Cortical Structure Alterations and Social Behavior Impairment in p50-Deficient Mice.

Alterations in genes that regulate neurodevelopment can lead to cortical malformations, resulting in malfunction during postnatal life. The NF-κB pathway has a key role during neurodevelopment by regulating the maintenance of the neural progenitor cell pool and inhibiting neuronal differentiation. In this study, we evaluated whether mice lacking the NF-κB p50 subunit (KO) present alterations in cortical structure and associated behavioral impairment. We found that, compared with wild type (WT), KO mice at postnatal day 2 present an increase in radial glial cells, an increase in Reelin protein expression levels, in addition to an increase of specific layer thickness. Moreover, adult KO mice display abnormal columnar organization in the somatosensory cortex, a specific decrease in somatostatin- and parvalbumin-expressing interneurons, altered neurite orientation, and a decrease in Synapsin I protein levels. Concerning behavior, KO mice, in addition to an increase in locomotor and exploratory activity, display impairment in social behaviors, with a reduction in social interaction. Finally, we found that risperidone treatment decreased hyperactivity of KO mice, but had no effect on defective social interaction. Altogether, these data add complexity to a growing body of data, suggesting a link between dysregulation of the NF-κB pathway and neurodevelopmental disorders pathogenesis.

Potential Role of Microtubule Stabilizing Agents in Neurodevelopmental Disorders.

Neurodevelopmental disorders (NDDs) are characterized by neuroanatomical abnormalities indicative of corticogenesis disturbances. At the basis of NDDs cortical abnormalities, the principal developmental processes involved are cellular proliferation, migration and differentiation. NDDs are also considered "synaptic disorders" since accumulating evidence suggests that NDDs are developmental brain misconnection syndromes characterized by altered connectivity in local circuits and between brain regions. Microtubules and microtubule-associated proteins play a fundamental role in the regulation of basic neurodevelopmental processes, such as neuronal polarization and migration, neuronal branching and synaptogenesis. Here, the role of microtubule dynamics will be elucidated in regulating several neurodevelopmental steps. Furthermore, the correlation between abnormalities in microtubule dynamics and some NDDs will be described. Finally, we will discuss the potential use of microtubule stabilizing agents as a new pharmacological intervention for NDDs treatment.

Cytoskeletal protection: acting on notch to prevent neuronal dysfunction.

Functional and structural plasticity is a fundamental property of the brain involving chemical, electrical, molecular and cellular responses and leading to reorganization of connections within a brain region and/or between brain regions. The Notch pathway has been recognized as one of the main contributors in regulating neural development and has been proposed as a key mediator in neuroplasticity. We supported this concept, demonstrating that Notch plays a role in determining the only possible 'cell fate' decisions in post-mitotic mature neurons: synaptic remodelling or neurite extension/retraction. We demonstrated that Notch pathway activation causes a decrease in neurite branching and a loss of varicosities, with consequent reduction in the release of neurotransmitters. Furthermore, in dysfunctional neurons that present Notch pathway hyper-activation, neuronal morphology was reverted by Notch-inhibiting agents. Potentially, a better understanding of the molecular events participating in neuroplasticity may provide relevant information for innovative therapeutic approaches in a variety of neurological disorders. Hence, we propose a Notch-signalling fine-tuned manipulation as a novel approach to modulate neuronal cytoskeleton plasticity in order to prevent dysfunctional structural plasticity in neurodegenerative diseases.

Nuclear factor κB-dependent neurite remodeling is mediated by Notch pathway.

In this study, we evaluated whether a cross talk between nuclear factor κB (NF-κB) and Notch may take place and contribute to regulate cell morphology and/or neuronal network in primary cortical neurons. We found that lack of p50, either induced acutely by inhibiting p50 nuclear translocation or genetically in p50(-/-) mice, results in cortical neurons characterized by reduced neurite branching, loss of varicosities, and Notch1 signaling hyperactivation. The neuronal morphological effects found in p50(-/-) cortical cells were reversed after treatment with the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl 1]-S-phenylglycine t-butyl ester) or Notch RNA interference. Together, these data suggested that morphological abnormalities in p50(-/-) cortical neurons were dependent on Notch pathway hyperactivation, with Notch ligand Jagged1 being a major player in mediating such effect. In this line, we demonstrated that the p50 subunit acts as transcriptional repressor of Jagged1. We also found altered distribution of Notch1 and Jagged1 immunoreactivity in the cortex of p50(-/-) mice compared with wild-type littermates at postnatal day 1. These data suggest the relevance of future studies on the role of Notch/NF-κB cross talk in regulating cortex structural plasticity in physiological and pathological conditions.

Comparison of the characteristics, morbidity and mortality of COVID-19 between first and second/third wave in a hospital setting in Lombardy: a retrospective cohort study.

Coronavirus disease 2019 (COVID-19) represents a major health problem in terms of deaths and long-term sequelae. We conducted a retrospective cohort study at Montichiari Hospital (Brescia, Italy) to better understand the determinants of outcome in two different COVID-19 outbreaks. A total of 634 unvaccinated patients admitted from local emergency room to the Internal Medicine ward with a confirmed diagnosis of SARS-CoV-2 infection and a moderate-to-severe COVID-19 were included in the study. A group of 260 consecutive patients during SARS-CoV-2 first wave (from February to May 2020) and 374 consecutive patients during SARS-CoV-2 2nd/3rd wave (from October 2020 to May 2021) were considered. Demographic data were not significantly different between waves, except a lower prevalence of female sex during first wave. Mortality was significantly higher during the 1st wave than in the following periods (24.2% vs. 11%; p < 0.001). Time from symptoms onset to hospital admission was longer during first wave (8 ± 6 vs. 6 ± 4 days; p < 0.001), while in-hospital staying was significantly shorter (10 ± 14 vs. 15 ± 11 days; p < 0.001). Other significant differences were a larger use of corticosteroids and low-molecular weight heparin as well less antibiotic prescription during the second wave. Respiratory, bio-humoral and X-ray scores were significantly poorer at the time of admission in first-wave patients. After a multivariate regression analysis, C-reactive protein and procalcitonin values, % fraction of inspired oxygen on admission to the Internal Medicine ward and length of hospital stay and duration of symptoms were the strongest predictors of outcome. Concomitant anti-hypertensive treatment (including ACE-inhibitors and angiotensin-receptor blockers) did not affect the outcome. In conclusion, our data suggest that earlier diagnosis, timely hospital admission and rational use of the therapeutic options reduced the systemic inflammatory response and were associated to a better outcome during the 2nd/3rd wave.

Short-Term Consequences of SARS-CoV-2-Related Pneumonia: A Follow Up Study.

The aim of the study was to assess the short-term consequences of SARS-CoV-2-related pneumonia, also in relation to radiologic/laboratory/clinical indices of risk at baseline. This prospective follow-up cohort study included 94 patients with confirmed COVID-19 admitted to a medical ward at the Montichiari Hospital, Brescia, Italy from February 28th to April 30th, 2020. Patients had COVID-19 related pneumonia with respiratory failure. Ninety-four patients out of 193 survivors accepted to be re-evaluated after discharge, on average after 4 months. In » of the patients an evidence of pulmonary fibrosis was detected, as indicated by an altered diffusing capacity of the lung for carbon monoxide (DLCO); in 6-7% of patients the alteration was classified as of moderate/severe degree. We also evaluated quality of life thorough a structured questionnaire: 52% of the patients still lamented fatigue, 36% effort dyspnea, 10% anorexia, 14% dysgeusia or anosmia, 31% insomnia and 21% anxiety. Finally, we evaluated three prognostic indices (the Brixia radiologic score, the Charlson Comorbidity Index and the 4C mortality score) in terms of prediction of the clinical consequences of the disease. All of them significantly predicted the extent of short-term lung involvement. In conclusion, our study demonstrated that SARS-CoV-2-related pneumonia is associated to relevant short-term clinical consequences, both in terms of persistence of symptoms and in terms of impairment of DLCO (indicator of a possible development of pulmonary fibrosis); some severity indices of the disease may predict short-term clinical outcome. Further studies are needed to ascertain whether such manifestations may persist long-term.

Generation of reactive oxygen species by beta amyloid fibrils and oligomers involves different intra/extracellular pathways.

A neuropathological characteristic of Alzheimer's disease is the extracellular accumulation of amyloid beta peptide (Abeta) in neuritic plaques. Recent evidences suggested that soluble Abeta oligomers are the predominant neurotoxic species for neurons. Thus, considerable attention has been paid to discriminate the cytotoxic pathways of Abeta pre-fibrillar aggregates and mature fibrils. We showed that the mechanisms by which Abeta oligomers and fibrils generated reactive oxygen species differ in terms of site of production and kinetics, suggesting the involvement of different intra/extracellular pathways.

Wild type but not mutant APP is involved in protective adaptive responses against oxidants.

This study points out different behaviour between HEK cells overexpressing wild-type or mutant APP when exposed to oxidative insult. Although apparently both APPwt and APPmut overexpression conferred resistance to oxidative insult, some differences in terms of degree of protection was observed in the two clones. We found that the two clones differed, especially, in terms of redox profile. HEK-APPmut cells were characterized by higher levels of oxidative markers in comparison with HEK-APPwt. In addition, SOD activity appeared more efficient in HEK-APPwt than in HEK-APPmut, thus justifying the differences in terms of cell survival in the two clones. We suggest that, according to "hormesis theory", in HEK-APPwt cells low amount of oxidative stress can exert a beneficial effect that at a higher intensity results harmful. In contrast, HEK-APPmut cells lost this stress resistance probably because the degree of oxidative stress is too high and the antioxidant enzymes are themselves compromised.

Mitochondria-targeted antioxidant effects of S(-) and R(+) pramipexole.

BACKGROUND: Pramipexole exists as two isomers. The S(-) enantiomer is a potent D3/D2 receptor agonist and is extensively used in the management of PD. In contrast, the R(+) enantiomer is virtually devoid of any of the DA agonist effects. Very limited studies are available to characterize the pharmacological spectrum of the R(+) enantiomer of pramipexole. RESULTS: Using differentiated SH-SY5Y neuroblastoma cells as an experimental model, here we show that S(-) and R(+) pramipexole are endowed with equipotent efficacy in preventing cell death induced by H2O2 and inhibiting mitochondrial reactive oxygen species generation. Both pramipexole enantiomers prevented mitochondrial ROS generation with a potency about ten times higher then that elicited for neuroprotection. CONCLUSIONS: These results support the concept of both S(-) and R(+) pramipexole enantiomers as mitochondria-targeted antioxidants and suggest that the antioxidant, neuroprotective activity of these drugs is independent of both the chiral 6-propylamino group in the pramipexole molecule and the DA receptor stimulation.

Prognostic factors and predictors of outcome in patients with COVID-19 and related pneumonia: a retrospective cohort study.

The aim of the present study was to simultaneously assess several potential predictors of outcome (co-morbidity, previous and in-hospital treatment, radiologic Brixia score) in patients with COVID-19. This retrospective cohort study included 258 consecutive patients with confirmed COVID-19 admitted to a medical ward at Montichiari Hospital, Brescia, Italy from February 28th to April 30rd, 2020. Patients had SARS-CoV-2 related pneumonia with respiratory failure, and were treated with hydroxychloroquine and lopinavir plus ritonavir. In some patients, additional treatment with tocilizumab, dexamethasone and enoxaparin was adopted. Outcomes (death or recovery) were assessed at the end of the discharge period or at the end of the follow-up (August 2020). During hospitalization, 59 patients died, while 6 died after discharge. The following variables were demonstrated to be associated with a worse prognosis: Radiologic Brixia score higher than 8, presence at baseline of hypertension, diabetes, chronic obstructive pulmonary disease, heart disease, cancer, previous treatment with ACE-inhibitors or anti-platelet drugs. Anticoagulant treatment during hospital admission with enoxaparin at a dose higher than 4000 U once daily was associated with a better prognosis. In conclusion, our study demonstrates that some co-morbidities and cardiovascular risk factors may affect prognosis. The radiologic Brixia score may be a useful tool to stratify the risk of death at baseline. Anticoagulant treatment with enoxaparin might be associated to a clinical benefit in terms of survival in patients with COVID-19.

Impaired adult neurogenesis associated with short-term memory defects in NF-kappaB p50-deficient mice.

Neurogenesis proceeds throughout adulthood in the brain of most mammalian species, but the molecular mechanisms underlying the regulation of stem/progenitor cell proliferation, survival, maturation, and differentiation have not been completely unraveled. We have studied hippocampal neurogenesis in NF-kappaB p50-deficient mice. Here we demonstrate that in absence of p50, the net rate of neural precursor proliferation does not change, but some of the steps leading to the final neuron differentiation status are hampered, resulting in approximately 50% reduction in the number of newly born neurons in the adult mutant hippocampus. Additionally, in p50(-/-) mice, we observed a selective defect in short-term spatial memory performance without impairment of hippocampal-dependent spatial long-term memory and learning. Our results highlight the role of NF-kappaB p50 in hippocampal neurogenesis and in short-term spatial memory.

Cannabinoids in health and disease: pharmacological potential in metabolic syndrome and neuroinflammation.

The use of different natural and/or synthetic preparations of Cannabis sativa is associated with therapeutic strategies for many diseases. Indeed, thanks to the widespread diffusion of the cannabinoidergic system in the brain and in the peripheral districts, its stimulation, or inhibition, regulates many pathophysiological phenomena. In particular, central activation of the cannabinoidergic system modulates the limbic and mesolimbic response which leads to food craving. Moreover, cannabinoid agonists are able to reduce inflammatory response. In this review a brief history of cannabinoids and the protagonists of the endocannabinoidergic system, i.e. synthesis and degradation enzymes and main receptors, will be described. Furthermore, the pharmacological effects of cannabinoids will be outlined. An overview of the involvement of the endocannabinoidergic system in neuroinflammatory and metabolic pathologies will be made. Finally, particular attention will also be given to the new pharmacological entities acting on the two main receptors, cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), with particular focus on the neuroinflammatory and metabolic mechanisms involved.

Favorable prognostic role of tropomodulins in neuroblastoma.

Neuroblastoma is a pediatric tumor of the sympatoadrenal lineage of the neural crest characterized by high molecular and clinical heterogeneity, which are the main causes of the poor response to standard multimodal therapy. The identification of new and selective biomarkers is important to improve our knowledge on the mechanisms of neuroblastoma progression and to find the targets for innovative cancer therapies. This study identifies a positive correlation among tropomodulins (TMODs) proteins expression and neuroblastoma progression. TMODs bind the pointed end of actin filaments, regulate polymerization and depolymerization processes modifying actin cytoskeletal dynamic and influencing neuronal development processes. Expression levels of TMODs genes were analyzed in 17 datasets comprising different types of tumors, including neuroblastoma, and it was demonstrated that high levels of tropomodulin1 (TMOD1) and tropomodulin 2 (TMOD2) correlate positively with high survival probability and with favorable clinical and molecular characteristics. Functional studies on neuroblastoma cell lines, showed that TMOD1 knockin induced cell cycle arrest, cell proliferation arrest and a mature functional differentiation. TMOD1 overexpression was responsible for particular cell morphology and biochemical changes which directed cells towards a neuronal favorable differentiation profile. TMOD1 downregulation also induced cell proliferation arrest but caused the loss of mature cell differentiation and promoted the development of neuroendocrine cellular characteristics, delineating an aggressive and unfavorable tumor behavior. Overall, these data indicated that TMODs are favorable prognostic biomarkers in neuroblastoma and we believe that they could contribute to unravel a new pathophysiological mechanism of neuroblastoma resistance contributing to the design of personalized therapeutics opportunities.

Microvascular Density and Circulating Endothelial Progenitor Cells Before and After Treatment with Incretin Mimetics in Diabetic Patients.

INTRODUCTION: Glucagon-like peptide 1-receptor agonists (incretin mimetics) and dipeptidyl peptidase-4 inhibitors (incretin enhancers) have been recently introduced in the treatment of diabetes mellitus. In particular, incretin mimetics seems to have ancillary antioxidant/antinflammatory properties that might be involved in endothelial protection. AIM: To investigate the effect of incretin mimetic therapy (liraglutide, exenatide) given to 11 patients with type 2 diabetes mellitus, on circulating endothelial progenitor cells (EPCs) (bone marrow-derived cells possibly participating in neovascularization and endothelial protection and repair) and capillary density. METHODS: Four diabetic patients were treated with exenatide (5 µg twice daily for 4 weeks and then 10 µg twice daily for 3 weeks) and 7 with liraglutide (0.6 mg per day for 1 week and then 1.2 mg per day for 3 weeks). Peripheral venous blood samples were obtained before treatment (basal) and after 4 week in patients treated with liraglutide, and after 4 and 7 weeks in patients treated with exenatide, since drug titration is usually longer. EPCs were evaluated by flow cytometry as CD34+/KDR+ cells. Capillary density was evaluated by videomicroscopy, before and after venous congestion, in the dorsum of the 4th finger. RESULTS: Patients treated with liraglutide (6 males 1 female, age 54 ± 12 years) showed a decrease in body mass index and blood pressure during treatment, while patients treated with exenatide (3 males 1 female, age 57 ± 6 years) did not show any relevant change. EPCs were significantly increased after treatment with exenatide, but not after treatment with liraglutide. Capillary density was slightly increased only after 4 weeks of treatment with exenatide, however the increase was no longer present at the final evaluation. CONCLUSIONS: Treatment with exenatide, but not with liraglutide, was able to increase the number of circulating EPCs, possibly through an antioxidative/antiinflammatory effect.

Blockade of the tumor necrosis factor-related apoptosis inducing ligand death receptor DR5 prevents beta-amyloid neurotoxicity.

We originally suggested that inhibition of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) death pathway could be taken into consideration as a potential therapeutic strategy for Alzheimer's disease (AD). However, because the critical role of TRAIL in immune surveillance, the neutralization of TRAIL protein by an antibody to prevent its binding to death receptors is definitely a risky approach. Here, we demonstrated that the blockade of the TRAIL death receptor DR5 with a specific antibody completely prevented amyloid beta peptide (A beta) neurotoxicity in both neuronal cell line and primary cortical neurons. DR5 was demonstrated to be a key factor in TRAIL death pathway. In fact, whereas TRAIL expression was enhanced dose-dependently by concentrations of beta amyloid ranging from 10 nM to 1 microM, only the highest toxic dose of A beta (25 microM) induced the increased expression of DR5 and neuronal cell death. In addition, the increased expression of DR5 receptor after beta amyloid treatment was sustained by p53 transcriptional activity, as demonstrated by the data showing that the p53 inhibitor Pifithrin alpha prevented both beta amyloid-induced DR5 induction and cell death. These data suggest a sequential activation of p53 and DR5 upon beta amyloid exposure. Further insight into the key role of DR5 in AD was suggested by data showing a significant increase of DR5 receptor in cortical slices of AD brain. Thus, these findings may give intracellular TRAIL pathway a role in AD pathophysiology, making DR5 receptor a possible candidate as a pharmacological target.

Pramipexole prevents neurotoxicity induced by oligomers of beta-amyloid.

Here we demonstrate that pramipexole, an antiparkinsonian dopamine receptor agonist drug, exerts neuroprotective effects against beta-amyloid neurotoxicity. Using a specific protocol to test individually oligomers, fibrils, or unaggregated amyloid beta-peptide, we found pramipexole able to protect cells against oligomers and fibrils. Unaggregated amyloid beta-peptide was found unable to cause cell death. Fibrils and oligomers were also found to produce elevated amount of free radicals, and this effect was prevented by pramipexole. We propose pramipexole may become in the future a coadjuvant in the treatment of neuropathologies, besides Parkinson's disease, where amyloid beta-peptide-mediated oxidative injury exerts a relevant role.

Long-lasting induction of Notch2 in the hippocampus of kainate-treated adult mice.

Notch proteins are involved in cell fate specification during development in tissues including brain. Little is known about their function in adulthood. Recently, Notch receptors have been hypothesized to play a role in neurodegeneration and in particular in Alzheimer's disease (Notch1) and CADASIL (Notch3). Here we show that another family member (Notch2) is constitutively expressed in adult mouse hippocampus in DG and not in CA1 and CA3 neurons. Treatment with kainic acid resulted in marked Notch2 induction in pyramidal neurons of CA1 and in a subpopulation of CA3 neurons surviving the lesion and protein expression was still detectable 6 weeks after drug treatment. These results suggest Notch2 involvement in the response of postmitotic neurons to excitotoxic stimuli.

TorsinA negatively controls neurite outgrowth of SH-SY5Y human neuronal cell line.

Early onset generalized dystonia is a severe form of primary dystonia linked to a mutation of the DYT1(TOR1A) gene on chromosome 9q34. DYT1 gene codifies for human torsinA, an AAA+ ATPase associated with the membranes of endoplasmic reticulum (ER) and the synaptic vesicles and proposed to be involved in trafficking of tubular-vesicular membrane through neuronal processes. In this study, the presence and the intracellular distribution of torsinA protein in SH-SY5Y neuroblastoma cells were evaluated by immunofluorescence and Western blot analysis following differentiation with retinoic acid and BDNF. Protein expression was then inhibited by transient antisense transfection and the possible effect on neurite outgrowth was observed. In SH-SY5Y cells torsinA, with an apparent MW of 38 kDa, is endogenously present and distributed, with a punctate pattern, in the cytosol and along the neurites. The protein showed high intensity of immunoreactivity in the neurite varicosities and was partially co-localized with vesicles markers. Terminally differentiated cells showed an increase of protein expression. Oligonucleotide antisense treatment induced a significant response to differentiating stimuli, lead to sprouting of longer neurites and increase in growth cone areas. A relationship between torsinA and tau protein, which is involved in axon elongation and establishment of neuronal polarity, was demonstrated by co-immunoprecipitation experiments. These findings suggest that torsinA, throughout the interaction with microtubule associated proteins, may contribute to control neurite outgrowth and could be involved in maintaining cell polarity.

Involvement of DNA damage and repair systems in neurodegenerative process.

The study summarizes some recent data from our and other groups underlining the contribution to neurodegeneration of two transcription factors known to be involved in DNA damage sensing and repairing: the tumour suppressor gene p53 and the component of the DNA repair system MSH2. Both proteins participate in the cancer prevention machinery for the body as well as in the neurodegenerative process, suggesting that cancer and neurodegenerative disease may share common genetic risk factors for the development and progression of the disease. Here we show that, in neuronal cells, divergent cellular insults, i.e. the exposure to glutamate, beta-amyloid (Abeta) or H(2)O(2), may converge to a common pathway that initiate with elevation of p53 protein levels. We also found that in SH-SY5Y neuronal cells H(2)O(2) induced the activation of DNA repair system with the nuclear translocation of MSH2, and PCNA. Differently no changes in MSH2 and PCNA cellular distribution were found in undifferentiating SH-SY5Y cells exposed to H(2)O(2). This argues that defects in the repair of, or response to, DNA damage impact significantly on brain function.

TorsinA, microtubules and cell polarity.

Early-onset primary dystonia is an inherited disorder characterized by involuntary twisting, repetitive movements and abnormal postures. It has recently been demonstrated that the DYT1 gene is the most relevant gene associated with primary generalized dystonia. The DYT1 gene product is a 332-aminoacid long protein, termed TorsinA, whose function is still not clear. Based on the results obtained in other species, we proposed that TorsinA, similarly to OOC-5 in nematodes, directs and/or stabilizes the subcellular localization of specific kinases, which may in turn phosphorylate microtubule associated proteins, such as tau. In this way, TorsinA may contribute to maintaining the appropriate site-directed polarization and control neurite outgrowth.