Activity deprivation induces neuronal cell death: mediation by tissue-type plasminogen activator.

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.

Activity-dependent survival of neurons in culture: a model of slow neurodegeneration.

Central neurons express persistent spontaneous electrical network activity both in the developing brain in vivo as well as in dissociated cultures. This electrical activity is important for the formation of connections among neurons, and for their survival. Prolonged suppression of the spontaneous activity using the sodium channel blocker tetrodotoxin (TTX) causes the death of the cultured neurons. In the present study, we investigated molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 6-7 days of exposure to TTX, neurons begin to express apoptotic vacuoles and shrunken dendrites. Eventually, neurons activate p53, caspase-3 and BAX, hallmarks of neuronal apoptosis, before they die. This death is restricted to neurons, and no parallel process is seen in glial cells that co-exist in the culture. These experiments may lead to a better understanding of slow neuronal death, akin to that found in neurodegenerative diseases of the brain.

Degeneration of cultured cortical neurons following prolonged inactivation: molecular mechanisms.

Networks of neurons express persistent spontaneous network activity when maintained in dissociated cultures. Prolonged blockade of the spontaneous activity with tetrodotoxin (TTX) causes the eventual death of the neurons. In this study, we investigated some molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 3-4 days of exposure to TTX, well before the neurons die, they began to express markers that lead to their eventual death, 7-10 days later. There was a reduction in glutamate receptor (GluR2) expression, a persistent increase in intracellular calcium concentration, activation of calpain, and an increase in spectrin breakdown products. At this point, blockade of GluR2-lacking GluR1 or calpain (either with a selective antagonist or through the natural regulator of calpain, calpastatin), protected cells from the toxic action of TTX. Subsequently, mitochondria lost their normal elongated shape as well as their membrane potential. Eventually, neurons activated caspase 3 and PUMA (p53 up-regulated modulator of apoptosis), hallmarks of neuronal apoptosis, and died. These experiments will lead to a better understanding of slow neuronal death, typical of neurodegenerative diseases.