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Allogeneic hematopoietic transplantation is a powerful treatment for hematologic malignancies. Posttransplant immune incompetence exposes patients to disease relapse and infections. We previously demonstrated that donor alloreactive natural killer (NK) cells ablate recipient hematopoietic targets, including leukemia. Here, in murine models, we show that infusion of donor alloreactive NK cells triggers recipient dendritic cells (DCs) to synthesize β-2-microglobulin (B2M) that elicits the release of c-KIT ligand and interleukin-7 that greatly accelerate posttransplant immune reconstitution. An identical chain of events was reproduced by infusing supernatants of alloreactive NK/DC cocultures. Similarly, human alloreactive NK cells triggered human DCs to synthesize B2M that induced interleukin-7 production by thymic epithelial cells and thereby supported thymocyte cellularity in vitro. Chromatography fractionation of murine and human alloreactive NK/DC coculture supernatants identified a protein with molecular weight and isoelectric point of B2M, and mass spectrometry identified amino acid sequences specific of B2M. Anti-B2M antibody depletion of NK/DC coculture supernatants abrogated their immune-rebuilding effect. B2M knock-out mice were unable to undergo accelerated immune reconstitution, but infusion of (wild-type) NK/DC coculture supernatants restored their ability to undergo accelerated immune reconstitution. Similarly, silencing the B2M gene in human DCs, before coculture with alloreactive NK cells, prevented the increase in thymocyte cellularity in vitro. Finally, human recombinant B2M increased thymocyte cellularity in a thymic epithelial cells/thymocyte culture system. Our studies uncover a novel therapeutic principle for treating posttransplant immune incompetence and suggest that, upon its translation to the clinic, patients may benefit from adoptive transfer of large numbers of cytokine-activated, ex vivo–expanded donor alloreactive NK cells.

Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression ofFoxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTPγS promotes rapid regeneration of the thymusin vivofollowing acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration.SUMMARYThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration.Damage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes.Elevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage.Extracellular ATP release promotesFoxn1expression in cTECs via activation of P2Y2Therapeutic targeting of the P2Y2 receptor promotes thymic regeneration.

The 10q24.33 locus is known to be associated with susceptibility to cutaneous malignant melanoma (CMM), but the mechanisms underlying this association have been not extensively investigated. We carried out an integrative genomic analysis of 10q24.33 using epigenomic annotations and in vitro reporter gene assays to identify regulatory variants. We found two putative functional single nucleotide polymorphisms (SNPs) in an enhancer and in the promoter of OBFC1, respectively, in neural crest and CMM cells, one, rs2995264, altering enhancer activity. The minor allele G of rs2995264 correlated with lower OBFC1 expression in 470 CMM tumors and was confirmed to increase the CMM risk in a cohort of 484 CMM cases and 1801 controls of Italian origin. Hi-C and chromosome conformation capture (3C) experiments showed the interaction between the enhancer-SNP region and the promoter of OBFC1 and an isogenic model characterized by CRISPR-Cas9 deletion of the enhancer-SNP region confirmed the potential regulatory effect of rs2995264 on OBFC1 transcription. Moreover, the presence of G-rs2995264 risk allele reduced the binding affinity of the transcription factor MEOX2. Biologic investigations showed significant cell viability upon depletion of OBFC1, specifically in CMM cells that were homozygous for the protective allele. Clinically, high levels of OBFC1 expression associated with histologically favorable CMM tumors. Finally, preliminary results suggested the potential effect of decreased OBFC1 expression on telomerase activity in tumorigenic conditions. Our results support the hypothesis that reduced expression of OBFC1 gene through functional heritable DNA variation can contribute to malignant transformation of normal melanocytes.

The capacity of T cells to recognize and mount an immune response against tumor antigens depends on the large diversity of the T-cell receptor (TCR) repertoire generated in the thymus during the process of T-cell development. However, this process is dramatically impaired by immunological insults, such as that caused by cytoreductive cancer therapies and infections, and by the physiological decline of thymic function with age. Defective thymic function and a skewed TCR repertoire can have significant clinical consequences. The presence of an adequate pool of T cells capable of recognizing specific tumor antigens is a prerequisite for the success of cancer immunotherapy using checkpoint blockade therapy. However, while this approach has improved the chances of survival of patients with different types of cancer, a large proportion of them do not respond. The limited response rate to checkpoint blockade therapy may be linked to a suboptimal TCR repertoire in cancer patients prior to therapy. Here, we focus on the role of the thymus in shaping the T-cell pool in health and disease, discuss how the TCR repertoire influences patients’ response to checkpoint blockade therapy and highlight approaches able to manipulate thymic function to enhance anti-tumor immunity.

Objective Spreading depolarization (SD) is a transient self-propagating wave of neuronal and glial depolarization coupled with large membrane ionic changes and a subsequent depression of neuronal activity. Spreading depolarization in the cortex is implicated in migraine, stroke, and epilepsy. Conversely, spreading depolarization in the striatum, a brain structure deeply involved in motor control and in Parkinson's disease (PD) pathophysiology, has been poorly investigated. Methods We characterized the participation of glutamatergic and dopaminergic transmission in the induction of striatal spreading depolarization by using a novel approach combining optical imaging, measurements of endogenous DA levels, and pharmacological and molecular analyses. Results We found that striatal spreading depolarization requires the concomitant activation of D1-like DA and N-methyl-d-aspartate receptors, and it is reduced in experimental PD. Chronic l-dopa treatment, inducing dyskinesia in the parkinsonian condition, increases the occurrence and speed of propagation of striatal spreading depolarization, which has a direct impact on one of the signaling pathways downstream from the activation of D1 receptors. Conclusion Striatal spreading depolarization might contribute to abnormal basal ganglia activity in the dyskinetic condition and represents a possible therapeutic target. © 2019 International Parkinson and Movement Disorder Society.

Parkinson’s disease (PD) is considered the most common disorder of synucleinopathy, which is characterised by intracellular inclusions of aggregated and misfolded α-synuclein (α-syn) protein in various brain regions, and the loss of dopaminergic neurons. During the early prodromal phase of PD, synaptic alterations happen before cell death, which is linked to the synaptic accumulation of toxic α-syn specifically in the presynaptic terminals, affecting neurotransmitter release. The oligomers and protofibrils of α-syn are the most toxic species, and their overexpression impairs the distribution and activation of synaptic proteins, such as the SNARE complex, preventing neurotransmitter exocytosis and neuronal synaptic communication. In the last few years, the role of the immune system in PD has been increasingly considered. Microglial and astrocyte activation, the gene expression of proinflammatory factors, and the infiltration of immune cells from the periphery to the central nervous system (CNS) represent the main features of the inflammatory response. One of the actors of these processes is α-syn accumulation. In light of this, here, we provide a systematic review of PD-related α-syn and inflammation inter-players.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma characterized by an impaired myogenic differentiation despite the expression of myogenic master genes MYOD and MYOG. Therefore, the restoration of differentiation is considered an anti-cancer therapy. SKP2 is an oncogenic E3-ubiquitin ligase that promotes cell proliferation by targeting the CDKi p21Cip1 and p27Kip1. Previous works showed that SKP2 overexpression is induced by the fusion oncoprotein PAX3-FOXO1 expressed in fusion positive (FP)-RMS cells, and promotes tumor cell proliferation through p27kip1 degradation. However, the role of SKP2 in fusion negative (FN)-RMS cells, devoid of any fusion gene, remains unclear. We report here that SKP2 transcript and protein levels are up-regulated in RMS patients and cell lines compared to normal tissue. Accordingly, we observed increased acetylation of H3K27 histone mark in RMS patients and cell lines compared to myoblasts and muscle tissue. We then show that in RMS cell lines SKP2 expression is induced by MYOD, which binds two SKP2 regulatory regions, an intronic and a distal enhancers, identified by Hi-C and 3C experiments. SKP2 knockdown in FN-RMS cells leads to p21Cip1 and p27Kip1 protein levels up-regulation coupled with G1/S cell cycle arrest. Rescue experiments showed that SKP2 promotes cell proliferation directly targeting p27Kip1. Moreover, SKP2 binds and promotes degradation of p57Kip2 and its silencing restores myogenic differentiation associated to MYOG and de novo MyHC expression in FN-RMS cells. SKP2 depletion also induces cell senescence and prevents anchorage-independent growth and stemness in vitro, and tumor growth in vivo. In turn, SKP2 forced expression partially rescued the anti-cancer effects preventing the increase of p21Cip1, p27Kip1, p57Kip2 and MYOG, promoting re-entry into cell cycle, inhibiting human myoblasts cell differentiation and restoring the tumorigenic potential in FN-RMS. Since neddylation is an essential step for the activity of SKP2, we used MLN4924, an inhibitor of the Nedd8 Activating Enzyme (NAE), under clinical investigation, to resume SKP2 knockdown features. MLN4924 induces p21Cip1 and p27Kip2 expression, promotes senescence and apoptosis, and hampers cell growth in vitro and in vivo both in FP- and FN-RMS. These results unveil an unprecedented role for SKP2 in governing both proliferation and myogenic differentiation in RMS, suggesting that targeting SKP2 functions through MLN4924 treatment might have clinical relevance in FP- and FN-RMS. The study has been founded by AIRC and 5xmille 2021/Ministero della Salute to RR. Citation Format: Silvia Pomella, Matteo Cassandri, Doris Phelps, Clara Perrone, Michele Pezzella, Marco Wachtel, Benjamin Sunkel, Antonella Cardinale, Zoe Walters, Cristina Cossetti, Sonia Rodriguez, Nadia Carlesso, Janet Shipley, Lucio Miele, Beat Schafer, Enrico Velardi, Peter Houghton, Berkley Gryder, Benjamin Stanton, Concetta Quintarelli, Biagio De Angelis, Franco Locatelli, Rossella Rota. A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 668.

Background In experimental models of Parkinson's disease (PD), different degrees of degeneration to the nigrostriatal pathway produce distinct profiles of synaptic alterations that depend on progressive changes in N-methyl-D-aspartate receptors (NMDAR)-mediated functions. Repetitive transcranial magnetic stimulation (rTMS) induces modifications in glutamatergic and dopaminergic systems, suggesting that it may have an impact on glutamatergic synapses modulated by dopamine neurotransmission. However, no studies have so far explored the mechanisms of rTMS effects at early stages of PD. Objectives We tested the hypothesis that in vivo application of rTMS with intermittent theta-burst stimulation (iTBS) pattern alleviates corticostriatal dysfunctions by modulating NMDAR-dependent plasticity in a rat model of early parkinsonism. Methods Dorsolateral striatal spiny projection neurons (SPNs) activity was studied through ex vivo whole-cell patch-clamp recordings in corticostriatal slices obtained from 6-hydroxydopamine-lesioned rats, subjected to a single session (acute) of iTBS and tested for forelimb akinesia with the stepping test. Immunohistochemical analyses were performed to analyze morphological correlates of plasticity in SPNs. Results Acute iTBS ameliorated limb akinesia and rescued corticostriatal long-term potentiation (LTP) in SPNs of partially lesioned rats. This effect was abolished by applying a selective inhibitor of GluN2B-subunit-containing NMDAR, suggesting that iTBS treatment could be associated with an enhanced activation of specific NMDAR subunits, which are major regulators of structural plasticity during synapse development. Morphological analyses of SPNs revealed that iTBS treatment reverted dendritic spine loss inducing a prevalence of thin-elongated spines in the biocytin-filled SPNs. Conclusions Taken together, our data identify that an acute iTBS treatment produces a series of plastic changes underlying striatal compensatory adaptation in the parkinsonian basal ganglia circuit. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Misfolding and aggregation of α-synuclein are specific features of Parkinson’s disease and other neurodegenerative diseases defined as synucleinopathies. Parkinson’s disease progression has been correlated with the formation and extracellular release of α-synuclein aggregates, as well as with their spread from neuron to neuron. Therapeutic interventions in the initial stages of Parkinson’s disease require a clear understanding of the mechanisms by which α-synuclein disrupts the physiological synaptic and plastic activity of the basal ganglia. For this reason, we identified two early time points to clarify how the intrastriatal injection of α-synuclein-preformed fibrils in rodents via retrograde transmission induces time-dependent electrophysiological and behavioural alterations. We found that intrastriatal α-synuclein-preformed fibrils perturb the firing rate of dopaminergic neurons in the substantia nigra pars compacta, while the discharge of putative GABAergic cells of the substantia nigra pars reticulata is unchanged. The α-synuclein-induced dysregulation of nigrostriatal function also impairs, in a time-dependent manner, the two main forms of striatal synaptic plasticity, long-term potentiation and long-term depression. We also observed an increased glutamatergic transmission measured as an augmented frequency of spontaneous excitatory synaptic currents. These changes in neuronal function in the substantia nigra pars compacta and striatum were observed before overt neuronal death occurred. In an additional set of experiments, we were able to rescue α-synuclein-induced alterations of motor function, striatal synaptic plasticity and increased spontaneous excitatory synaptic currents by subchronic treatment with l-DOPA, a precursor of dopamine widely used in the therapy of Parkinson’s disease, clearly demonstrating that a dysfunctional dopamine system plays a critical role in the early phases of the disease., Tozzi et al. show that intrastriatal injection of α-synuclein fibrils, modelling the early phase of Parkinson’s disease, alters motor behaviour and nigral and striatal neuron function in mice. These alterations can be rescued by l-DOPA, demonstrating the importance of the dopaminergic system in the early stages of Parkinson’s disease.

Introduction Plasticity at corticostriatal synapses is a key substrate for a variety of brain functions – including motor control, learning and reward processing – and is often disrupted in disease conditions. Despite intense research pointing toward a dynamic interplay between glutamate, dopamine (DA), and serotonin (5-HT) neurotransmission, their precise circuit and synaptic mechanisms regulating their role in striatal plasticity are still unclear. Here, we analyze the role of serotonergic raphe-striatal innervation in the regulation of DA-dependent corticostriatal plasticity. Methods Mice (males and females, 2–6 months of age) were housed in standard plexiglass cages at constant temperature (22 ± 1 °C) and maintained on a 12/12 h light/dark cycle with food and demineralized water ad libitum. In the present study, we used a knock-in mouse line in which the green fluorescent protein reporter gene (GFP) replaced the I Tph2 exon (Tph2GFP mice), allowing selective expression of GFP in the whole 5-HT system, highlighting both somata and neuritis of serotonergic neurons. Heterozygous, Tph2+/GFP, mice were intercrossed to obtain experimental cohorts, which included Wild-type (Tph2+/+), Heterozygous (Tph2+/GFP), and Mutant serotonin-depleted (Tph2GFP/GFP) animals. Results Using male and female mice, carrying on different Tph2 gene dosages, we show that Tph2 gene modulation results in sex-specific corticostriatal abnormalities, encompassing the abnormal amplitude of spontaneous glutamatergic transmission and the loss of Long Term Potentiation (LTP) in Tph2GFP/GFP mice of both sexes, while this form of plasticity is normally expressed in control mice (Tph2+/+). Once LTP is induced, only the Tph2+/GFP female mice present a loss of synaptic depotentiation. Conclusion We showed a relevant role of the interaction between dopaminergic and serotonergic systems in controlling striatal synaptic plasticity. Overall, our data unveil that 5-HT plays a primary role in regulating DA-dependent corticostriatal plasticity in a sex-related manner and propose altered 5-HT levels as a critical determinant of disease-associated plasticity defects.

A previous genome-wide association study (GWAS) identified common variation at the BARD1 locus as being highly associated with susceptibility to high-risk neuroblastoma, but the mechanisms underlying this association have been not extensively investigated. Here, we performed a fine mapping analysis of BARD1 locus (2q35) using GWAS data from 556 high-risk neuroblastoma patients and 2,575 controls of European-American ancestry, and identified two independent genome-wide neuroblastoma-associated loci. Functional single-nucleotide polymorphism (SNP) prioritization identified two causative variants that independently contributed to neuroblastoma risk, and each replicated robustly in multiple independent cohorts comprising 445 high-risk cases and 3,170 controls (rs17489363: combined p = 1.07 × 10-31 , OR:1.79, 95% CI:1.62-1.98 and rs1048108: combined p = 7.27 × 10-14 , OR:0.65, 95% CI:0.58-0.73). Particularly, the T risk allele of rs17489363 in the canonical promoter region of full-length BARD1 altered binding site of the transcription factor HSF1 and correlated with low expression of full-length BARD1 mRNA and protein. Low-level expression of full-length BARD1 associated with advanced neuroblastoma. In human neuroblastoma cells, attenuating full-length BARD1 increased proliferation and invasion capacity. In conclusion, we have identified two potentially causative SNPs at the BARD1 locus associated with predisposition to high-risk neuroblastoma, and have shown that full-length BARD1 may act as tumor suppressor.

Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10−8). We also detected a suggestive (P < 10−7) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10−4 demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1.

BARD1 is associated with the development of high-risk neuroblastoma patients. Particularly, the expression of full length (FL) isoform, FL BARD1, correlates to high-risk neuroblastoma development and its inhibition is sufficient to induce neuroblastoma cells towards a worst phenotype. Here we have investigated the mechanisms of FL BARD1 in neuroblastoma cell lines depleted for FL BARD1 expression. We have shown that FL BARD1 expression protects the cells from spontaneous DNA damage and from damage accumulated after irradiation. We demonstrated a role for FL BARD1 as tumor suppressor to prevent unscheduled mitotic entry of DNA damaged cells and to lead to death cells that have bypassed cell cycle checkpoints. FL BARD1-depleted cells that have survived to checkpoints acquire features of aggressiveness. Overall, our results show that FL BARD1 may defend cells against cancer and prevent malignant transformation of cells.

The genetic aetiology and the molecular mechanisms that characterize high‐risk neuroblastoma are still little understood. The majority of high‐risk neuroblastoma patients do not take advantage of current induction therapy. So far, one of the main reasons liable for cancer therapeutic failure is the acquisition of resistance to cytotoxic anticancer drugs, because of the DNA repair system of tumour cells. PARP1 is one of the main DNA damage sensors involved in the DNA repair system and genomic stability. We observed that high PARP1 mRNA level is associated with unfavourable prognosis in 3 public gene expression NB patients’ datasets and in 20 neuroblastomas analysed by qRT‐PCR. Among 4983 SNPs in PARP1, we selected two potential functional SNPs. We investigated the association of rs907187, in PARP1 promoter, and rs2048426 in non‐coding region with response chemotherapy in 121 Italian patients with high‐risk NB. Results showed that minor G allele of rs907187 associated with induction response of patients (P = .02) and with decrease PARP1 mRNA levels in NB cell line (P = .003). Furthermore, rs907187 was predicted to alter the binding site of E2F1 transcription factor. Specifically, allele G had low binding affinity with E2F1 whose expression positively correlates with PARP1 expression and associated with poor prognosis of patients with NB. By contrast, we did not find genetic association for the SNP rs2048426. These data reveal rs907187 as a novel potential risk variant associated with the failure of induction therapy for high‐risk NB.

In the past years, genome wide association studies (GWAS) have provided evidence that inter-individual susceptibility to diverse pathological conditions can reveal a common genetic architecture. Through the analysis of congenital heart disease (CHD) and neuroblastoma (NB) GWAS data, we aimed to dissect the genetic susceptibility shared between these conditions, which are known to arise from neural crest cell (NCC) migration or development abnormalities, via identification and functional characterization of common regions of association. Two loci (2q35 and 3q25.32) harbor single nucleotide polymorphisms (SNPs) that are associated at a p-value &lt, 10&minus, 3 with conotruncal malformations and ventricular septal defect respectively, as well as with NB. In addition, the lead SNP in 4p16.2 for atrial septal defect and the lead SNP in 3q25.32 for tetralogy of Fallot are less than 250 Kb distant from the lead SNPs for NB at the same genomic regions. Some of these shared susceptibility loci regulate the expression of relevant genes involved in NCC formation and developmental processes (such as BARD1, MSX1, and SHOX2) and are enriched in several epigenetic markers from NB and fetal heart cell lines. Although the clinical correlation between NB and CHD is unclear, our exploration of a possible common genetic basis between NB and a subset of cardiac malformations can help shed light on their shared embryological origin and pathogenetic mechanisms.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) that contributes to the main motor symptoms of the disease. At present, even if several advancements have been done in the last decades, the molecular and cellular mechanisms involved in the pathogenesis are far to be fully understood. Accordingly, the establishment of reliable in vitro experimental models to investigate the early events of the pathogenesis represents a key issue in the field. However, to mimic and reproduce in vitro the complex neuronal circuitry involved in PD-associated degeneration of DAergic neurons still remains a highly challenging issue. Here we will review the in vitro PD models used in the last 25 years of research, ranging from cell lines, primary rat or mice neuronal cultures to the more recent use of human induced pluripotent stem cells (hiPSCs) and, finally, the development of 3D midbrain organoids.

Inflammation significantly impacts the progression of Huntington's disease (HD) and the mutant HTT protein determines a pro‐inflammatory activation of microglia. Mesenchymal stem/stromal cells (MSC) from the amniotic membrane (hAMSC), and their conditioned medium (CM‐hAMSC), have been shown to possess protective effects in vitro and in vivo in animal models of immune‐based disorders and of traumatic brain injury, which have been shown to be mediated by their immunomodulatory properties. In this study, in the R6/2 mouse model for HD we demonstrate that mice treated with CM‐hAMSC display less severe signs of neurological dysfunction than saline‐treated ones. CM‐hAMSC treatment significantly delayed the development of the hind paw clasping response during tail suspension, reduced deficits in rotarod performance, and decreased locomotor activity in an open field test. The effects of CM‐hAMSC on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal atrophy and the formation of striatal neuronal intranuclear inclusions. In addition, while no significant increase was found in the expression of BDNF levels after CM‐hAMSC treatment, a significant decrease of microglia activation and inducible nitric oxide synthase levels were observed. These results support the concept that CM‐hAMSC could act by modulating inflammatory cells, and more specifically microglia.

Background Prolonged dopaminergic replacement therapy in PD results in pulsatile dopamine receptors stimulation in both dorsal and ventral striatum causing wearing off, motor fluctuations, and nonmotor side effects such as behavioral addictions. Among impulse control disorders, binge eating can be easily modeled in laboratory animals. Objectives We hypothesize that manipulation of dopamine levels in a 6-hydroxydopamine-lesioned rats, as a model of PD characterized by a different extent of dopamine denervation between dorsal and ventral striatum, would influence both synaptic plasticity of the nucleus accumbens and binge-like eating behavior. Methods Food preference, food intake, and weight gain were monitored in sham-operated and unilaterally lesioned rats, subjected to a modified version of Corwin's limited access protocol, modelling binge eating disorder. Electrophysiological properties and long-term potentiation of GABAergic spiny projection neurons of the nucleus accumbens core were studied through ex vivo intracellular and patch-clamp recordings from corticostriatal slices of naive and l-dopa-treated rats. Results Sham-operated animals with intact nucleus accumbens core plasticity reliably developed food-addiction-like behavior when exposed to intermittent access to a highly palatable food. In contrast, parkinsonian rats were unresponsive to such restriction regimens, and also plasticity was lost in ventral spiny neurons. Chronic l-dopa reestablished long-term potentiation and compulsive eating, but with a different temporal dynamic that follows that of drug administration. Conclusions Our data indicate that endogenous and exogenous dopamine drive binge-like consumption of a palatable food in healthy and parkinsonian rats with distinct temporal dynamics, providing new insights into the complexity of l-dopa effects on the mesolimbic dopaminergic system. © 2019 International Parkinson and Movement Disorder Society.

Huntington's disease (HD) is a fatal neurodegenerative condition, due to a mutation in the IT15 gene encoding for huntingtin. Currently, disease-modifying therapy is not available for HD, and only symptomatic drugs are administered for the management of symptoms. In the last few years, preclinical and clinical studies have indicated that pharmacological strategies aimed at inhibiting cyclic nucleotide phosphodiesterase (PDEs) may develop into a novel therapeutic approach in neurodegenerative disorders. PDEs are a family of enzymes that hydrolyze cyclic nucleotides into monophosphate isoforms. Cyclic nucleotides are second messengers that transduce the signal of hormones and neurotransmitters in many physiological processes, such as protein kinase cascades and synaptic transmission. An alteration in their balance results in the dysregulation of different biological mechanisms (transcriptional dysregulation, immune cell activation, inflammatory mechanisms, and regeneration) that are involved in neurological diseases. In this review, we discuss the action of phosphodiesterase inhibitors and their role as therapeutic agents in HD.

Terminal osseous dysplasia with pigmentary defects (TODPD) is an X-linked dominant syndrome with distal limb anomalies, pigmentary skin defects, digital fibromas, and generalized bone involvement due to a recurrent mutation in the filamin A (FLNA) gene. We here report the mutation c.5217G>A in FLNA in three families with TODPD and we found possible germline and somatic mosaicism in two out of the three families. The occurrence of somatic and germline mosaicism for TODPD indicates that caution should be taken in counseling recurrence risks for these conditions upon presentation of an isolated case.

Neuroglobin (Ngb) is expressed in the central and peripheral nervous system, cerebrospinal fluid, retina, and endocrine tissues where it is involved in binding O2 and other gasotransmitters. Several studies have highlighted its endogenous neuroprotective function. Huntington’s disease (HD), a dominant hereditary disease, is characterized by the gradual loss of neurons in discrete areas of the central nervous system. We analyzed the expression of Ngb in the brain tissue of a mouse model of HD, in order to define the role of Ngb with respect to individual cell type vulnerability in HD and to gender and age of mice. Our results showed different expressions of Ngb among neurons of a specific region and between different brain regions. We evidenced a decreased intensity of Ngb at 13 weeks of age, compared to 7 weeks of age. The double immunofluorescence and fluorescence resonance energy transfer (FRET) experiments showed that the co-localization between Ngb and huntingtin at the subcellular level was not close enough to account for a direct interaction. We also observed a different expression of Ngb in the striatum, depending on the sex and age of animals. These findings provide the first experimental evidence for an adaptive response of Ngb in HD, suggesting that Ngb may exert neuroprotective effects in HD beyond its role in reducing sensitivity to oxidative stress.

Among several mechanisms underlying the well-known trophic and protective effects of 17β-estradiol (E2) in the brain, we recently reported that E2 induces the up-regulation of two anti-apoptotic and neuroprotectant proteins: huntingtin (HTT) and neuroglobin (NGB). Here, we investigate the role of this up-regulation. The obtained results indicate that E2 promotes NGB-HTT association, induces the localization of the complex at the mitochondria, and protects SK-N-BE neuroblastoma cells and murine striatal cells, which express wild-type HTT (i.e., polyQ7), against H2O2-induced apoptosis. All E2 effects were completely abolished in HTT-knocked out SK-N-BE cells and in striatal neurons expressing the mutated form of HTT (mHTT; i.e., polyQ111) typical of Huntington’s disease (HD). As a whole, these data provide a new function of wild-type HTT which drives E2-induced NGB in mitochondria modulating NGB anti-apoptotic activity. This new function is lost by HTT polyQ pathological expansion. These data evidence the existence of a novel E2/HTT/NGB neuroprotective axis that may play a relevant role in the development of HD therapeutics.

Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in physiological processes as DNA repair, genomic stability, and apoptosis. Moreover, published studies demonstrated that PARP-1 mediates necrotic cell death in response to excessive DNA damage under certain pathological conditions. In Huntington’s disease brains, PARP immunoreactivity was described in neurons and in glial cells, thereby suggesting the involvement of apoptosis in HD. In this study, we sought to determine if the PARP-1 inhibitor exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD. Transgenic mice were treated with the PARP-1 inhibitor INO-1001 mg/Kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that INO 1001-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as striatal atrophy, morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. INO-1001 was effective in significantly increasing activated CREB and BDNF in the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that PARP-1 inhibition could be considered as a valid therapeutic approach for HD.

Background: Diuretics, have been accepted as first-line treatment in refractory heart failure, but a lack of response is a frequent event. A randomised single blind study was performed to evaluate the effects of the combination of high-dose furosemide and small-volume hypertonic saline solution (HSS) infusion in the treatment of refractory NYHA class IV congestive heart failure (CHF). Materials and methods: Sixty patients (21 F/39 M) with refractory CHF (NYHA class IV) of different etiologies, unresponsive to high oral doses of furosemide, ACE-inhibitors, digitalis, and nitrates, aged 65–90 years, were enrolled. They had to have an ejection fraction (EF) < 35%, serum creatinine < 2 mg/dl, BUN ≤60 mg/dl, a reduced urinary volume and a low natriuresis. The patients were randomised in two groups (single blind): group 1 (11 F/19 M) received an i.v. infusion of furosemide (500–1000 mg) plus HSS (150 ml of 1.4–4.6% NaCl) b.i.d. in 30 min. Group 2 (10 F/20 M) received an i.v. bolus of furosemide (500–1000 mg) b.i.d., without HSS, during a period lasting 6–12 days. Both groups received KCl (20–40 mEq.) i.v. to prevent hypokalemia. All patients underwent at entry a physical examination, measurement of body weight (BW), blood pressure (BP), heart rate (HR), evaluation of signs of CHF, and controls of serum Na, K, Cl, bicarbonate, albumin, uric acid, creatinine, urea and glycemia and daily during hospitalization, as well as the daily output of urine for, Na, K and Cl measurements. Chest X-ray, ECG and echocardiogram were obtained at entry during and at the discharge. During the treatment and after discharge the daily dietary Na intake was 120 mmol with a drink fluid intake of 1000 ml daily. An assessment of BW and 24-h urinary volume, serum and urinary laboratory parameters, until reaching a compensated state, were performed daily, when i.v. furosemide was replaced with oral administration (250–500 mg/day). After discharge, patients were followed as outpatients weekly for the first 3 months and subsequently once per month. Results: The groups were similar for age, sex, EF, risk factors, treatment and etiology of CHF. All patients showed a clinical improvement. Six patients in both groups had hyponatremia (from 120 to 128 mEq./l) at entry. A significant increase in daily diuresis in both groups was observed (from 390±155 to 2100±626, and from 433±141 to 1650±537 ml/24 h, P < 0.05). Natriuresis (from 49±15 to 198±28 mEq./24 h) was higher in group 1 vs. group 2 (from 53.83±12 to 129±39 mEq./24 h, P < 0.05). Serum Na (from 135.9±6.8 to 142.2±3.8 mEq./l, P < 0.05) increased in the group 1 and decreased in the group 2 (from 134.7±7.9 to 130.1±4.3 mEq./l). Serum K was decreased (from 4.4±0.6 to 3.9±0.6, and 4.6±9 to 3.6±0.5 mEq./l, P < 0. 05) in both groups. BW was reduced (from 73.8±9.1 to 63.8±8.8, and from 72.9±10.2 to 64.5±7.5 kg, P < 0.05) in both groups. Group 2 showed more patients in NYHA class III than group 1 (18 vs. 2 patients, P < 0.05). Group 2 showed an increase of serum creatinine. Serum uric acid increased in both groups. BP values decreased, and HR was corrected to normal values in both groups. Group 2 showed a longer hospitalization time than group receiving HHS infusion (11.67±1.8 vs. 8.57±2.3 days, P < 0.001). In the follow-up (6–12 months), none of the patients from group 1 were readmitted to the hospital and they maintained the NYHA class achieved at the discharge. Group 2 showed 12 patients readmitted to hospital and a higher class than at discharge. Conclusion: Our data suggest that the combination of furosemide with HSS is feasible and it appears that this combination produces an improvement of hemodynamic and clinical parameters, reduces the hospitalization time and maintains the obtained results over time in comparison with those receiving high-dose furosemide as bolus.