Abstract
Spontaneous activity is an essential attribute of neuronal networks and plays a critical role in their development and maintenance. Upon blockade of activity with tetrodotoxin (TTX), neurons degenerate slowly and die in a manner resembling neurodegenerative diseases-induced neuronal cell death. The molecular cascade leading to this type of slow cell death is not entirely clear. Primary post-natal cortical neurons were exposed to TTX for up to two weeks, followed by molecular, biochemical and immunefluorescence analysis. The expression of the neuronal marker, neuron specific enolase (NSE), was down-regulated, as expected, but surprisingly, there was a concomitant and striking elevation in expression of tissue-type plasminogen activator (tPA). Immunofluorescence analysis indicated that tPA was highly elevated inside affected neurons. Transfection of an endogenous tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), protected the TTX-exposed neurons from dying. These results indicate that tPA is a pivotal player in slowly progressing activity deprivation-induced neurodegeneration.
Highlights
Spontaneous ongoing network activity plays a critical role in neuronal development and survival, and regulates neuronal functionality in many neural systems [1,2,3]
We have demonstrated the irreversibility of the death process, which starts after 4 days of exposure to TTX and proceeds even after TTX removal, through which neuron specific enolase (NSE) continues to decrease (Fig. 1A, n = 5 independent experiments, p = 261029 - one way ANOVA, p = 0.97 - Shapiro-Wilk normality test)
TPA, maintained its high expression level when the cells were dying after removal of TTX (4-fold and 5-fold, respectively, Fig. 1C, n = 5 independent experiments, p,0.05 - one way ANOVA, p = 0.52 - Shapiro-Wilk normality test), indicating that it may be involved in neuronal death caused by activity deprivation (Fig. 1A)
Summary
Spontaneous ongoing network activity plays a critical role in neuronal development and survival, and regulates neuronal functionality in many neural systems [1,2,3]. Suppression of spontaneous ongoing network activity in culture using the voltage-gated sodium channel blocker TTX, can have fatal consequences to the exposed network which die following a prolonged period of activity deprivation [4,5,6]. This protracted cell death is caused by a gradual activation of cell death cascade and is accompanied by an increase in miniature excitatory post-synaptic currents (mEPSC) [4], proposed recently to be mediated by glial tumor necrosis factor (TNF) [7], another acknowledged regulator of cell death. We propose tPA as a crucial mediator of the toxic effects exerted by neuronal activity deprivation
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