SKELETAL MUSCLE REGENERATION IN THE ADULT MOUSE AND RAT: STUDY ON CONNEXIN EXPRESSION AND ROLE IN NORMAL AND REGENERATING SKELETAL MUSCLE AND ON LOW

The first aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) Cx37, Cx39, Cx40, Cx43, and Cx45 expression in developing skeletal muscle and in the adult regenerating skeletal muscle in response to crush injury. Among the Cxs examined, only the Cx39, Cx43 and Cx45 were found expressed during embryonic life and progressively reduced during early postnatal life to become dramatically expressed at very low levels like Cx43 and Cx45, or to be undetectable like Cx39 in the adult muscle. Cx37 and Cx40 were found expressed at low levels and were localized in the endothelial cells. In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers, or to repair the damaged one. Within 24h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesionforming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofibers. The second aim of this study was to examine how low-intensity endurance exercise affects the regeneration process in dystrophin-deficient skeletal muscle. The lack of dystrophin in mdx mice, an animal model for Duchenne muscular dystrophy, leads to cycles of muscle degeneration and regeneration processes. Male adult mdx and wild type mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 v Dott.ssa M. Frinchi days/week, for 4 weeks at progressively increasing loads. Exercised mdx mice showed a trend to lower body weight gain and positive effect on the degree of fatigue. Histomorphological analysis showed a significant reduction of both muscle necrosis foci and regeneration processes in the gastrocnemius and quadriceps muscles of exercised mdx mice. The reduction of regeneration process was also evaluated by examining the protein expression of Cx39, as a specific gene expressed during regeneration process of injured muscles. While Cx39 was not expressed both in wild type exercised nor in sedentary mice, it was markedly increased in sedentary mdx mice, because of active degeneration/regenerating process, and dropped to very low levels in exercised mdx mice, suggesting a reduction of muscle regeneration process. This study has shown that specific low intensity endurance exercise induces a strong beneficial effect on the regeneration of dystrophic muscle and may have therapeutic value at least to decrease progression of muscular dystrophy and in less extend for strengthening dystrophic muscle

Manipulation of HSP70-SOD1 Expression Modulates SH-SY5Y Differentiation and Susceptibility to Oxidative Stress-Dependent Cell Damage: Involvement in Oxotremorine-M-Mediated Neuroprotective Effects

: The differentiation of neural progenitors is a complex process that integrates different signals to drive transcriptional changes, which mediate metabolic, electrophysiological, and morphological cellular specializations. Understanding these adjustments is essential within the framework of stem cell and cancer research and therapy. Human neuroblastoma SH-SY5Y cells, widely used in neurobiology research, can be differentiated into neuronal-like cells through serum deprivation and retinoic acid (RA) supplementation. In our study, we observed that the differentiation process triggers the expression of Heat Shock Protein 70 (HSP70). Notably, inhibition of HSP70 expression by KNK437 causes a dramatic increase in cell death. While undifferentiated SH-SY5Y cells show a dose-dependent decrease in cell survival following exposure to hydrogen peroxide (H2O2), differentiated cells become resistant to H2O2-induced cell death. Interestingly, the differentiation process enhances the expression of SOD1 protein, and inhibition of HSP70 expression counteracts this effect and increases the susceptibility of differentiated cells to H2O2-induced cell death, suggesting that the cascade HSP70-SOD1 is involved in promoting survival against oxidative stress-dependent damage. Treatment of differentiated SH-SY5Y cells with Oxotremorine-M (Oxo), a muscarinic acetylcholine receptor agonist, enhances the expression of HSP70 and SOD1 and counteracts tert-Butyl hydroperoxide-induced cell death and reactive oxygen species (ROS) generation. It is worth noting that co-treatment with KNK437 reduces SOD1 expression and Oxo-induced protection against oxidative stress damage, suggesting the involvement of HSP70/SOD1 signaling in this beneficial effect. In conclusion, our findings demonstrate that manipulation of the HSP70 signal modulates SH-SY5Y differentiation and susceptibility to oxidative stress-dependent cell death and unravels novel mechanisms involved in Oxo neuroprotective functions. Altogether these data provide novel insights into the mechanisms underlying neuronal differentiation and preservation under stress conditions

Connexin36 (Cx36) expression and protein detection in the mouse carotid body and myenteric plexus

Although connexin36 (Cx36) has been studied in several tissues, it is notable that no data are available on Cx36 expression in the carotid body and the intestine. The present study was undertaken to evaluate using immunohistochemistry, PCR and Western blotting procedures, whether Cx36 was expressed in the mouse carotid body and in the intestine at ileum and colon level. In the carotid body, Cx36 was detected as diffuse punctate immunostaining and as protein by Western blotting and mRNA by RT-PCR. Cx36 punctate immunostaining was also evident in the intestine with localization restricted to the myenteric plexus of both the ileum and the colon, and this detection was also confirmed by Western blotting and RT-PCR. All the data obtained were validated using Cx36 knockout mice. Taken together the present data on localization of Cx36 gap-junctions in two tissues of neural crest-derived neuroendocrine organs may provide an anatomical basis for future functional investigations

Bidirectional Control between Cholesterol Shuttle and Purine Signal at the Central Nervous System

: Recent studies have highlighted the mechanisms controlling the formation of cerebral cholesterol, which is synthesized in situ primarily by astrocytes, where it is loaded onto apolipoproteins and delivered to neurons and oligodendrocytes through interactions with specific lipoprotein receptors. The "cholesterol shuttle" is influenced by numerous proteins or carbohydrates, which mainly modulate the lipoprotein receptor activity, function and signaling. These molecules, provided with enzymatic/proteolytic activity leading to the formation of peptide fragments of different sizes and specific sequences, could be also responsible for machinery malfunctions, which are associated with neurological, neurodegenerative and neurodevelopmental disorders. In this context, we have pointed out that purines, ancestral molecules acting as signal molecules and neuromodulators at the central nervous system, can influence the homeostatic machinery of the cerebral cholesterol turnover and vice versa. Evidence gathered so far indicates that purine receptors, mainly the subtypes P2Y2, P2X7 and A2A, are involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's and Niemann-Pick C diseases, by controlling the brain cholesterol homeostasis; in addition, alterations in cholesterol turnover can hinder the purine receptor function. Although the precise mechanisms of these interactions are currently poorly understood, the results here collected on cholesterol-purine reciprocal control could hopefully promote further research

Reduction of mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised versus sedentary mdx mice

In our recent study was shown a significant recovery of damaged skeletal muscle of mice with x-linked muscular dystrophy (mdx) following low-intensity endurance exercise, probably by reducing the degeneration of dystrophic muscle. Consequently, in the present work we aimed to identify proteins involved in the observed reduction of degenerating fibers. To this end, we used proteomic analysis to evaluate changes in the protein profile of quadriceps dystrophic muscles of exercised versus sedentary mdx mice. Four protein spots were found to be significantly changed and were identified as three isoforms of Carbonic anhydrase 3 (CA3) and superoxide dismutase [Cu-Zn] (SODC). Protein levels of CA3 isoforms were significantly up-regulated in quadriceps of sedentary mdx mice and were completely restored to wild type mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Protein levels of SODC were down-regulated in quadriceps of sedentary mdx mice and were significantly restored to wild type mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Western blot data were in agreement with those obtained using proteomic analysis and revealed the presence of one more CA3 isoform that was significantly changed. Based on data found in the present study, it seems that low-intensity endurance exercise may in part contribute to reduce cell degeneration process in mdx muscles, by counteracting oxidative stress

Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated

Small airways in sedentary and endurance-trained dystrophic (mdx) mice.

The effects of mild endurance exercise training on the small airways in mdx mice are unknown. We compared epithelial thickness and turnover, apoptosis, and stress marker expression in small airways of mdx mice and wild-type (WT) controls, at rest and during exercise training. Mdx and WT mice were randomly assigned to sedentary (mdx-S, n=17; WT-S, n=19) or trained (mdx- EX, n=14; WT-EX, n=16) groups. Low-intensity endurance training (running on a wheel) was done 5 d/wk for 6 wk at progressively increasing speed (rpm from 16 to 24) and time (15 min to 1 h). Lungs were processed for light microscopy and periodic acid Schiff (PAS) staining. Hsp60 and PCNA were quantified by immunohistochemistry. Apoptosis was assessed by TUNEL. Bronchial epithelial thickness decreased over time in WT mice irrespective of training (linear regression for time trends: WT-S: R2=0.43, r= -0.65; WT-EX: R2=0.68, r= -0.82, p<0.0005 for both); conversely, no significant change occurred in mdx mice. The number of PAS+ goblet cells was much lower in the bronchiolar epithelium of mdx compared to WT mice in all conditions. At 30 days, PCNA positivity was higher in EX than S animals in both groups; however, at 45 days it sharply decreased in mdx-S and -EX, but not in WT mice. The percentage of TUNEL+ cells was higher in mdx-EX than WT-EX mice at 45 days. In mdx mice, expression of Hsp60 progressively decreased (p<0.01), and was inversely related to the percentage of TUNEL+ cells (R2=0.44, r=-0.66, p=0.01). In conclusion, bronchiolar epithelium in mdx mice is poor of goblet cells, and progressively deteriorates over time possibly because of loss of stress-related protective mechanism. Mild training did not cause any additional damage

Uncovering the signaling pathway behind extracellular guanine-induced activation of NO System: New perspectives in memory-related disorders

Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Guanosine (GUO) and guanine (GUA) are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP) and guanine deaminase (GDA). Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment). This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i) in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii) the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders

No effects of low-intensity endurance exercise on muscle necrosis in the diaphragm of mdx mice

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle weakness. We have previously shown that low-intensity endurance training prevented muscle damage (Frinchi et al, Int J Sports Med 2014). Since the effects of low-intensity endurance training on the the diaphragm in the mdx mouse model are unknown, in the same animals we investigated Cx39 protein levels (Western blotting) in homogenates of the diaphragm before and after training. Mdx and wild-type (WT) mice were randomly assigned to sedentary (mdx-S, n=17; WT-S, n=19) or trained (mdx-EX, n=14; WT-EX, n=16) groups. Low-intensity endurance training (running on a wheel) was done 5 days/week for 6 weeks at progressively increasing time (15 min to 1 h) and speed (rpm from 16 to 24, distance covered during training sessions from 48 to 288 m). Compared to our previous analysis of skeletal muscles changes in gastrocnemius and quadriceps, showing decreased muscle damage in trained vs sedentary mdx mice, analysis of protein level of Cx39 showed similar values in diaphragm homogenates from sedentary and trained mdx mice. These preliminary data suggest that prevention of muscle necrosis after mild training does not occur in the diaphragm. As a speculation, continuous work of diaphragm vs intermittent work of skeletal muscle might at least partly account for the different results obtained in respiratory and locomotor muscle

The Guanine-Based Purinergic System: The Tale of An Orphan Neuromodulation

Guanine-based purines (GBPs) have been recently proposed to be not only metabolic agents but also extracellular signaling molecules that regulate important functions in the central nervous system. In such way, GBPs-mediated neuroprotection, behavioral responses and neuronal plasticity have been broadly described in the literature. However, while a number of these functions (i.e., GBPs neurothophic effects) have been well-established, the molecular mechanisms behind these GBPs-dependent effects are still unknown. Furthermore, no plasma membrane receptors for GBPs have been described so far, thus GBPs are still considered orphan neuromodulators. Interestingly, an intricate and controversial functional interplay between GBPs effects and adenosine receptors activity has been recently described, thus triggering the hypothesis that GBPs mechanism of action might somehow involve adenosine receptors. Here, we review recent data describing the GBPs role in the brain. We focus on the involvement of GBPs regulating neuronal plasticity, and on the new hypothesis based on putative GBPs receptors. Overall, we expect to shed some light on the GBPs world since although these molecules might represent excellent candidates for certain neurological diseases management, the lack of putative GBPs receptors precludes any high throughput screening intent for the search of effective GBPs-based drugs