CELL BIOLOGY AND NEUROANATOMY   

Prof. Silvia De Biasi, Prof. Laura Vitellaro Zuccarello, Dr. Alida  Amadeo, Dr. Maria Enrica Pasini.

The research activity relies on a wide spectrum of modern morphological techniques of analysis (high resolution microscopic imaging, fluorescence and confocal microscopy, electron microscopy) integrated with biochemical and molecular biology approaches. In general, our research topics include the study, from the microscopic to the molecular level, of: a) neuronal and glial subpopulations, b) synaptic circuits; c) components of the extracellular matrix in different areas of the the mammalian central nervous system (spinal cord, thalamus, neocortex, hippocampus, cerebellum) in normal and pathological conditions. To this end we employ neuroanatomical techniques at light, confocal and electron microscopical level that allow also to draw functional indications. Neurons, glia and synapses are identified by the expression of several molecular markers (cytoplasmic, membranous or extracellular) whereas extracellular matrix components are identified  using specific ligands.

Research topics:

1. Amyotrophic Lateral Sclerosis  (ALS)

De Biasi, Vitellaro, Gioria, Pasini, Fontana

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease arising from a progressive loss of motor neurons that leads to atrophy of voluntary muscles. The cause of motor neuron degeneration remains largely unknown and to date there is no potent therapy. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1), a crucial enzyme for cellular antioxidant defence mechanisms. Overexpression of SOD1 gene mutations in transgenic mice and rats recapitulates the clinical and pathological characteristics of ALS and therefore these rodents represent useful models in which to investigate the mechanisms leading to motor neurons degeneration. In collaboration with Dr. C. Bendotti (Laboratorio di Neurobiologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Milano) and with Prof. A. Poletti (Centro di Eccellenza per lo Studio delle Malattie Neurodegenerative, Università di Milano), we are using transgenic mice expressing mutant human SOD1 with a glycine-to-alanine mutation at position 93 (Tg SOD1G93A) to specifically investigate the following issues:

2.1. Role of altered proteolysis in the degeneration of motor neurons in a mouse model of familial amyotrophic lateral sclerosis

Like other neurodegenerative diseases, ALS is characterized by accumulation of intracellular aggregates of abnormal proteins, whose formation and impaired degradation may be relevant for degeneration. A contribution of the dysfunction of two proteolytic routes, the ubiquitin-proteasome and the autophagy-lysosome pathways, has been suggested for some neurodegenerative diseases, but the exact role played by the two mechanisms in ALS motor neuron degeneration is still incompletely understood. We are therefore investigating, in Tg SOD1G93A mice at different times of disease progression, the involvement of the two different intracellular proteolytic pathways in the development of spinal cord lesions typical of ALS and their relevance to motor neuron loss.

2.2. Role of altered mitochondria in the degeneration of motor neurons in a mouse model of familial amyotrophic lateral sclerosis

Several lines of evidence indicate that mitochondria are involved in the development and progression of ALS. In spinal motor neurons of Tg SOD1G93A mice we have previously described the presence of mitochondrial alterations at ages preceeding disease onset. We are now investigating the time course of these mitochondrial alterations during disease progression by examining also bulbar nuclei whose motor neurons are affected by ALS.

FIG.1. Electron micrograph of an altered mitochondrion (arrow) in the soma of a motor neuron  in a transgenic mouse used as a model of ALS.

2. Traumatic Spinal Cord Injury  (TSCI)

De Biasi, Vitellaro, Fontana

Traumatic spinal cord injury (TSCI) is an unfortunately very frequent and devastating human pathology for which there are currently no effective therapies. To contribute to the understanding of the secondary neurodegenerative mechanisms involved in this pathology we are using a rat model of contusive spinal cord injury developed in the laboratory of prof. A. Gorio (Dipartimento di Medicina, Chirurgia e Odontoiatria, Università di Milano). Specifically, the research on this issue deals with the characterization of the fate of grafted stem cells and of the neural parenchyma at the lesion site in an experimental model of traumatic spinal cord injury (TSCI). Stem cells represent a potentially powerful tool to restore function to injured nervous tissue provided that they survive, migrate to the lesion site and integrate functionally with host nervous tissue. The specific aims of this investigation are therefore to 1) perform a detailed morphological analysis to determine the localization, extent of engraftment and differentiation of injected or transplanted neural stam cells (NSCs) in the traumatically injured spinal cord of rodents, and 2) characterize the cellular and extracellular molecules expressed in the area of injured spinal cord surrounding the grafted NSCs.

FIG. 2. Immunocytochemical localization of a parvalbumin-containing interneuron (IN, green) and of a motor neuron (MN, blu) surrounded by extracellular matrix (red) in the rat spinal cord. Confocal microscopy. 

3. Cholinergic Innervation of Adult and Developing Rodent Brain

Amadeo, Pasini

The cerebral cholinergic system is well known to regulate the transitions between the brain states with different levels of attention. In addition, a body of relatively recent work indicates that the cholinergic innervation of the cerebral cortex and other brain regions is involved in a variety of cerebral functions, with relevant implications for neuropathologies such as schizophrenia, Alzheimer's disease and some epileptic forms. Despite the increasing use of murine models in fundamental neuroscience and neuropathology, large gaps remain in our understanding of the organization of the cerebral cholinergic system in the mouse. The few comparative studies reveal significant differences between the anatomical structure and the synaptic circuit organization in different mammalian species. This poses a problem when interpreting the results obtained on transgenic mice used as models of human neurologic pathologies. On these bases our research team aims at combining  neuroanatomical methods, by applying cytochemical and immunocytochemical procedures, and gene expression profiling to obtain a broad picture of the murine cholinergic system and to investigate in more detail some specific regions, such as cerebral cortex, thalamus and brainstem in adult and developing mouse.

FIG. 3. Biocytin-labeled neuron in the mouse neocortex characterized by a pyramid-like soma and by apical and basal dendrites with numerous spines. Scale bar: 20 um

Publications

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