Dottoressa di ricerca

ciclo: XXXIV

relatore: Maria Egle De Stefano
co-supervisore: Giuseppe Lupo

Titolo della tesi: Duchenne muscular dystrophy: a study of D- and L- aminoacids expression in the central nervous system and novel alterations in the sensory-motor innervation of skeletal muscles

PART1 Amino acids are important neurotransmitters, especially in the CNS, with both excitatory (glutamate, aspartate) and inhibitory function (glycine, GABA). A balanced spontaneous and induced neuronal transmission plays a fundamental role during neuronal development, so much so that its alterations have been associated with the onset of various neuronal pathologies and neurodevelopment. Early glutamatergic neurotransmission involves the amino acid D-Aspartate (D-Asp), agonist of the glutamate receptor NMDA, whose signaling is involved in various aspects of neural circuit development. In pre- and neonatal stages of various organisms, D-Asp is one of the most abundant, expressed in different districts, including the brain. In order to analyze how levels of D-Asp and other D/L amino acids may affect neurodevelopment, a large-scale study was conducted on two animal models. The first study, object of a publication, concerned the phenotypic characterization of the murine model (R26Ddo/Ddo) (generated in the laboratory of Prof. Usiello), which is knock-in for the enzyme D-Asp oxidase (DDO). This enzyme is relevantly expressed from the first post-natal stages and induces a drastic and significant decrease in D-Asp levels. The results obtained highlighted the onset of specific alterations in the brains of mice deprived of D-Asp prematurely due to excess DDO.. The second study was conducted on the well-known Duchenne Muscular Dystrophy (DMD) mdx mouse model. DMD is characterized by progressive muscle degeneration and neurological disorders of various degrees that originate from morpho-functional alterations in brain areas that characteristically express high levels of D-Asp during embryonic development (hippocampus, cerebellum and prefrontal cortex). These alterations are stablished during embryonic development, and mainly involve the differentiation of certain neuronal circuits and the stabilization of specific receptor subtypes, as the GABAergic, cholinergic and glutamatergic ones. Analysis by HPLC of the levels of different D- and L-amino acids at pre- and postnatal dates showed that, predominantly in the hippocampus, mdx mice are characterized by reduced prenatal levels of one or more amino acid compared to wild type mice, variations that in some cases persists in adult mice. A reduction of D-Asp also occurs in the spinal cord of adult mdx mice. These data are innovative and for the first time correlate a DMD phenotype with alterations in D/L amino acids’ metabolism, which will need to be thoroughly investigated.   PART2 Duchenne muscular dystrophy (DMD) is a recessive genetic disease associated with the X chromosome, characterized by the lack of dystrophin (Dp427), a cytoskeletal protein abundantly expressed in skeletal muscles, but also in other types of muscle and non-muscle cells, including some neurons of the central nervous system (CNS) and peripheral (PNS). Clinically, DMD is characterized by progressive and lethal muscle wasting. However, DMD patients also exhibit neurological disorders and autonomic dysfunctions of various kinds and degrees. In the nervous system of both patients and mouse models of the disease, such as the DMDmdx mouse, Dp427 has been described predominantly localized in some post-synaptic specializations, where it contributes to the stabilization of specific receptor subtypes, such as GABA type A receptors (GABAA) and nicotinic acetylcholine receptor subtypes. In the nervous system, as well as in other tissues, Dp427 can coexist, or be replaced, by one or more shorter isoforms. In particular, glial cells such as oligondendrocytes in the CNS and Schwann cells (SCs) in the SNP express Dp427 and its Dp116 isoform, respectively. Both cell types are responsible for the intense glia-neuron cross-talk, crucial for axon growth and activity, as well as for glial differentiation and myelination. This signaling occurs through complex autocrine and paracrine circuits involving GABA, glutamate, acetylcholine and their receptors. In the PNS, SCs are also important for axonal regeneration and maintenance of the neuromuscular junctions that motor neurons (MNs) establish on skeletal muscle fibers. To date, compared to the numerous studies conducted on skeletal muscles, little is known about the possible morpho-functional effects that this pathology can exert on MNs and their intraspinal connections. This part of the PhD thesis, reports on the data obtained in the first set of exeperiments aimed at characterizing alterations that the lack of Dp427 in the mdx mouse model induces on both motor and sensory innervation associated with skeletal muscles. This study has analyzed both expression levels and localization of myelin proteins, and of GABAA and GABAB receptor subunits, expressed by SCs and demonstrated to be involved in the signaling with the ensheated peripheral sensory and motor fibers. At this aim, transcriptomic (in collaboration with Prof. Magnaghi), biochemical and immunohistochemical approaches were used. The results obtained are innovative and indicate that the lack of Dp427 alone has a significant impact on both expression and localization of all proteins analyzed, focusing on possible motor-sensory problems never taken into consideration in DMD.

Produzione scientifica

11573/1396063 - 2020 - Prenatal expression of d‑aspartate oxidase causes early cerebral d‑aspartate depletion and influences brain morphology and cognitive functions at adulthood
De Rosa, Arianna; Mastrostefano, Francesca; Di Maio, Anna; Nuzzo, Tommaso; Saitoh, Yasuaki; Katane, Masumi; Isidori, Andrea M.; Caputo, Viviana; Marotta, Pina; Falco, Geppino; De Stefano, Maria Egle; Homma, Hiroshi; Usiello, Alessandro; Errico, Francesco - 01a Articolo in rivista
rivista: AMINO ACIDS (Wien: Springer.) pp. - - issn: 1438-2199 - wos: WOS:000520693600001 (13) - scopus: 2-s2.0-85081909051 (13)

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