Inhibition of ERK1/2 or CRMP2 Disrupts Alcohol Memory Reconsolidation and Prevents Relapse in Rats.

Relapse to alcohol abuse, often caused by cue-induced alcohol craving, is a major challenge in alcohol addiction treatment. Therefore, disrupting the cue-alcohol memories can suppress relapse. Upon retrieval, memories transiently destabilize before they reconsolidate in a process that requires protein synthesis. Evidence suggests that the mammalian target of rapamycin complex 1 (mTORC1), governing the translation of a subset of dendritic proteins, is crucial for memory reconsolidation. Here, we explored the involvement of two regulatory pathways of mTORC1, phosphoinositide 3-kinase (PI3K)-AKT and extracellular regulated kinase 1/2 (ERK1/2), in the reconsolidation process in a rat (Wistar) model of alcohol self-administration. We found that retrieval of alcohol memories using an odor-taste cue increased ERK1/2 activation in the amygdala, while the PI3K-AKT pathway remained unaffected. Importantly, ERK1/2 inhibition after alcohol memory retrieval impaired alcohol-memory reconsolidation and led to long-lasting relapse suppression. Attenuation of relapse was also induced by post-retrieval administration of lacosamide, an inhibitor of collapsin response mediator protein-2 (CRMP2)-a translational product of mTORC1. Together, our findings indicate the crucial role of ERK1/2 and CRMP2 in the reconsolidation of alcohol memories, with their inhibition as potential treatment targets for relapse prevention.

FGF2 is an endogenous regulator of alcohol reward and consumption.

Alcohol use disorder (AUD) is a chronic, relapsing disorder, characterized by escalating alcohol drinking and loss of control, with very limited available treatments. We recently reported that the expression of fibroblast growth factor 2 (Fgf2) is increased in the striatum of rodents following long-term excessive alcohol drinking and that the systemic or intra-striatal administration of recombinant FGF2 increases alcohol consumption. Here, we set out to determine whether the endogenous FGF2 plays a role in alcohol drinking and reward, by testing the behavioural phenotype of Fgf2 knockout mice. We found that Fgf2 deficiency resulted in decreased alcohol consumption when tested in two-bottle choice procedures with various alcohol concentrations. Importantly, these effects were specific for alcohol, as a natural reward (sucrose) or water consumption was not affected by Fgf2 deficiency. In addition, Fgf2 knockout mice failed to show alcohol-conditioned place preference (CPP) but showed normal fear conditioning, suggesting that deletion of the growth factor reduces alcohol's rewarding properties. Finally, Fgf2 knockout mice took longer to recover from the loss of righting reflex and showed higher blood alcohol concentrations when challenged with an intoxicating alcohol dose, suggesting that their ethanol metabolism might be affected. Together, our results show that the endogenous FGF2 plays a critical role in alcohol drinking and reward and indicate that FGF2 is a positive regulator of alcohol-drinking behaviours. Our findings suggest that FGF2 is a potential biomarker for problem alcohol drinking and is a potential target for pharmacotherapy development for AUD.

Elevated and dysregulated bone morphogenic proteins in immune cells of patients with relapsing-remitting multiple sclerosis.

The abundance of neural stem cells (NSCs) in multiple sclerosis (MS) lesions with extensive astrogliosis suggests that fate factors of NSCs, such as the bone morphogenic protein (BMP) signaling maybe defective in MS. We found an elevated mRNA expression and protein secretion of BMP-2,4,5 but not of BMP-7. This was primarily in T cells. Cell stimulation with anti-CD3/CD28 antibodies or with IFN-γ induced expression of BMP-2,4,5 mRNA in untreated RR-MS patients, indicating that proinflammatory processes in MS may play a role in the BMP-2,4,5 productions in T cells. These results contribute to the understanding of the negligible extent of neurogenesis and oligodendrogenesis with extensive astrogliogenesis and the failure of adequate tissue repair in MS lesions.

Low and dysregulated production of follistatin in immune cells of relapsing-remitting multiple sclerosis patients.

One of the mechanisms known to play a key role in neuronal and oligodendroglial fate specification of neural stem cells (NSCs) is restriction of bone morphogenic proteins (BMP) signaling by BMP antagonists. Here, we demonstrate that follistatin mRNA and protein secreted levels in peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RR-MS) patients are significantly reduced compared to healthy controls (HC). We also observed a different profile of regulation mechanisms. Follistatin was similarly expressed and secreted by T lymphocytes and monocytes among the PBMCs of HC, and follistatin upregulation of HC was subjected to stimulation with both LPS and TNF-α. Among PBMCs of RR-MS patients, however, follistatin was found to be downregulated in their monocytes and unresponsive to stimulation with either LPS or TNF-α. Our results may shed some light on the mechanisms involved in remyelination failure in MS, which may be related to the inability of RR-MS patients' immune cells to provide a sufficient pro-neurogenic and oligodendrogenic niche, by expressing and secreting follistatin, in addition to the previously described noggin reduced expression. Our results indicate that the low expression of follistatin in immune cells of patients with RR-MS is a result of the altered immunoregulation of monocytes in these patients.

Reduced production of noggin by immune cells of patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) plaques are characterized by neurodegeneration, astrogliolis, the presence of immature oligodendrocytes and infiltrating immune cells. Recent studies revealed a putative role for noggin in both neurogenesis and oligodenrocytes development. In order to study the potential of peripheral immune cells to contribute to neurogenesis in MS, we studied the mRNA expression, protein secretion and regulation profile of noggin in peripheral blood mononuclear cells (PBMCs) of untreated patients with relapsing-remitting MS (RR-MS), interferon-ß (IFN-ß) treated RR-MS patients compared to matched healthy controls (HC). Basal levels of noggin mRNA expression, determined by quantitative real-time PCR were lower in untreated patients than in HC. No differences were found between untreated patients and IFN-ß treated patients. Similarly, the secreted levels of noggin, detected in 24h PBMCs supernatants by ELISA, were decreased in untreated RR-MS patients than in HC. Again no significant differences were found between untreated patients and IFN-ß treated patients. Stimulation with anti-CD3/CD28 mAbs increased noggin mRNA expression in untreated patients but not in HC. However, noggin mRNA levels in untreated patients PBMCs stimulated with anti-CD3/CD28 did not reach noggin levels in unstimulated PBMCs of HC. Purification of monocytes (CD14+) and T cells (CD3+ cells) by magnet-activated cell separation has demonstrated that noggin mRNA is predominantly expressed in CD3(+) cells in both HC and in RR-MS patients. This pattern also appeared in protein level of noggin, tested by Western blot. The incubation of the PBMCs with TNF-α increased the expression of noggin only in HC group. In conclusion, T cells possess the potential to participate in the induction of neurogeneration by the production of noggin. This potential seems to be defective in immune cells of RR-MS patients as there is reduced mRNA expression and protein secretion levels of noggin, insufficient stimulatory effect of CD3/CD28 stimulation and unresponsiveness to TNF-α in these patients PBMCs.

Dysregulated neurotrophin mRNA production by immune cells of patients with relapsing remitting multiple sclerosis.

Recent studies have suggested a neuroprotective activity of the lesion-infiltrating immune cells in multiple sclerosis (MS) by secretion of neurotrophins. We had earlier reported that immune cells from relapsing remitting MS (RR-MS) patients secrete low levels of brain-derived neurotrophic factor (BDNF), and that its secretion is dysregulated after CD40 stimulation. Here, we measured mRNA levels for BDNF, NT3 and NGF-beta mRNA in unstimulated PBMCs and found levels lower in untreated RR-MS patients than in healthy controls (HC). T-cell stimulation with anti-CD3/CD28 mAb up-regulated neurotrophin mRNA expression in untreated RR-MS patients and not in HC, whereas stimulation of PBMCs with anti-CD40 mAb up-regulated neurotrophin mRNA expression in HC and not in RR-MS patients. Further cellular analyses of the production of the neurotrophin mRNA in individual cells revealed that T cells were the main producers of the neurotrophin mRNA in RR-MS patients, and that monocytes were the main producers of NT3 and NGF-beta mRNA in HC. BDNF mRNA was similarly produced in monocytes and T cells in the HC group. The cytokines TNF-alpha and IL-17 up-regulated the expression of neurotrophin mRNA in HC but not in RR-MS patients. The neuroprotective activity of PBMCs appeared to be dysregulated in untreated patients with RR-MS, while the differences between the IFN-beta-treated RR-MS patients and the HC were smaller.

Interferon-beta therapy up-regulates BDNF secretion from PBMCs of MS patients through a CD40-dependent mechanism.

We had reported that immune cells from relapsing remitting multiple sclerosis (RR-MS) patients secrete low levels of BDNF and that there is a defective regulation of its secretion via DC40. We now studied the effect of interferon-beta (IFN-beta1) on the secretion and regulation of BDNF from immune cells in patients with RR-MS. The PBMCs of IFN-beta1a treated RR-MS patients secreted higher BDNF levels vs. untreated patients. Anti CD40 mAb stimulation of PBMCs of IFN-beta1a treated patients upregulated the BDNF levels. There was no significant effect of CD40 stimulation on PBMCs of untreated patients. CD40(+) expression on CD14(+) cells was higher in IFN-beta treated patients vs. untreated patients. In vitro treatment with IFN-beta1a of PBMCs from healthy controls and untreated patients led to a significant increase in CD40 expression on CD14(+) cells in both groups. The addition of IFN-beta1a to CD40 stimulated PBMCs of untreated patients restored the up regulatory effect of CD40 stimulation on BDNF levels. Therefore, reduced BDNF secretion from PBMCs and defective regulation effect of CD40 stimulation on BDNF levels in untreated RR-MS are reversible by therapy with IFN-beta1a.