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

Impaired Sprouting and Axonal Atrophy in Cerebellar Climbing Fibres following In Vivo Silencing of the Growth-Associated Protein GAP-43

The adult mammalian central nervous system has a limited ability to establish new connections and to recover from traumatic or degenerative events. The olivo-cerebellar network represents an excellent model to investigate neuroprotection and repair in the brain during adulthood, due to its high plasticity and ordered synaptic organization. To shed light on the molecular mechanisms involved in these events, we focused on the growth-associated protein GAP-43 (also known as B-50 or neuromodulin). During development, this protein plays a crucial role in growth and in branch formation of neurites, while in the adult it is only expressed in a few brain regions, including the inferior olive (IO) where climbing fibres (CFs) originate. Following axotomy GAP-43 is usually up-regulated in association with regeneration. Here we describe an in vivo lentiviral-mediated gene silencing approach, used for the first time in the olivo-cerebellar system, to efficiently and specifically downregulate GAP-43 in rodents CFs. We show that lack of GAP-43 causes an atrophy of the CF in non-traumatic conditions, consisting in a decrease of its length, branching and number of synaptic boutons. We also investigated CF regenerative ability by inducing a subtotal lesion of the IO. Noteworthy, surviving CFs lacking GAP-43 were largely unable to sprout on surrounding Purkinje cells. Collectively, our results demonstrate that GAP-43 is essential both to maintain CFs structure in non-traumatic condition and to promote sprouting after partial lesion of the IO

The effect of TTX and conditioned medium on the cell survival and tPA expression level in neuronal cultures.

<p>(a) Mean cell survival rates of primary neuronal cultures following the application of 3 days TTX-conditioned medium or fresh TTX for 7 days. (b) Cell survival rates of primary neuronal cultures following the application of conditioned medium of cells that were treated for 4 days with TTX (‘wash conditioning’) and left for 3 more days in fresh medium, with or without the addition of fresh TTX, for 7 days (c) The change in tPA mRNA expression level in primary neuronal cultures following a week of incubation in TTX- or wash-conditioned medium (means±SEM; * P<0.05).</p

Intrinsic blockade of tPA protects neurons from TTX-mediated neuronal cell death.

<p>(a) Representative images of control and TTX-treated neuronal cultures stained for the neuronal marker NeuN (red), and for the tPA inhibitor PAI-1 (green), transfected with either GFP, PAI-1 or non-transfected controls (bar = 100 µm). (b) Control and TTX-treated neurons were stained for NeuN and for PAI-1. Cell survival rates of the NeuN-positive cells and of the PAI-1-positive cells out of the NeuN-positive population were estimated. (c) Cell survival rates of double-labeled cells of the three groups - Non-transfected, GFP and PAI-1, demonstrating the protection exerted by the over-expression of PAI-1.</p