Abstract: Synaptic dysfunction is responsible for the cognitive deficits associated with Alzheimer's disease (AD). Increasing evidence suggests that recurrent Herpes Simplex virus type-1 (HSV-1) infection in the brain is a risk factor for AD but the underlying mechanisms have not been fully elucidated yet. Aim of the present study is to check whether HSV-1 infection alters synaptic function and to identify the underlying molecular mechanisms. In cultured mouse cortical neurons, HSV-1 (5 MOI) triggered amyloid precursor protein phosphorylation at T668 (pAPP; +185±8% vs. mock-infected cells at 24 hours post infection [hpi]; P<0.01). This effect primarily depended on activation of Glicogen Sinthase Kinase (GSK)-3 with smaller contribution of cyclin-dependent kinase 5 and c-Jun N-terminal kinase, as demonstrated by experiments with the specific inhibitors SB216763 (10 µM), roscovitine (10 µM) and Inhibitor I (5 µM) that reduced pAPP immunoreactivity by 54%, 39% and 20%, respectively (P<0.01 vs. HSV-1 alone). pAPP is a key event for A? production and HSV-1-infected neurons exhibited intracellular accumulation of A? assessed by Western Blot and immunofluorescence. HSV-1- infected neurons (24 hpi) had reduced expression of the presynaptic proteins synapsin-1 and synaptophysin (-58% and -39% vs. mock, respectively; P<0.05) that was paralleled by a marked reduction of the frequency of spontaneous miniature excitatory post-synaptic currents (from 7.6 to 3.7 Hz; P<0.01). These effects were significantly reversed by SB216763 and depended on A? accumulation. Indeed, an antibody recognizing A? and being uploaded intracellularly (4G8) rescued synaptic protein expression and synaptic transmission from the effects of HSV-1. In HSV-1-infected neurons derived from APP KO mice GSK-3 activation was not associated with A? accumulation and significant changes in synaptic protein expression, thus suggesting that GSK-3 alone is not sufficient to induce synaptic alterations in infected cells. Among the downstream components of the molecular pathways potentially responsible for the HSV-1-induced synaptic dysfunction, we studied cAMP responsive element-binding protein (CREB) because it is target of GSK-3 and is modulated by A?. We found that 24 hpi the activatory phosphorylation of CREB (at S133) was reduced whereas the inhibitory one (at S129) was increased (-25±2% and +35±4% vs. mock, respectively; P<0.01). Either SB216763 or 4G8 completely reversed these effects. Our data indicate that HSV-1 infections may contribute to the pathophysiology of AD by a mechanisms likely involving GSK-3 activation, A? accumulation and inhibition of CREB-dependent transcriptional program.
Synaptic function alterations induced by Herpes Simplex Virus type-1 infection in vitro depend on Glycogen Synthase Kinase 3 activation and intraneuronal accumulation of amyloid-beta protein
2015
Abstract
Abstract: Synaptic dysfunction is responsible for the cognitive deficits associated with Alzheimer's disease (AD). Increasing evidence suggests that recurrent Herpes Simplex virus type-1 (HSV-1) infection in the brain is a risk factor for AD but the underlying mechanisms have not been fully elucidated yet. Aim of the present study is to check whether HSV-1 infection alters synaptic function and to identify the underlying molecular mechanisms. In cultured mouse cortical neurons, HSV-1 (5 MOI) triggered amyloid precursor protein phosphorylation at T668 (pAPP; +185±8% vs. mock-infected cells at 24 hours post infection [hpi]; P<0.01). This effect primarily depended on activation of Glicogen Sinthase Kinase (GSK)-3 with smaller contribution of cyclin-dependent kinase 5 and c-Jun N-terminal kinase, as demonstrated by experiments with the specific inhibitors SB216763 (10 µM), roscovitine (10 µM) and Inhibitor I (5 µM) that reduced pAPP immunoreactivity by 54%, 39% and 20%, respectively (P<0.01 vs. HSV-1 alone). pAPP is a key event for A? production and HSV-1-infected neurons exhibited intracellular accumulation of A? assessed by Western Blot and immunofluorescence. HSV-1- infected neurons (24 hpi) had reduced expression of the presynaptic proteins synapsin-1 and synaptophysin (-58% and -39% vs. mock, respectively; P<0.05) that was paralleled by a marked reduction of the frequency of spontaneous miniature excitatory post-synaptic currents (from 7.6 to 3.7 Hz; P<0.01). These effects were significantly reversed by SB216763 and depended on A? accumulation. Indeed, an antibody recognizing A? and being uploaded intracellularly (4G8) rescued synaptic protein expression and synaptic transmission from the effects of HSV-1. In HSV-1-infected neurons derived from APP KO mice GSK-3 activation was not associated with A? accumulation and significant changes in synaptic protein expression, thus suggesting that GSK-3 alone is not sufficient to induce synaptic alterations in infected cells. Among the downstream components of the molecular pathways potentially responsible for the HSV-1-induced synaptic dysfunction, we studied cAMP responsive element-binding protein (CREB) because it is target of GSK-3 and is modulated by A?. We found that 24 hpi the activatory phosphorylation of CREB (at S133) was reduced whereas the inhibitory one (at S129) was increased (-25±2% and +35±4% vs. mock, respectively; P<0.01). Either SB216763 or 4G8 completely reversed these effects. Our data indicate that HSV-1 infections may contribute to the pathophysiology of AD by a mechanisms likely involving GSK-3 activation, A? accumulation and inhibition of CREB-dependent transcriptional program.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
