IGF2 prevents dopaminergic neuronal loss and decreases intracellular alpha-synuclein accumulation in Parkinson's disease models.

Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.

The c-Abl/p73 pathway induces neurodegeneration in a Parkinson's disease model.

Parkinson's disease is the second most common neurodegenerative disorder. Although it is clear that dopaminergic neurons degenerate, the underlying molecular mechanisms are still unknown, and thus, successful treatment is still elusive. One pro-apoptotic pathway associated with several neurodegenerative diseases is the tyrosine kinase c-Abl and its target p73. Here, we evaluated the contribution of c-Abl and p73 in the degeneration of dopaminergic neurons induced by the neurotoxin 6-hydroxydopamine as a model for Parkinson's disease. First, we found that in SH-SY5Y cells treated with 6-hydroxydopamine, c-Abl and p73 phosphorylation levels were up-regulated. Also, we found that the pro-apoptotic p73 isoform TAp73 was up-regulated. Then, to evaluate whether c-Abl tyrosine kinase activity is necessary for 6-hydroxydopamine-induced apoptosis, we co-treated SH-SY5Y cells with 6-hydroxydopamine and Imatinib, a c-Abl specific inhibitor, observing that Imatinib prevented p73 phosphorylation, TAp73 up-regulation, and protected SH-SY5Y cells treated with 6-hydroxydopamine from apoptosis. Interestingly, this observation was confirmed in the c-Abl conditional null mice, where 6-hydroxydopamine stereotaxic injections induced a lesser reduction of dopaminergic neurons than in the wild-type mice significantly. Finally, we found that the intraperitoneal administration of Imatinib prevented the death of dopaminergic neurons induced by injecting 6-hydroxydopamine stereotaxically in the mice striatum. Thus, our findings support the idea that the c-Abl/p73 pathway is involved in 6-hydroxydopamine degeneration and suggest that inhibition of its kinase activity might be used as a therapeutical drug in Parkinson's disease.

Dynamics of Central Remyelination and Treatment Evolution in a Model of Multiple Sclerosis with Optic Coherence Tomography.

The need for remyelinating drugs is essential for healing disabling diseases such as multiple sclerosis (MS). One of the reasons for the lack of this class of therapies is the impossibility to monitor remyelination in vivo, which is of utmost importance to perform effective clinical trials. Here, we show how optical coherence tomography (OCT), a cheap and non-invasive technique commonly used in ophthalmology, may be used to assess remyelination in vivo in MS patients. Our pioneer approach validates OCT as a technique to study remyelination of the optic nerve and reflects what is occurring in non-accessible central nervous system (CNS) structures, like the spinal cord. In this study we used the orally bioavailable small molecule VP3.15, confirming its therapeutical potential as a neuroprotective, anti-inflammatory, and probably remyelinating drug for MS. Altogether, our results confirm the usefulness of OCT to monitor the efficacy of remyelinating therapies in vivo and underscore the relevance of VP3.15 as a potential disease modifying drug for MS therapy.

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the UPR in models of neurodegeneration.

Alteration to endoplasmic reticulum (ER) proteostasis is observed in a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR target genes. In this study, we designed an ATF6f/XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has a stronger effect in reducing the abnormal aggregation of mutant huntingtin and α-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson's disease and Huntington's disease. These results support the concept in which directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

Myeloid-derived suppressor cells support remyelination in a murine model of multiple sclerosis by promoting oligodendrocyte precursor cell survival, proliferation, and differentiation.

The most frequent variant of multiple sclerosis (MS) is the relapsing-remitting form, characterized by symptomatic phases followed by periods of total/partial recovery. Hence, it is possible that these patients can benefit from endogenous agents that control the inflammatory process and favor spontaneous remyelination. In this context, there is increasing interest in the role of myeloid-derived suppressor cells (MDSCs) during the clinical course of experimental autoimmune encephalomyelitis (EAE). MDSCs speed up infiltrated T-cell anergy and apoptosis. In different animal models of MS, a milder disease course is related to higher presence/density of MDSCs in the periphery, and smaller demyelinated lesions in the central nervous system (CNS). These observations lead us to wonder whether MDSCs might not only exert an anti-inflammatory effect but might also have direct influence on oligodendrocyte precursor cells (OPCs) and remyelination. In the present work, we reveal for the first time the relationship between OPCs and MDSCs in EAE, relationship that is guided by the distance from the inflammatory core. We describe the effects of MDSCs on survival, proliferation, as well as potent promoters of OPC differentiation toward mature phenotypes. We show for the first time that osteopontin is remarkably present in the analyzed secretome of MDSCs. The ablation of this cue from MDSCs-secretome demonstrates that osteopontin is the main MDSC effector on these oligodendroglial cells. These data highlight a crucial pathogenic interaction between innate immunity and the CNS, opening ways to develop MDSC- and/or osteopontin-based therapies to promote effective myelin preservation and repair in MS patients.

The proportion of myeloid-derived suppressor cells in the spleen is related to the severity of the clinical course and tissue damage extent in a murine model of multiple sclerosis.

Multiple Sclerosis (MS) is the second cause of paraplegia among young adults, after all types of CNS traumatic lesions. In its most frequent relapsing-remitting form, the severity of the disease course is very heterogeneous, and its reliable evaluation remains a key issue for clinicians. Myeloid-Derived sSuppressor Cells (MDSCs) are immature myeloid cells that suppress the inflammatory response, a phenomenon related to the resolution or recovery of the clinical symptoms associated with experimental autoimmune encephalomyelitis (EAE), the most common model for MS. Here, we establish the severity index as a new parameter for the clinical assessment in EAE. It is derived from the relationship between the maximal clinical score and the time elapsed since disease onset. Moreover, we relate this new index with several histopathological hallmarks in EAE and with the peripheral content of MDSCs. Based on this new parameter, we show that the splenic MDSC content is related to the evolution of the clinical course of EAE, ranging from mild to severe. Indeed, when the severity index indicates a severe disease course, EAE mice display more intense lymphocyte infiltration, demyelination and axonal damage. A direct correlation was drawn between the MDSC population in the peripheral immune system, and the preservation of myelin and axons, which was also correlated with T cell apoptosis within the CNS (being these cells the main target for MDSC suppression). The data presented clearly indicated that the severity index is a suitable tool to analyze disease severity in EAE. Moreover, our data suggest a clear relationship between circulating MDSC enrichment and disease outcome, opening new perspectives for the future targeting of this population as an indicator of MS severity.

The presence and suppressive activity of myeloid-derived suppressor cells are potentiated after interferon-ß treatment in a murine model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the human central nervous system (CNS), mainly affecting young adults. Among the immunomodulatory disease modifying treatments approved up to date to treat MS, IFN-ß remains to be one of the most widely prescribed for the Relapsing-Remitting (RR) variant of the disease, although its mechanism of action is still partially understood. RR-MS variant is characterized by phases with increasing neurological symptoms (relapses) followed by periods of total or partial recovery (remissions), which implies the existence of immunomodulatory agents to promote the relapsing-to-remitting transition. Among these agents, it has been described the immunosuppressive role of a heterogeneous population of immature myeloid cells, namely the myeloid-derived suppressor cells (MDSCs) during the clinical course of the experimental autoimmune encephalomyelitis (EAE), the most used MS model to study RRMS. However, it is still unknown how the current MS disease modifying treatments, e.g. IFN- ß, affects to MDSCs number or activity. Our present results show that a single injection of IFN-ß at the onset of the clinical course reduces the severity of the EAE, enhancing the presence of MDSCs within the smaller demyelinated areas. Moreover, the single dose of IFN-ß promotes MDSC immunosuppressive activity both in vivo and in vitro, augmenting T cell apoptosis. Finally, we show that IFN-ß preserves MDSC immaturity, preventing their differentiation to mature and less suppressive myeloid cell subsets. Taking together, all these data add new insights into the mechanism of IFN-ß treatment in EAE and point to MDSCs as a putative endogenous mediator of its beneficial role in this animal model of MS.

Disulfide cross-linked multimers of TDP-43 and spinal motoneuron loss in a TDP-43A315T ALS/FTD mouse model.

Tar DNA binding protein 43 (TDP-43) is the principal component of ubiquitinated protein inclusions present in nervous tissue of most cases of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies described a TDP-43A315T transgenic mouse model that develops progressive motor dysfunction in the absence of protein aggregation or significant motoneuron loss, questioning its validity to study ALS. Here we have further characterized the course of the disease in TDP-43A315T mice using a battery of tests and biochemical approaches. We confirmed that TDP-43 mutant mice develop impaired motor performance, accompanied by progressive body weight loss. Significant differences were observed in life span between genders, where females survived longer than males. Histopathological analysis of the spinal cord demonstrated a significant motoneurons loss, accompanied by axonal degeneration, astrogliosis and microglial activation. Importantly, histopathological alterations observed in TDP-43 mutant mice were similar to some characteristic changes observed in mutant SOD1 mice. Unexpectedly, we identified the presence of different species of disulfide-dependent TDP-43 aggregates in cortex and spinal cord tissue. Overall, this study indicates that TDP-43A315T transgenic mice develop key features resembling key aspects of ALS, highlighting its relevance to study disease pathogenesis.

A diet enriched with plant sterols prevents the memory impairment induced by cholesterol loss in senescence-accelerated mice.

Cholesterol reduction at the neuronal plasma membrane has been related to age-dependent cognitive decline. We have used senescent-accelerated mice strain 8 (SAMP8), an animal model for aging, to examine the association between cholesterol loss and cognitive impairment and to test strategies to revert this process. We show that the hippocampus of SAMP8 mice presents reduced cholesterol levels and enhanced amount of its degrading enzyme Cyp46A1 (Cyp46) already at 6 months of age. Cholesterol loss accounts for the impaired long-term potentiation in these mice. Plant sterol (PSE)-enriched diet prevents long-term potentiation impairment and cognitive deficits in SAMP8 mice without altering cholesterol levels. PSE diet also reduces the abnormally high amyloid peptide levels in SAMP8 mice brains and restores membrane compartmentalization of presenilin1, the catalytic component of the amyloidogenic γ-secretase. These results highlight the influence of cholesterol loss in age-related cognitive decline and provide with a noninvasive strategy to counteract it. Our results suggest that PSE overtake cholesterol functions in the brain contributing to reduce deleterious consequences of cholesterol loss during aging.

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.

The intersection between growth factors, autophagy and ER stress: A new target to treat neurodegenerative diseases?

One of the salient features of most neurodegenerative diseases is the aggregation of specific proteins in the brain. This proteostasis imbalance is proposed as a key event triggering the neurodegenerative cascade. The unfolded protein response (UPR) and autophagy pathways are emerging as critical processes implicated in handling disease-related misfolded proteins. However, in some conditions, perturbations in the buffering capacity of the proteostasis network may be part of the etiology of the disease. Thus, pharmacological or gene therapy strategies to enhance autophagy or UPR responses are becoming an attractive target for disease intervention. Here, we discuss current evidence depicting the complex involvement of autophagy and ER stress in brain diseases. Novel pathways to modulate protein misfolding are discussed including the relation between aging and growth factor signaling. This article is part of a Special Issue entitled SI:Autophagy.

Myeloid derived suppressor cells in inflammatory conditions of the central nervous system.

The knowledge of the immune system elements and their relationship with other tissues, organs and systems are key approximations for the resolution of many immune-related disorders. The control of the immune response and/or its modulation from the pro-inflammatory to the anti-inflammatory response is being deeply studied in the field. In the last years, the study of myeloid-derived suppressor cells (MDSCs), a group of immature myeloid cells with a high suppressive activity on T cells has been extensively addressed in cancer. In contrast, their role in neuroimmune diseases is far from being totally understood. In this review, we will summarize data about MDSCs coming from the study of neuroinflammatory diseases in general and their potential role in multiple sclerosis, in order to introduce the putative use of this extraordinary promising cell type for future cell-based therapies. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

The synthetic retinoid Am80 delays recovery in a model of multiple sclerosis by modulating myeloid-derived suppressor cell fate and viability.

Relapsing-remitting multiple sclerosis (RR-MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). It is characterized by relapsing phases with ongoing neurological affectation that are followed by a remitting period in which inflammatory events are controlled and the patients partially recover. Experimental Autoimmune Encephalomyelitis (EAE) is the animal model most often used to study the inflammatory component of MS. Several cell types are involved in controlling the immune response in EAE and immature myeloid-derived suppressor cells (MDSCs) have emerged as important actors in the immunomodulation that occurs in EAE due to their ability to suppress inflammatory responses by inducing T cell apoptosis. In this study, we assessed whether MDSC differentiation may have consequences on the clinical course of EAE by treating mice around the peak of the clinical course EAE with the MDSC-differentiating agent Am80, an analogue of retinoid acid. Am80 administration abrogates the immunomodulation that occurs in EAE mice through different MDSC-related mechanisms: i) induction of MDSC apoptosis; ii) polarization of MDSCs to mature subsets of myeloid cells (dendritic cells/macrophages/neutrophils); and iii) altering their immunosuppressor phenotype. Consequently, T cell density increases and their viability is promoted, delaying the animal's recovery. Therefore, our data point to MDSC behaviour as a crucial factor in facilitating the transition from the relapsing to the remission phase in EAE, which should be considered for future immune-related therapies for MS.

The effect of glia-glia interactions on oligodendrocyte precursor cell biology during development and in demyelinating diseases.

Oligodendrocyte precursor cells (OPCs) originate in specific areas of the developing central nervous system (CNS). Once generated, they migrate towards their destinations where they differentiate into mature oligodendrocytes. In the adult, 5-8% of all cells in the CNS are OPCs, cells that retain the capacity to proliferate, migrate, and differentiate into oligodendrocytes. Indeed, these endogenous OPCs react to damage in demyelinating diseases, like multiple sclerosis (MS), representing a key element in spontaneous remyelination. In the present work, we review the specific interactions between OPCs and other glial cells (astrocytes, microglia) during CNS development and in the pathological scenario of MS. We focus on: (i) the role of astrocytes in maintaining the homeostasis and spatial distribution of different secreted cues that determine OPC proliferation, migration, and differentiation during CNS development; (ii) the role of microglia and astrocytes in the redistribution of iron, which is crucial for myelin synthesis during CNS development and for myelin repair in MS; (iii) how microglia secrete different molecules, e.g., growth factors, that favor the recruitment of OPCs in acute phases of MS lesions; and (iv) how astrocytes modify the extracellular matrix in MS lesions, affecting the ability of OPCs to attempt spontaneous remyelination. Together, these issues demonstrate how both astroglia and microglia influence OPCs in physiological and pathological situations, reinforcing the concept that both development and neural repair are complex and global phenomena. Understanding the molecular and cellular mechanisms that control OPC survival, proliferation, migration, and differentiation during development, as well as in the mature CNS, may open new opportunities in the search for reparative therapies in demyelinating diseases like MS.