioned by COS cells transfected with the human NGF gene. The human NGF behaved similar to mouse NGF in a sympathetic ganglion bioassay. The monoclonal antibody 27/21 to mouse NGF was shown to effectively block the activity of both the human recombinant NGF and mouse native NGF. A two-site EIA using monoclonal antibody 27/21 was optimized. Under the conditions used, the EIA detected the human recombinant NGF with the same sensitivity (1 pg/ml) as shown for the mouse NGF. It should now be possible to test this EIA also on homogenized tissue to examine human NGF in brain samples from Alzheimer patients and age-matched controls.

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Release of nerve growth factor by human glial cells in culture

Cited by 60 publications

References 44 publications

Impaired NGF/TrkA Signaling Causes Early AD-Linked Presynaptic Dysfunction in Cholinergic Primary Neurons

Impaired NGF/TrkA Signaling Causes Early AD-Linked Presynaptic Dysfunction in Cholinergic Primary Neurons

Two‐site enzyme immunoassay for βNGF applied to human patient sera

Recombinant human β‐nerve growth factor (NGF): Biological activity and properties in an enzyme immunoassay

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