Gianluca Fulgenzi

GABAergic miniature spontaneous activity is increased in the CA1 hippocampal region of dystrophic mdx mice

Duchenne muscular dystrophy (DMD), a genetic disease due to dystrophin gene mutation and characterised by skeletal muscle failure, is associated with non-progressive cognitive deficits. In human and mouse brain, full-length dystrophin is localised postsynaptically in neocortical, hippocampal and cerebellar neurons. Evidence obtained in the CNS of dystrophic mice (mdx) suggested alterations of the GABAergic system. However, a direct functional evaluation of GABAergic synaptic transmission in mdx mice has not been conducted in the hippocampus, which is involved in cognitive processes and is rich in full-length dystrophin. Here, we investigated evoked and miniature inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons of mdx mice with patch clamp recording techniques. Results showed an increased frequency of miniature spontaneous IPSCs in mdx mice compared with controls, whereas evoked IPSCs did not show significant variations. Paired-pulse facilitation (PPF) analysis showed lack of facilitation at short intervals in mdx mice compared with that in wild-type mice. Analysis of density of synapses that innervate CA1 pyramidal cell bodies did not indicate significant differences between mdx mice and controls. Therefore, we suggest that increased miniature spontaneous IPSC frequency is due to altered pre-synaptic release probability. The present findings are discussed in the light of the accrued evidence for alterations of inhibitory synaptic transmission in the brain of dystrophic mice.

The effect of chronic skeletal muscle stimulation on capillary growth in the rat: are sensory nerve fibres involved?

Indirect chronic electrical stimulation of skeletal muscle activates not only efferent but also afferent nerve fibres. To investigate effects specific to this on capillary growth, one of the earliest changes, cell proliferation and capillary ultrastructure were studied in ankle flexors of rats with and without deafferentation of the stimulated side. Two weeks after preganglionic section of dorsal roots L4‐L6, the peroneal nerve was stimulated (10 Hz, 8 h day−1) for 2 or 7 days. Proliferating nuclei labelled by bromodeoxyuridine or proliferating cell nuclear antigen staining were colocalized to alkaline phosphatase‐stained capillaries (Lc) or other interstitial nuclei (Li) in frozen sections of extensor digitorum longus. Capillary fine structure was examined in extensor hallucis proprius by transmission electron microscopy. The stimulation‐induced increase in capillary and interstitial proliferation (Lc 9.9 ± 1.9 %, Li 8.8 ± 2.1 % vs. Lc 2.6 ± 0.4 %, Li 1.9 ± 0.3 % in controls, P < 0.05) was depressed at 2 days by dorsal root section (Lc 4.8 ± 0.7 %, Li 3.2 ± 0.9 %, P < 0.05), an effect likely to be mainly on fibroblasts; no depression was seen at 7 days. Dorsal root section reduced stimulation‐induced capillary endothelial swelling at both time points. In contralateral muscles of intact rats, stimulation increased interstitial cell proliferation and capillary swelling, both effects being eliminated by dorsal root section. Capillary growth induced by stimulation (24 % increase in capillary : fibre ratio at 7 days) was unaffected by deafferentation. The reduction in capillary ultrastructural changes and interstitial proliferation in both stimulated and contralateral muscles implies that stimulation of afferent fibres leads directly to release of humoral factors and/or activation via dorsal roots of fibres that release humoral substances. Contralateral muscles are an inadequate control for the effects of chronic stimulation in the intact animal.

BDNF modulates heart contraction force and long-term homeostasis through truncated TrkB.T1 receptor activation

BDNF exerts inotropic function in the adult mammalian heart through TrkB.T1 receptor and loss of this ligand/receptor system in cardiomyocytes impairs calcium signaling and causes cardiomyopathy, suggesting an essential physiological role for this pathway in cardiac function.

Gamma irradiation can reduce muscle damage in mdx dystrophic mice.

Abstract We report the effects of a single gamma irradiation delivered to the soleus muscle of one limb of normal and mdx mice at the age of 16–20 days. At 45, 75 and 90 days of age transverse cryostat sections from the mid-belly of the muscles were used for microscopic examination. In normal mice the growth of fibres was appreciably reduced by irradiation without fibre loss. In the irradiated soleus of mdx mice the number of the regenerated centrally nucleated fibres was very small and the total number of fibres was remarkably reduced. The number of the peripherally nucleated fibres, presumably surviving since the birth of the animal, was almost consistently larger than in the contralateral non-irradiated limb. The cross-sectional area of the irradiated fibres was smaller. It is well known that proliferation and fusion of satellite cells are required both for regeneration after fibre damage and for the normal postnatal growth of muscle fibres: irradiation appears to reduce regeneration and growth. It is suggested that irradiation reduces damage by reducing fusion associated with growth. Our hypothesis indirectly indicates a significant link between dystrophin deficiency and fibre necrosis and accounts well for many features of mouse dystrophy under natural and experimental conditions.

Acetylcholine, GABA and neuronal networks: A working hypothesis for compensations in the dystrophic brain

Duchenne muscular dystrophy (DMD), a genetic disease arising from a mutation in the dystrophin gene, is characterized by muscle failure and is often associated with cognitive deficits. Studies of the dystrophic brain on the murine mdx model of DMD provide evidence of morphological and functional alterations in the central nervous system (CNS) possibly compatible with the cognitive impairment seen in DMD. However, while some of the alterations reported are a direct consequence of the absence of dystrophin, others seem to be associated only indirectly. In this review we reevaluate the literature in order to formulate a possible explanation for the cognitive impairments associated with DMD. We present a working hypothesis, demonstrated as an integrated neuronal network model, according to which within the cascade of events leading to cognitive impairments there are compensatory mechanisms aimed to maintain functional stability via perpetual adjustments of excitatory and inhibitory components. Such ongoing compensatory response creates continuous perturbations that disrupt neuronal functionality in terms of network efficiency. We have theorized that in this process acetylcholine and network oscillations play a central role. A better understating of these mechanisms could provide a useful diagnostic index of the diseases progression and, perhaps, the correct counterbalance of this process might help to prevent deterioration of the CNS in DMD. Furthermore, the involvement of compensatory mechanisms in the CNS could be extended beyond DMD and possibly help to clarify other physio-pathological processes of the CNS.

Dystrophin Is Required for the Normal Function of the Cardio-Protective KATP Channel in Cardiomyocytes

Duchenne and Becker muscular dystrophy patients often develop a cardiomyopathy for which the pathogenesis is still unknown. We have employed the murine animal model of Duchenne muscular dystrophy (mdx), which develops a cardiomyopathy that includes some characteristics of the human disease, to study the molecular basis of this pathology. Here we show that the mdx mouse heart has defects consistent with alteration in compounds that regulate energy homeostasis including a marked decrease in creatine-phosphate (PC). In addition, the mdx heart is more susceptible to anoxia than controls. Since the cardio-protective ATP sensitive potassium channel (KATP) complex and PC have been shown to interact we investigated whether deficits in PC levels correlate with other molecular events including KATP ion channel complex presence, its functionality and interaction with dystrophin. We found that this channel complex is present in the dystrophic cardiac cell membrane but its ability to sense a drop in the intracellular ATP concentration and consequently open is compromised by the absence of dystrophin. We further demonstrate that the creatine kinase muscle isoform (CKm) is displaced from the plasma membrane of the mdx cardiac cells. Considering that CKm is a determinant of KATP channel complex function we hypothesize that dystrophin acts as a scaffolding protein organizing the KATP channel complex and the enzymes necessary for its correct functioning. Therefore, the lack of proper functioning of the cardio-protective KATP system in the mdx cardiomyocytes may be part of the mechanism contributing to development of cardiac disease in dystrophic patients.

Determination of high-energy phosphate compounds and inorganic phosphate by reversed-phase high-performance liquid chromatography: evaluation of myocardial metabolic status in aerobically perfused and hypoxic mouse heart.

The present paper describes a simple HPLC method designed for measuring high-energy phosphate (HEP) compounds in a single run and inorganic phosphate (Pi) in an other short run under the same HPLC conditions. Inorganic phosphate was estimated by using thymidine phosphorylase (EC 2.4.2.4) which catalyzes a reaction involving inorganic phosphate to produce 2-deoxyribose 1-phosphate and thymine. The thymine/Pi stoichiometry was 1. The method provides a reproducible instrument for evaluating myocardial high-energy metabolism under physiological and pathological conditions.

Deletion of the endogenous TrkB.T1 receptor isoform restores the number of hippocampal CA1 parvalbumin-positive neurons and rescues long-term potentiation in pre-symptomatic mSOD1(G93A) ALS mice

ABSTRACT Amyotrophic lateral sclerosis (ALS) causes rapidly progressive paralysis and death within 5 years from diagnosis due to degeneration of the motor circuits. However, a significant population of ALS patients also shows cognitive impairments and progressive hippocampal pathology. Likewise, the mutant SOD1(G93A) mouse model of ALS (mSOD1), in addition to loss of spinal motor neurons, displays altered spatial behavior and hippocampal abnormalities including loss of parvalbumin‐positive interneurons (PVi) and enhanced long‐term potentiation (LTP). However, the cellular and molecular mechanisms underlying these morpho‐functional features are not well understood. Since removal of TrkB.T1, a receptor isoform of the brain‐derived neurotrophic factor, can partially rescue the phenotype of the mSOD1 mice, here we tested whether removal of TrkB.T1 can normalize the number of PVi and the LTP in this model. Stereological analysis of hippocampal PVi in control, TrkB.T1−/−, mSOD1, and mSOD1 mice deficient for TrkB.T1 (mSOD1/T1−/−) showed that deletion of TrkB.T1 restored the number of PVi to physiological level in the mSOD1 hippocampus. The rescue of PVi neuron number is paralleled by a normalization of high‐frequency stimulation‐induced LTP in the pre‐symptomatic mSOD1/T1−/− mice. Our experiments identified TrkB.T1 as a cellular player involved in the homeostasis of parvalbumin expressing interneurons and, in the context of murine ALS, show that TrkB.T1 is involved in the mechanism underlying structural and functional hippocampal degeneration. These findings have potential implications for hippocampal degeneration and cognitive impairments reported in ALS patients at early stages of the disease. HighlightsDeletion of TrkB.T1 (T1) increases parvalbumin‐positive interneurons (PVi) number in the CA3 hippocampal subfield.T1 contributes to the loss of PVi occurring in pre‐symptomatic mSOD1(G93A) ALS mice.Deletion of T1 restores physiological levels of high‐frequency stimulus‐induced LTP in pre‐symptomatic ALS mice.T1 may be involved in the mechanism causing cognitive impairments the mSOD1 mouse model of ALS.

A Poloxamer-407 modified liposome encapsulating epigallocatechin-3-gallate in the presence of magnesium: Characterization and protective effect against oxidative damage

ABSTRACT Epigallocatechin‐3‐gallate (EGCG) is a polyphenolic catechin from green tea, well known for being bioactive in age‐associated pathologies where oxidative stress plays a preeminent role. The activity of this molecule is however contrasted by its high chemical and metabolic instability that determines a poor concentration of the antioxidant within the biological system after administration. In order to protect the molecule and increase its delivery efficiency, we have encapsulated EGCG inside anionic liposomes made of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine and cholesteryl hemisuccinate. To maximize EGCG internalization, magnesium salt was added in the preparation. However stable nanodispersions suitable for drug delivery were obtained only after treatment with Poloxamer‐407, a polyethylene–propylene glycol copolymer. The structural and morphological properties of the produced dispersion were studied by X‐ray diffraction, which showed a multilamellar structure even after EGCG addition and an ordering effect of Poloxamer‐407; Dynamic Light Scattering demonstrated serum stability of the liposomes. The characterization was completed by evaluating both encapsulation efficiency (100%, in the final formulation) and in vitro EGCG release. Since oxidative stress is involved in numerous retinal degenerative diseases, such as age‐related macular degeneration, the ability of these liposomes to contrast H2O2‐induced cell death was assessed in human retinal cells. Morphological changes at the subcellular level were analyzed by Transmission Electron Microscopy, which showed that mitochondria were better preserved in cells treated with liposomes then those treated with free EGCG. In conclusion, the results demonstrated that the produced formulation enhances the efficacy of EGCG under stress conditions, thus representing a potential formulation for the intracellular delivery of EGCG in diseases caused by oxidative damage.