Minerva Giménez y Ribotta

PREVENTION OF MOTONEURON DEATH BY ADENOVIRUS-MEDIATED NEUROTROPHIC FACTORS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons, and has no effective treatment. Experimental studies in rodents have shown that motoneurons respond to a variety of molecules including brain‐derived neurotrophic factor (BDNF), and the glial‐cell line‐derived neurotrophic factor (GDNF). Here we investigated the neuroprotective effect of these growth factors, encoded by an adenovirus, on the death of axotomized facial motoneurons in newborn rats. We used a new gene therapy strategy that involves gene transfer to motoneurons by intramuscular injection of an adenoviral vector, which is retrogradely transported from injected target muscle (Finiels et al.,: NeuroReport 7:373–378, 1995). A significant increased survival of motoneurons was observed in animals pretreated with adenovirus encoding BDNF (34.5%, P < 0.05) or GDNF (41.9%, P < 0.05) 1 week after axotomy.

Cerebellar defect and impaired motor coordination in mice lacking Vimentin

Vimentin belongs to the family of intermediate filament (IF) proteins. During the nervous system development in mammals, it is transiently expressed in precursor cells of neuronal and glial lineages, and then it is progressively replaced by other types of IF proteins. Surprisingly, mice knock‐out for vimentin develop and reproduce without any apparent defects (Colucci‐Guyon et al. Cell 79:679–694, 1994). In adult rodents, Bergmann glia (BG) of the cerebellum continue to express vimentin together with glial fibrillary acidic protein (GFAP). A careful analysis of cerebellar morphology and ultrastructure in mutants showed poorly developed and highly abnormal BG, whereas the migration of granular neurons proceeded normally. Moreover, many Purkinje cells (PC) appeared stunted with a loss of spiny branchlets, and some of them were necrotic. Finally, impaired motor coordination was evidenced by behavioral tests. These observations demonstrate a role for vimentin in contributing to the normal development and morphology of BG and reveal a hitherto unreported functional relationship between BG and PC. GLIA 25:33–43, 1999.

GFAP null astrocytes are a favorable substrate for neuronal survival and neurite growth.

During the development of the CNS, astrocytes play a key role as a substrate for neuronal migration and axonal growth. These neuron‐astrocyte interactions could be regulated, in part, by the astrocytic cytoskeleton. Nestin, vimentin, and glial fibrillary acidic protein (GFAP) are the three identified proteins constitutive of intermediate filaments present in astrocytes. In the present study, we used mice deficient in GFAP to define the influence of the major protein of the astrocytic cytoskeleton on neuron survival and axonal growth in a model of neuron‐astrocyte coculture. We observed that GFAP null astrocytes are a better substrate for neuronal survival and neurite outgrowth than wild‐type astrocytes. This may be correlated with the relatively late occurrence of GFAP expression in astrocyte maturation when the early steps of neurogenesis are completed. GLIA 31:267–272, 2000.

Specific and efficient gene transfer strategy offers new potentialities for the treatment of motor neurone diseases.

Several growth factors are candidates for the therapy of motor neurone diseases. However, there is no efficient, safe, and practicable administration route which hampers the clinical use of these potentially therapeutic agents. We show that specific and high yield gene transfer into motor neurones can be obtained by peripheral intramuscular injections of recombinant adenoviruses. These vectors are retrogradely transported from muscular motor units to motor neurone cell bodies. Gene transfer can thus be specifically targeted to particular regions of the spinal cord by appropriate choice of the injected muscle. The efficiency of gene transfer is high, with 58–100% of the motor neurones afferent to the injected muscle expressing the transgene. This new therapeutic protocol allows specific targeting of motor neurones without lesioning the spinal cord, and should avoid undesirable side effects associated with systemic administration of therapeutic factors.

Glial scar and axonal regeneration in the CNS: lessons from GFAP and vimentin transgenic mice.

Astrocytes play an active role in the brain and spinal cord. For example, they have a function in formation and maintenance of the blood-brain barrier, ion homeostasis, neurotransmitter transport, production of extracellular matrix, and neuromodulation. Moreover, they play a role in preserving or even restoring the structural and physiological integrity after tissue injury. Currently, the function of astrocytes was studied with regard to the controversially discussed aspects of permissivity on the one-hand-side and inhibition of the other side exerted by reactive astrocytes for axonal regrowth in the adult CNS. Accordingly, knock-out mice deficient in vimentin (VIM) and/or glial fibrillary acidic protein (GFAP), the two major IF-proteins of astrocytes, were investigated. In addition, in vitro studies were carried out, on whether the absence of one or both proteins (VIM, GFAP) influences axonal regeneration. In experimental animals, a hemisection of the spinal cord was performed utilizing the above mentioned double-mutant mice. The knock-out mice were generated by gene targeting. Double-mutants were obtained by crossing single null mice. The in vitro results indicate that both VIM and GFAP were absent in astrocytic cultures obtained from double-mutant mice. On the other side, the proteins were detected in more than 85%, of cultured cells from wild types. Co-culture of mutant mice astrocytes with neurons revealed that the neuronal density was different from that obtained in culture with wild type astrocytes. On the other side, there was a marked increase in neuronal density in co-cultures utilizing both GFAP knock-out- or double-mutant mice astrocytes again as compared to co-cultures with wild type astrocytes. Moreover, the neurite length of neurons was significantly increased in experiments with neurons growing on astrocytes from GFAP-knock-out or double-mutant mice. The in vivo experiments demonstrate an increase of nestin (NES) immunoreactivity at three days in the sectioned side of the spinal cord, in the perikaryon and astroglial processes. In double-mutant mice only a slight increase in NES-immunoreactivity was found in the lesion side, albeit confined to the perikaryon of astrocytes. Below the lesion, serotonin immunostaining was dramatically reduced three days after the insult in both sides, particularly in the lesion side. The decrease was more pronounced in double-mutant than in wild type mice. On the other side, double-mutant mice had a much higher density of serotonergic fibers in the ventral horn in the lesioned side. In conclusion, the findings demonstrate that in the absence of important astrocytic proteins as VIM and GFAP, the astroglial response to injury is significantly modified underlying reduced scar formation. Attenuation of scar formation may enhance axonal sprouting of serotonergic axons below the lesion, which specifically reinnervate motoneuron pools.

Recovery of Locomotion following Transplantation of Monoaminergic Neurons in the Spinal Cord of Paraplegic Ratsa

Abstract: Severe traumatic lesions of the spinal cord yield a permanent deficit of motricity in adult mammals and specifically a loss of locomotor activity of hindlimbs when the lesion is located at the lower thoracic level. To restore this function, we have developed a paradigm of transplantation in rats based on a transection model of the spinal cord and the subsequent injection at the sublesional level of a suspension of embryonic brainstem monoaminergic neurons which play a key role in the modulation of locomotion. A genuine locomotion was characterized in transplanted animals by electromyographic and electroneurographic recordings. This correlated with a specific reinnervation pattern of targets, where typical synapses were found, and with the normalization of biochemical parameters.

Biological interventions for spinal cord injury.

Spinal cord injury is frequently followed by the loss of supraspinal control of sensory, autonomus and motor functions at sublesional level. To enhance recovery in patients with spinal cord injuries, three fundamental strategies have been developed in experimental models. These strategies involve three different time points for postlesional intervention in the spinal cord. Neuroprotection soon after injury uses pharmacological tools to reduce the progressive secondary injury processes that follow during the first week after the initial lesion occurs, in order to limit tissue damage. A second strategy, which is initiated shortly after the lesion occurs, aims at promoting axonal regeneration by acting pharmacologically on inhibitors or barriers of regeneration, or by the application of cell or gene therapy as a source of neurotrophic factors or as a bridge or support to enhance the regeneration of lesioned axons. Finally, a mid-term substitutive strategy is the management of the sublesional spinal cord by sensorimotor stimulation or the supply of missing key afferents, such as monoaminergic systems. These three strategies are reviewed. Only a combination of these different approaches can provide an optimal basis for potential therapeutic interventions aimed at functional recovery after spinal cord injury.

Comparative anatomy of the cerebellar cortex in mice lacking vimentin, GFAP, and both vimentin and GFAP

In the cerebellum of adult mammals, glial fibrillary acidic protein (GFAP) and vimentin (VIM) are coexpressed in Golgi epithelial cells (GEC), also known as Bergmann glia. In this study we used three transgenic knockout mice (GFAP, VIM and double GFAP and VIM) to analyze the involvement of these proteins in the building of glial filaments and in neuron‐glia interactions. The cerebella of VIM, GFAP, and GFAP/VIM mutant mice were processed by the rapid Golgi method and also for electron microscopy. In VIM mutant mice, Bergmann fibers are hypertrophic with thickened appendages. In the electron microscope they appear as large glial profiles devoid of glial filaments, with embedded dendritic thorns and parallel fiber boutons. In addition, signs of degeneration are observed in Purkinje cells. In GFAP mutant mice, GEC exhibit fine, delicate processes, as those seen in wild‐type animals, however, a large accumulation of lamellae and granular appendages was observed along their surfaces, which came into contact with each other. The electron microscope exhibited fine and scarce astroglial profiles containing some glial filaments, a stunted glia limitans, and the presence of large extracellular spaces. In double mutant mice, the two phenotypes are expressed but appear attenuated, with a total absence of glial filaments and the general appearance of immaturity for GEC. In conclusion, it appears that the absence of each of the proteins yields a specific phenotype and that the defects are not necessarily additive. GLIA 31:69–83, 2000.

Chapter 15 Strategies for regeneration and repair in spinal cord traumatic injury

Spinal cord injury is frequently followed by the loss of supraspinal control of sensory, autonomic and motor functions at the sublesional level. In order to enhance recovery in spinal cord-injured patients, we have developed three fundamental strategies in experimental models. These strategies define in turn three chronological levels of postlesional intervention in the spinal cord. Neuroprotection soon after injury using pharmacological tools to reduce the progressive secondary injury processes that follow during the first week after the initial lesion. This strategy was conducted up to clinical trials, showing that a pharmacological therapy can reduce the permanent neurological deficit that usually follows an acute injury of the central nervous system (CNS). A second strategy, which is initiated not long after the lesion, aims at promoting axonal regeneration by acting on the main barrier to regeneration of lesioned axons: the glial scar. Finally a mid-term substitutive strategy is the management of the sublesional spinal cord by sensorimotor stimulation and/or supply of missing key afferents, such as monoaminergic systems. These three strategies are reviewed. Only a combination of these different approaches will be able to provide an optimal basis for potential therapeutic interventions directed to functional recovery after spinal cord injury.

Influence of hypergravity on the development of monoaminergic systems in the rat spinal cord

We have investigated in this study the influence of a moderate hypergravity (1.8 G) on the development of monoaminergic projections to the spinal cord in the rat. Pregnant dams and their offspring were submitted to hypergravity from day 11 of gestation to postnatal day 15. Some animals were sacrificed at birth, other at postnatal day 15 and other after 8 months of normal gravity. In newborn animals, a substantial delay of the development of monoaminergic projections to the spinal cord was evidenced. In 15 days and 8 months animals, the pattern of innervation appeared anarchic, with numerous dystrophic profiles, mainly of serotonergic system. Ultrastructural examination of serotonergic projections revealed a paucity of synapses, and the frequent enveloping of serotonergic boutons by thin astrocytic profiles. We conclude that rats submitted to hypergravity during the critical period of onset of monoaminergic projections to the spinal cord are affected durably in the organization and the ultrastructure of these projections. Future studies are directed to the functional analysis of hypergravity animals, and to the influence of microgravity on the same system.