Glial cells are affected more than interneurons by the loss of Engrailed 2 gene in the mouse cerebellum.

Glial cells play a pivotal role in the inflammatory processes, which are common features of several neurodevelopmental and neurodegenerative disorders. Their major role in modulating neuroinflammation underscores their significance in these conditions. Engrailed-2 knockout mice (En2-/- ) are considered a valuable model for autism spectrum disorder (ASD) due to their distinctive neuroanatomical and behavioral traits. Given the higher prevalence of ASD in males, our objective was to investigate glial and interneuron alterations in the cerebellum of En2-/- mice compared with wild-type (WT) mice in both sexes. We employed immunohistochemical analysis to assess cell density for all cell types studied and analyzed the area (A) and shape factor (SF) of microglia cell bodies. Our findings revealed the following: (a) In WT mice, the density of microglia and astrocytes was higher in females than in males, while interneuron density was lower in females. Notably, in En2-mutant mice, these differences between males and females were not present. (b) In both male and female En2-/- mice, astrocyte density exceeded that in WT mice, with microglia density being greater only in females. (c) In WT females, microglia cell bodies exhibited a larger area and a lower shape factor compared to WT males. Remarkably, the En2 mutation did not appear to influence these sex-related differences. (d) In both male and female En2-/- mice, we observed a consistent pattern: microglia cell bodies displayed a larger area and a smaller shape factor. Given the ongoing debate surrounding the roles of glia and sex-related factors in ASD, our observations provide valuable insights into understanding how an ASD-associated gene En2 affects specific cell types in the cerebellum.

New antiproliferative agents derived from tricyclic 3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazine scaffold: Synthesis and pharmacological effects.

A series of novel 3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazine (BIT) derivatives were designed and synthesized. In vitro antiproliferative activity was detected toward two human colorectal adenocarcinoma cell lines (CaCo-2 and HT-29) and one human dermal microvascular endothelial cell line (HMVEC-d). The most active compounds, namely 2-4 and 8, were further investigated to clarify the mechanism behind their biological activity. Through immunofluorescence assay, we identified the target of these molecules to be the microtubule cytoskeleton with subsequent formation of dense microtubule accumulation, particularly at the periphery of the cancer cells, as observed in paclitaxel-treated cells. Overall, these results highlight BIT derivatives as robust and feasible candidates deserving to be further developed in the search for novel potent antiproliferative microtubule-targeting agents.

Morphological alterations of the reticular thalamic nucleus in Engrailed-2 knockout mice.

The reticular thalamic nucleus (Rt) is a sheet of neurons that surrounds the dorsal thalamus laterally, along its dorso-ventral and rostro-caudal axes. It consists of inhibitory neurons releasing gamma-aminobutyric acid (GABA). This nucleus participates in the circuitry between the thalamus and the cerebral cortex, and its impairment is associated with neuro-psychiatric disorders. In this study, we investigated the Rt anatomy of Engrailed-2 knockout mice (En2-/- ), a mouse model of autism spectrum disorder (ASD), using parvalbumin as an immunohistochemical marker. We compared 4- and 6-week-old wild type (WT) and En2-/- mice using various morphometric parameters: cell area, shape factor, circularity and cell density. Significant differences were present in 6-week-old male mice with different genetic background (WT vs. En2-/- ): the Rt neurons of En2-/- mice showed a bigger cell area, shape factor and circularity when compared with WT. Age (4 weeks vs. 6 weeks) influenced the shape factor of WT females, the circularity and cell density of En2-/- males, and the shape factor and circularity of En2-/- females. Gender affected cell density in 4-week-old WT mice, shape factor and cellularity of 6-week-old WT mice, and cell area, shape factor and cell density of En2-/- at 6 weeks. Intrasubject (left-right) asymmetry of Rt was never observed. These results show for the first time that sex- and age-related changes occur in the Rt GABAergic neurons of the En2-/- ASD mouse model.

Dopamine-mediated autocrine inhibition of insulin secretion.

The aim of the present research was to explore the mechanisms underlying the role of dopamine in the regulation of insulin secretion in beta cells. The effect of dopamine on insulin secretion was investigated on INS 832/13 cell line upon glucose and other secretagogues stimulation. Results show that dopamine significantly inhibits insulin secretion stimulated by both glucose and other secretagogues, while it has no effect on the basal secretion. This effect requires the presence of dopamine during incubation with the various secretagogues. Both electron microscopy and immunohistochemistry indicate that in beta cells the D2 dopamine receptor is localized within the insulin granules. Blocking dopamine entry into the insulin granules by inhibiting the VMAT2 transporter with tetrabenazine causes a significant increase in ROS production. Our results confirm that dopamine plays an important role in the regulation of insulin secretion by pancreatic beta cells through a regulated and precise compartmentalization mechanisms.

In-vitro Approaches to Investigate the Detrimental Effect of Light on Dopaminergic Neurons.

Our recent study revealed that fluorescent lamp light can penetrate deep into the brain of mice and rats leading to the development of typical histological characteristics associated with Parkinson's disease such as the loss of dopamine neurons in the substantia nigra. Monochromatic LED lights were thus used in this work to deepen our knowledge on the effects of the major wavelength peaks of fluorescent light on mouse and human dopaminergic cells. In particular, we exposed immortalized dopaminergic MN9D neuronal cells, primary cultures of mouse mesencephalic dopaminergic cells and human dopaminergic neurons differentiated from induced pluripotent stem cells (hiPSC) to different LED light wavelengths. We found that chronic exposure to LED light reduced overall undifferentiated MN9D cell number, with the most significant effects observed at wavelengths of 485 nm and 610 nm. Moreover, LED light especially at 610 nm was able to negatively impact on the survival of mouse mesencephalic dopaminergic cells and of human dopaminergic neurons derived from hiPSC. Notably, differentiated MN9D dopaminergic cells, which closely resemble mature dopamine neuronal phenotype, acutely exposed for 3 h at 610 nm, showed a clear increase in ROS production and cytotoxicity compared to controls undifferentiated MN9D cells. These increases were even more pronounced by the co-treatment with the oxidative agent H2O2. Collectively, these findings suggest that specific wavelengths, particularly those capable of penetrating deep into the brain, could potentially pose an environmental hazard in relation to Parkinson's disease.

Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib.

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Pharmacological Strategies for Bipolar Disorders in Acute Phases and Chronic Management with a Special Focus on Lithium, Valproic Acid, and Atypical Antipsychotics.

Bipolar disorders (BDs) are a heterogeneous group of severe affective disorders generally described by the alternation of (hypo)manic, depressive, and mixed phases, with euthymic intervals of variable duration. BDs are burdened with high psychiatric and physical comorbidity, increased suicide risk and reduced life expectancy. In addition, BDs can progress into complicated forms (e.g., mixed states, rapid/irregular cycling), which are more difficult to treat and often require personalized pharmacological combinations. Mood stabilizers, particularly Lithium and Valproic acid (VPA), still represent the cornerstones of both acute and chronic pharmacotherapies of BDs. Lithium is the gold standard in BD-I and BDII with typical features, while VPA seems more effective for atypical forms (e.g., mixed-prevalence and rapid-cycling). However, despite appropriate mood stabilization, many patients show residual symptoms, and more than a half recur within 1-2 years, highlighting the need of additional strategies. Among these, the association of atypical antipsychotics (AAPs) with mood stabilizers is recurrent in the treatment of acute phases, but it is also being growingly explored in the maintenance pharmacotherapy. These combinations are clinically more aggressive and often needed in the acute phases, whereas simplifying pharmacotherapies to mood stabilizers only is preferable in the long-term, whenever possible. When mood stabilizers are not enough for maintenance treatment, Quetiapine and, less consistently, Aripiprazole have been proposed as the most advisable adjunctive strategies, for their safety and tolerability profiles. However, in view of the increased risk of serious adverse effects, a careful patient-centered balance between costs and benefits is mandatory.

ß-Cells Different Vulnerability to the Parkinsonian Neurotoxins Rotenone, 1-Methyl-4-phenylpyridinium (MPP+) and 6-Hydroxydopamine (6-OHDA).

Neurotoxins such as rotenone, 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) are well known for their high toxicity on dopaminergic neurons and are associated with Parkinson's disease (PD) in murine models and humans. In addition, PD patients often have glucose intolerance and may develop type 2 diabetes (T2D), whereas T2D patients have higher risk of PD compared to general population. Based on these premises, we evaluated the toxicity of these three toxins on pancreatic ß-cell lines (INS-1 832/13 and MIN6) and we showed that rotenone is the most potent for reducing ß-cells viability and altering mitochondrial structure and bioenergetics in the low nanomolar range, similar to that found in dopaminergic cell lines. MPP+ and 6-OHDA show similar effects but at higher concentration. Importantly, rotenone-induced toxicity was counteracted by α-tocopherol and partially by metformin, which are endowed with strong antioxidative and cytoprotective properties. These data show similarities between dopaminergic neurons and ß-cells in terms of vulnerability to toxins and pharmacological agents capable to protect both cell types.

Rho-inhibition and neuroprotective effect on rotenone-treated dopaminergic neurons in vitro.

Mesencephalic cell cultures are a good model to study the vulnerability of dopaminergic neurons and reproduce, in vitro, experimental models of Parkinson's disease. Rotenone associated as an environmental neurotoxin related to PD, is able to provoke dopaminergic neuron degeneration by inhibiting complex I of the mitochondrial respiratory chain and by inducing accumulation of α-synuclein. Recently, rotenone has been described to activate RhoA, a GTPase protein. In the present study we evaluated a possible neuroprotective effect of Rho-inhibitor molecules on rotenone-damaged dopaminergic (DA) neurons obtained from mouse primary mesencephalic cell culture. Our results showed that Clostridium Botulinum C3 toxin (C3) and simvastatin, as RhoA inhibitors, were able to protect DA neurons from rotenone damages. In fact, pretreatment with C3 or simvastatin significantly prevented the reduction of [3H]dopamine uptake, neurites injury and the expression patterns of proteins like α-syn, actin and connexin 43.

Parkinson's disease and light: The bright and the Dark sides.

Light exerts a major influence on human behaviour and health, mainly owing to the importance of sight in our lives, but also due to its entrainment of daily rhythms via the suprachiasmatic nucleus, the master pacemaker. Light may also be a useful clinical medium, as in lumino-therapy for the improvement of depressed mood. Further, as discussed herein, local application of near infrared light to the substantia nigra exerts neuroprotective properties in models of Parkinson's disease. However, light also has a darker side. In general, as regards the growing problem to human health - and the natural world - of excess exposure to artificial light: both urban glow and ubiquitous screens. Moreover, over-exposure to light, in particular fluorescent light, disrupts circadian rhythms and sleep, and may damage dopaminergic neurons. Is it, then, a neglected risk factor for Parkinson's disease? The present article discusses epidemiological and experimental evidence supporting beneficial and potentially deleterious impact of light on dopaminergic neurons and highlights the mechanisms whereby light might influence neuronal tissue.

Apomorphine offers new insight into dopaminergic neuron vulnerability in mesencephalic cultures.

The mechanism by which the dopamine neurons of the substantia nigra pars compacta degenerate in Parkinson's disease, is partly unknown. Dopamine could be implicated in this phenomenon, and in order to explain its toxicity several hypotheses have been suggested. The similarity between apomorphine and dopamine as regards their chemical, pharmacological and toxicological properties provided a basis for investigating the nature of the toxicity of the former agent. In this study we describe some effects of apomorphine on mouse mesencephalic cell cultures at relatively low concentrations (from 0.5 to 2.5microM), apomorphine produced a neurotrophic effect, consisting of a 60% increase in dopaminergic neuron survival as measured by [(3)H] dopamine uptake. At high concentrations (over 20microM), however, apomorphine induced an increasing cytotoxic effect, as measured by the marked decrease in [(3)H] dopamine uptake, and by the direct observation of the dopaminergic neurons after TH immunostaining. This study may offer a new strategy for investigating the mechanisms underlying DA neuron vulnerability.

Role of CYP2E1 in the mouse model of MPTP toxicity.

It has been shown that diethyldithiocarbamate (DDC) potentiates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice as a result of increased levels of 1-methyl-4-phenylpyridinium ion (MPP(+)) in the striatum. Brain CYP2E1 inhibition by DDC in C57Bl mice was responsible for increased toxicity and striatal MPP(+) accumulation. However, CYP2E1-null mice did not show any enhanced sensitivity to MPTP or any MPP(+) accumulation. This unexpected finding suggested that the CYP2E1-null mice compensate with other isozymes as already described for acetaminophen-induced liver damage. MPP(+) intoxication of mesencephalic cell cultures from CYP2E1-null mice indicated a reduced sensitivity of dopaminergic (DA) neurons from knockout animals. Surprisingly, MPP(+) cell distribution under these conditions indicated that the toxin accumulates more intracellularly in knockout cultures, suggesting further that CYP2E1 has a role in MPP(+) storage and efflux.

Melanocortin Receptor-4 and Glioblastoma Cells: Effects of the Selective Antagonist ML00253764 Alone and in Combination with Temozolomide In Vitro and In Vivo.

Currently, no description of melanocortin receptor-4 (MC4R) expression or activity is available in human cancer cells, including glioblastoma (GBM). The aim of this study is to evaluate the presence of MC4Rs in GBM cells and the selective inhibition of their activity through the MC4R antagonist ML00253764 alone and in association with temozolomide in vitro and in vivo. MC4R genotyping and gene expression were performed on human GBM cells (U-87 and U-118) with real-time PCR. MC4R western blotting, immunohistochemistry, and immunofluorescence were obtained in both cell lines and in human tissues. Proliferation, cell cycle, and apoptotic assays were performed with ML00253764, whereas the synergism of the simultaneous combination with temozolomide was evaluated by the combination index method. ERK1/2 and Akt phosphorylation were quantified by ELISA. In vivo experiments were performed in U-87 xenografted nude mice. Both GBM cell lines and tumor tissues expressed MC4R receptors. The selective antagonist ML00253764 determined an antiproliferative and proapoptotic activity through the inhibition of the phosphorylation of ERK1/2 and Akt. Moreover, the simultaneous combination of temozolomide and ML00253764 determined a highly synergistic effect on GBM cells. The same combination in vivo showed a strong and significant decrease of GBM tumor volumes if compared to the single drug treatments, with an excellent tolerability profile. In conclusion, MC4R is present in GBM cells and its selective inhibition determined antiproliferative and proapoptotic effects, through the inhibition of ERK1/2 and Akt phosphorylation, and the synergistic enhancement of temozolomide effects in vitro and in vivo.

The role of mitochondria in stem cell biology.

This mini-review summarizes the current literature on the role of mitochondrial DNA mutations and mitochondrial metabolism in stem cell biology. The possible uses of stem cells as a therapeutic tool in mitochondrial disorders are also reported.

Recovery from experimental parkinsonism by intrastriatal application of erythropoietin or EPO-releasing neural precursors.

An extensive literature has shown a powerful neuroprotective action of Erythropoietin (EPO) both in vivo and in vitro. This study shows that EPO, whether ectopically administered or released by neural precursors, does reverse MPTP-induced parkinsonism in mice. Unilateral stereotaxic injection of 2.5 × 105 erythropoietin-releasing neural precursor cells (Er-NPCs) rescued degenerating striatal dopaminergic neurons and promoted behavioral recovery as shown by three independent behavioral tests. These effects were replicated through direct intrastriatal administration of recombinant human EPO. At the end of the observational period, most of the transplanted Er-NPCs were vital and migrated via the striatum to reach Substantia Nigra. The restorative effects appear to be mediated by EPO since co-injection of anti-EPO or anti-EPOR antibodies antagonized the positive outcomes. Furthermore, this report supports the neuroprotective action of EPO, which may also be achieved via administration of EPO-releasing cells such as Er-NPCs.

Fluorescent light induces neurodegeneration in the rodent nigrostriatal system but near infrared LED light does not.

We investigated the effects of continuous artificial light exposure on the mouse substantia nigra (SN). A three month exposure of C57Bl/6J mice to white fluorescent light induced a 30% reduction in dopamine (DA) neurons in SN compared to controls, accompanied by a decrease of DA and its metabolites in the striatum. After six months of exposure, neurodegeneration progressed slightly, but the level of DA returned to the basal level, while the metabolites increased with respect to the control. Three month exposure to near infrared LED light (∼710nm) did not alter DA neurons in SN, nor did it decrease DA and its metabolites in the striatum. Furthermore mesencephalic cell viability, as tested by [3H]DA uptake, did not change. Finally, we observed that 710nm LED light, locally conveyed in the rat SN, could modulate the firing activity of extracellular-recorded DA neurons. These data suggest that light can be detrimental or beneficial to DA neurons in SN, depending on the source and wavelength.

Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue.

Tissue clarification has been recently proposed to allow deep tissue imaging without light scattering. The clarification parameters are somewhat arbitrary and dependent on tissue type, source and dimension: every laboratory has its own protocol, but a quantitative approach to determine the optimum clearing time is still lacking. Since the use of transgenic mouse lines that express fluorescent proteins to visualize specific cell populations is widespread, a quantitative approach to determine the optimum clearing time for genetically labeled neurons from thick murine brain slices using CLARITY2 is described. In particular, as the main objective of the delipidation treatment is to clarify tissues, while limiting loss of fluorescent signal, the "goodness" of clarification was evaluated by considering the bulk tissue clarification index (BTCi) and the fraction of the fluorescent marker retained in the slice as easily quantifiable macroscale parameters. Here we describe the approach, illustrating an example of how it can be used to determine the optimum clearing time for 1 mm-thick cerebellar slice from transgenic L7GFP mice, in which Purkinje neurons express the GFP (green fluorescent protein) tag. To validate the method, we evaluated confocal stacks of our samples using standard image processing indices (i.e., the mean pixel intensity of neurons and the contrast-to-noise ratio) as figures of merit for image quality. The results show that detergent-based delipidation for more than 5 days does not increase tissue clarity but the fraction of GFP in the tissue continues to diminish. The optimum clearing time for 1 mm-thick slices was thus identified as 5 days, which is the best compromise between the increase in light penetration depth due to removal of lipids and a decrease in fluorescent signal as a consequence of protein loss: further clearing does not improve tissue transparency, but only leads to more protein removal or degradation. The rigorous quantitative approach described can be generalized to any clarification method to identify the moment when the clearing process should be terminated to avoid useless protein loss.

Grafted Neural Precursors Integrate Into Mouse Striatum, Differentiate and Promote Recovery of Function Through Release of Erythropoietin in MPTP-Treated Mice.

Erythropoietin-releasing neural precursor cells (Er-NPCs) are a subclass of subventricular zone-derived neural progenitors, capable of surviving for 6 hr after death of donor. They present higher neural differentiation. Here, Er-NPCs were studied in animal model of Parkinson's disease. Dopaminergic degeneration was caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperitoneal administration in C57BL/6 mice. The loss of function was evaluated by specific behavioral tests. Er-NPCs (2.5 × 105) expressing the green fluorescent protein were administered by stereotaxic injection unilaterally in the left striatum. At the end of observational research period (2 weeks), most of the transplanted Er-NPCs were located in the striatum, while several had migrated ventrally and caudally from the injection site, up to ipsilateral and contralateral substantia nigra. Most of transplanted cells had differentiated into dopaminergic, cholinergic, or GABAergic neurons. Er-NPCs administration also promoted a rapid functional improvement that was already evident at the third day after cells administration. This was accompanied by enhanced survival of nigral neurons. These effects were likely promoted by Er-NPCs-released erythropoietin (EPO), since the injection of Er-NPCs in association with anti-EPO or anti-EPOR antibodies had completely neutralized the recovery of function. In addition, intrastriatal administration of recombinant EPO mimics the effects of Er-NPCs. We suggest that Er-NPCs, and cells with similar properties, may represent good candidates for cellular therapy in neurodegenerative disorders of this kind.

Serotonin abnormalities in Engrailed-2 knockout mice: New insight relevant for a model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a congenital neurodevelopmental behavioral disorder that appears in early childhood. Recent human genetic studies identified the homeobox transcription factor, Engrailed 2 (EN2), as a possible ASD susceptibility gene. En2 knockout mice (En2-/-) display subtle cerebellar neuropathological changes and reduced levels of tyrosine hydroxylase, noradrenaline and serotonin in the hippocampus and cerebral cortex similar to those ones which have been observed in the ASD brain. Furthermore other similarities link En2 knockout mice to ASD patients. Several lines of evidence suggest that serotonin may play an important role in the pathophysiology of the disease. In the present study we measured, by using an HPLC, the 5-HT levels in different brain areas and at different ages in En2-/- mice. In the frontal and occipital cortex, the content of 5HT was reduced in En2-/- 1 and 3 months old mice; in 6 month old mice, the difference was still present, but it was not statistically significant. The 5-HT content of cerebellar cortex was significantly reduced at 1 month old but significantly high when the KO mice reached 3 months of age. The increase was present even at 6 months of age. A similar trend was highlighted by SERT immunolabeling in En2-/- mice compared to control in the same areas and age analyzed. Our findings, in agreement with the current knowledge on the 5-HT system alterations in ASD, confirm the early neurotransmitter deficit with a late compensatory recovery in En2 KO-mice further suggesting that this experimental animal may be considered a good predictive model for the human disease.

Effect of itraconazole on mouse mesencephalic neurons.

Neuronal cells have complex geometrical shapes, long processes such as axons and dendrites, and as a response to specific stimuli, they go through polarized neuronal migration that influences connectivity and information processing. Recently, it has been discovered that itraconazole, a widely used systemic antifungal drug, has an effect on cell morphology, acting as an inhibitor of the morphogen Sonic Hedgehog (Shh) and of the mammalian target of rapamycin mTOR pathways. In this paper we evaluated the effect of itraconazole on mouse mesencephalic dopaminergic neurons following their neurite outgrowth and functional activity by [(3)H] DA uptake. Furthermore the expression of several neural markers, the activation of the mTOR and of the morphogenic Shh pathways in the neuronal population was examined. Our results show for the first time a strong alteration of neurons morphology and an inhibitory effect of differentiation by itraconazole, probably due to cholesterol trafficking reduction, mTOR and Shh pathways inhibition. The inhibition of mTOR and Shh pathways by this drug has also been found in other cellular systems such as endothelial cells and lung cancer cells, suggesting a conserved mechanism of intercellular communication. As itraconazole is currently involved in multiple human clinical trials as a prospective anticancer agent, the effect on neuronal differentiation should be taken into account.