Marco Feligioni

Inflammation Subverts Hippocampal Synaptic Plasticity in Experimental Multiple Sclerosis

Abnormal use-dependent synaptic plasticity is universally accepted as the main physiological correlate of memory deficits in neurodegenerative disorders. It is unclear whether synaptic plasticity deficits take place during neuroinflammatory diseases, such as multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). In EAE mice, we found significant alterations of synaptic plasticity rules in the hippocampus. When compared to control mice, in fact, hippocampal long-term potentiation (LTP) induction was favored over long-term depression (LTD) in EAE, as shown by a significant rightward shift in the frequency–synaptic response function. Notably, LTP induction was also enhanced in hippocampal slices from control mice following interleukin-1β (IL-1β) perfusion, and both EAE and IL-1β inhibited GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) without affecting glutamatergic transmission and AMPA/NMDA ratio. EAE was also associated with selective loss of GABAergic interneurons and with reduced gamma-frequency oscillations in the CA1 region of the hippocampus. Finally, we provided evidence that microglial activation in the EAE hippocampus was associated with IL-1β expression, and hippocampal slices from control mice incubated with activated microglia displayed alterations of GABAergic transmission similar to those seen in EAE brains, through a mechanism dependent on enhanced IL-1β signaling. These data may yield novel insights into the basis of cognitive deficits in EAE and possibly of MS.

c-Jun N-terminal kinase regulates soluble Aβ oligomers and cognitive impairment in AD mouse model.

Background: Neuropathological mechanisms in Alzheimer disease (AD) are partially unknown. Results: Chronic JNK inhibition with a cell-permeable peptide (CPP) rescues memory deficits, LTP impairment, and reduces Aβ oligomers in a mouse model that mimics AD. Conclusion: JNK is crucial in AD neurodegenerative mechanisms. Significance: CPPs offer an important tool to interfere with neurodegeneration. JNK is a promising target against AD. Alzheimer disease (AD) is characterized by cognitive impairment that starts with memory loss to end in dementia. Loss of synapses and synaptic dysfunction are closely associated with cognitive impairment in AD patients. Biochemical and pathological evidence suggests that soluble Aβ oligomers correlate with cognitive impairment. Here, we used the TgCRND8 AD mouse model to investigate the role of JNK in long term memory deficits. TgCRND8 mice were chronically treated with the cell-penetrating c-Jun N-terminal kinase inhibitor peptide (D-JNKI1). D-JNKI1, preventing JNK action, completely rescued memory impairments (behavioral studies) as well as the long term potentiation deficits of TgCRND8 mice. Moreover, D-JNKI1 inhibited APP phosphorylation in Thr-668 and reduced the amyloidogenic cleavage of APP and Aβ oligomers in brain parenchyma of treated mice. In conclusion, by regulating key pathogenic mechanisms of AD, JNK might hold promise as innovative therapeutic target.

Crosstalk between JNK and SUMO Signaling Pathways: deSUMOylation Is Protective against H2O2-Induced Cell Injury

Background Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown. Methodology/Principal Findings With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H2O2 induced-oxidative stress. Our data show that H2O2 modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNKs action with the D-JNKI1 peptide rescued cells from death. Following H2O2, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H2O2 induced death. Importantly, in our model, direct interaction between these proteins can occur. Conclusions/Significance Taken together our results show that SUMOylation may significantly contribute to modulation of JNK activation and contribute to cell death in oxidative stress conditions.

N‐Methyl‐d‐aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV‐1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [3H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 µm or 1 mm) plus glycine (1 µm)‐elicited [3H]NA, but not [3H]DA release. The NMDA (100 µm)‐evoked release of [3H]NA and [3H]DA was similar and concentration‐dependently enhanced by glycine or d‐serine (0.1–1 µm); in contrast, the HIV‐1 envelope protein gp120 potently (30–100 pm) enhanced the NMDA‐evoked release of [3H]NA, but not that of [3H]DA. Gp120 also potentiated quinolinate‐evoked [3H]NA release. Ifenprodil (0.1–0.5 µm) or CP‐101,606 (0.1–10 µm) inhibited the NMDA plus glycine‐evoked release of both [3H]catecholamines. Zinc (0.1–1 µm) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [3H]NA elicited by NMDA plus glycine, whereas the release of [3H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 µm) or H7 (10 µm) selectively prevented the effect of NMDA plus glycine on the release of [3H]NA. GF 109203X also blocked the release of [3H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.

SUMO: a (Oxidative) Stressed Protein

Redox species are produced during the physiological cellular metabolism of a normal tissue. In turn, their presence is also attributed to pathological conditions including neurodegenerative diseases. Many are the molecular changes that occur during the unbalance of the redox homeostasis. Interestingly, posttranslational protein modifications (PTMs) play a remarkable role. In fact, several target proteins are modified in their activation, localization, aggregation, and expression after the cellular stress. Among PTMs, protein SUMOylation represents a very important molecular modification pathway during “oxidative stress”. It has been reported that this ubiquitin-like modification is a fine sensor for redox species. Indeed, SUMOylation pathway efficiency is affected by the exposure to oxidative species in a different manner depending on the concentration and time of application. Thus, we here report updated evidence that states the role of SUMOylation in several pathological conditions, and we also outline the key involvement of c-Jun N-terminal kinase and small ubiquitin modifier pathway cross talk.

Potentiation of NMDA receptor function through somatostatin release: a possible mechanism for the cognition-enhancing activity of GABAB receptor antagonists

CGP 36742 is a weak GABA(B) receptor antagonist. However, it improves cognitive performances at low doses; it blocks GABA(B) receptors potently and selectively on somatostatinergic terminals; it prevents kynurenate from antagonising NMDA-induced release of noradrenaline from rat brain slices potently. We here investigated whether and how somatostatin plays a role in the CGP 36742 activity. CGP 36742 increased the somatostatin-like immunoreactivity (SRIF-LI) release from hippocampal slices exposed to NMDA. In the kynurenate test with rat hippocampal slices SRIF-14 mimicked the effect of CGP 36742. CGP 36742 lost its activity in rats whose somatostatin content had been depleted with cysteamine. Exogenous SRIF-14 reverted kynurenate antagonism in somatostatin-depleted slices. L362855, an sst(5) receptor agonist, but not the selective sst(1)-sst(4) agonists, L797591, L779976, L796778 and L803087, displayed activity in the kynurenate test. The effects of CGP 36742, SRIF-14 and L362855 were antagonised by the sst(5)-preferring antagonist BIM-23056. The protein kinase C inhibitor GF 109203X prevented the reversal of the kynurenate antagonism by CGP 36742 or SRIF-14. In conclusion, by selectively blocking GABA(B) receptors on somatostatinergic terminals, CGP 36742 may disinhibit somatostatin release; the consequent activation of sst(5) receptors would potentiate the function of NMDA receptors coexisting with sst(5) receptors on noradrenergic neurons.

The Human Immunodeficiency Virus-1 Protein Transactivator of Transcription Up-Regulates N-Methyl-d-aspartate Receptor Function by Acting at Metabotropic Glutamate Receptor 1 Receptors Coexisting on Human and Rat Brain Noradrenergic Neurones

We investigated the effects of the human immunodeficiency virus-1 transactivator of transcription (Tat) on the release of norepinephrine (NE) from human and rat brain synaptosomes. Tat could not evoke directly release of [3H]NE. In the presence of Tat (1 nM), N-methyl-d-aspartate (NMDA) concentrations unable to release (human synaptosomes) or slightly releasing (rat synaptosomes) [3H]NE became very effective. The NMDA/Tat-evoked release depends on NMDA receptors (NMDARs) since it was abolished by MK-801 (dizocilpine). Tat binding at NMDARs was excluded. The NMDA-induced release of [3H]NE in the presence of glycine was further potentiated by Tat. The release evoked by NMDA/glycine/Tat depends on metabotropic glutamate receptor 1 (mGluR1) activation, since it was halved by mGluR1 antagonists. Tat seems to act at the glutamate recognition site of mGluR1. Recently, Tat was shown to release [3H]acetylcholine from human cholinergic terminals; here, we demonstrate that this effect is also mediated by presynaptic mGluR1. The peptide sequence Tat41–60, but not Tat61–80, mimicked Tat. Phospholipase C, protein kinase C, and cytosolic tyrosine kinase are involved in the NMDA/glycine/Tat-evoked [3H]NE release. To conclude, Tat can represent a potent pathological agonist at mGlu1 receptors able to release acetylcholine from human cholinergic terminals and up-regulate NMDARs mediating NE release from human and rat noradrenergic terminals.

Age-related changes of protein SUMOylation balance in the AβPP Tg2576 mouse model of Alzheimer's disease

Alzheimers disease (AD) is a complex disorder that affects the central nervous system causing a severe neurodegeneration. This pathology affects an increasing number of people worldwide due to the overall aging of the human population. In recent years SUMO protein modification has emerged as a possible cellular mechanism involved in AD. Some of the proteins engaged in the physiopathological process of AD, like BACE1, GSK3-β tau, AβPP, and JNK, are in fact subject to protein SUMO modifications or interactions. Here, we have investigated the SUMO/deSUMOylation balance and SUMO-related proteins during the onset and progression of the pathology in the Tg2576 mouse model of AD. We examined four age-stages (1.5, 3, 6, 17 months old) and observed shows an increase in SUMO-1 protein conjugation at 3 and 6 months in transgenic mice with respect to WT in both cortex and hippocampus. Interestingly this is paralleled by increased expression levels of Ubc9 and SENP1 in both brain regions. At 6 months of age also the SUMO-1 mRNA resulted augmented. SUMO-2-ylation was surprisingly decreased in old transgenic mice and was unaltered in the other time windows. The fact that alterations in SUMO/deSUMOylation equilibrium occur from the early phases of AD suggests that global posttranslational modifications may play an important role in the mechanisms underlying disease pathogenesis, thus providing potential targets for pharmacological interventions.

In vitro exposure to nicotine induces endocytosis of presynaptic AMPA receptors modulating dopamine release in rat nucleus accumbens nerve terminals.

Here we provide functional and immunocytochemical evidence supporting the presence on Nucleus Accumbens (NAc) dopaminergic terminals of cyclothiazide-sensitive, alfa-amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors, which activation causes Ca²⁺-dependent [³H]dopamine ([³H]DA) exocytosis. These AMPA receptors cross-talk with co-localized nicotinic receptors (nAChRs), as suggested by the finding that in vitro short-term pre-exposure of synaptosomes to 30 μM nicotine caused a significant reduction of both the 30 μM nicotine and the 100 μM AMPA-evoked [³H]DA overflow. Entrapping pep2-SVKI, a peptide known to compete for the binding of GluA2 subunit to scaffolding proteins involved in AMPA receptor endocytosis, in NAC synaptosomes prevented the nicotine-induced reduction of AMPA-mediated [³H]DA exocytosis, while pep2-SVKE, used as negative control, was inefficacious. Immunocytochemical studies showed that a significant percentage of NAc terminals were dopaminergic and that most of these terminals also posses GluA2 receptor subunits. Western blot analysis of GluA2 immunoreactivity showed that presynaptic GluA2 proteins in NAc terminals were reduced in nicotine-pretreated synaptosomes when compared to the control. The nACh-AMPA receptor-receptor interaction was not limited to dopaminergic terminals since nicotine pre-exposure also affected the presynaptic AMPA receptors controlling hippocampal noradrenaline release, but not the presynaptic AMPA receptors controlling GABA and acetylcholine release. These observations could be relevant to the comprehension of the molecular mechanisms at the basis of nicotine rewarding.

Synaptic plasticity in multiple sclerosis and in experimental autoimmune encephalomyelitis

Approximately half of all patients with multiple sclerosis (MS) experience cognitive dysfunction, including learning and memory impairment. Recent studies suggest that hippocampal pathology is involved, although the mechanisms underlying these deficits remain poorly understood. Evidence obtained from a mouse model of MS, the experimental autoimmune encephalomyelitis (EAE), suggests that in the hippocampus of EAE mice long-term potentiation (LTP) is favoured over long-term depression in response to repetitive synaptic activation, through a mechanism dependent on enhanced IL-1β released from infiltrating lymphocytes or activated microglia. Facilitated LTP during an immune-mediated attack might underlie functional recovery, but also cognitive deficits and excitotoxic neurodegeneration. Having identified that pro-inflammatory cytokines such as IL-1β can influence synaptic function and integrity in early MS, it is hoped that new treatments targeted towards preventing synaptic pathology can be developed.