Silvio Varon - US grants

The long-term objectives of this research group continues to be the investigation of i) neuro(no)trophic factors that support maintenance, growth, function and repair of selected nerve cells, ii) neurite modulating factors that promote (or inhibit) and guide the growth of neuronal axons in development and adult regeneration, and iii) adult rat models that test the competence of neurotrophic and neurite modulating factors to perform in vivo within the context of adult mammalian central and peripheral nervous systems. The underlying expectation -- now widely shared -- is that such factors may ultimately serve as therapeutic tools in degenerative human diseases (e.g. Alzheimer's, Parkinson's, ALS, etc.) and support long-track axonal regeneration after spinal cord or optic nerve lesions, among others. No information is yet available on what trophic and other factors may influence maintenance and repair of corticospinal (CS) motor neurons: the project will address this question by use of both an in vitro model of developing CS cells and an in vivo model of lesioned adult CNS neurons. Another question concerns nerve growth factor (NGF, the prototype for neurotrophic factors), namely the location and identity of NGF producer cells: this project will explore the possible identity between NGF producers and certain subsets of CNS GABAergic neurons. NGF-containing cells have also been found in the hypothalamus and the anterior pituitary, increasing the perception of important links between NGF and thyroid hormones -- and this project will investigate the nature and regulation of hypothalamic and pituitary NGF. Lastly, even when CNS axons are made to regenerate successfully to their innervation territory, the problem remains of what may regulate their distribution there: this project seeks to characterize the newly recognized occurrence of sedimentable NGF in adult brain extracts and the detection of anchored NGF in adult brain sections and to correlate them with pathways and end stations of regenerating cholinergic CNS afferents.

In the adult rat chamber model for PNS regeneration, the two stumps of a resected sciatic nerve are inserted into opposite ends of a cylindrical silicone tube leaving a 10 mm, 25 u1 gap between them. In a defined sequence--fluid exudate accumulation, acellular matrix formation, cell immigration and axonal regrowth- a nerve structure forms de novo in the chamber and sensory and motor recoveries eventually take place. This research will complete the development of a continuous perfusion system for the experimental administration of exogenous agents, in particular: neuronotrophic factors, growth factors for Schwann and other cells, and neurite-promoting extracellular matrix components.

Neuronal survival and neurite elongation, two key components of neural regeneration, are likely to depend on the availability to the neurons of appropriate tropic stimuli. Nerve Growth Factor (NGF) has served for 30 years as a model for such neuronotrophic factors. Neuronotrophic activities other than NGF have been observed in several tissue extracts and culture-conditioned media, which address neurons from several peripheral ganglia and--more rarely--from various CNS tissues. We have identified and purified a new ciliary neuronotrophic factor CNTF, directed to the cholinergic neurons of chick embryo ciliary ganglia, in the extract of intraocular tissues that are innervated by these neurons. CNTF activity has also been recognized in extracts of peripheral nerve and CNS tissues, and found to accumulate in the fluid surrounding damaged nerve or brain. We have purified, on a pilot scale, the CNTF from mammalian (rat) sciatic nerve with similar biological but distinct physicochemical properties as those of the chick eye CNTF. We plan to continue the investigation of these several factors, particularly with regard to i) scaled-up production of both avian and mammalian CNTFs for both in vivo and in vitro uses, ii) generation of anti-CNTF antibodies, iii) use of molecular biology techniques to sequence the factor proteins, clone their genes and messenger RNAs, and attempt bioengineering production of the factors, iv) screening of other CNTFs by a new blot-and-culture technique and v) exploration of structural and biological properties of the purified CNTF.

In the adult rat septo-hippocampal model for acute experimental lesions, a complete transection of the fimbria fornix causes the ipsilateral disappearance of most of the medial septum cholinergic (MSC) neurons. The apparent loss of MSC neurons is prevented by continuous intraventricular infusion of Nerve Growth Factor (NGF), and is largely reversed by delayed NGF treatments. In addition, NGF treatments cause an increase in body size and ChAT immunostaining in cholinergic neurons of an undamaged neostriatum. This proposal has two major aims. One is to expand-our still modest information concerning the action of NGF on adult cholinergic neurons by, for example, defining dose-responsive relationships, understanding the limits of a delayed NGF rescue, identifying additional responses to NGF with or without experimental lesions. and evaluating chronic cholinergic deficits upon treatment with NGF antibodies. The other is to examine the ability of selected agents other than NGF, alone or in surgery with NGF, to elicit responses from cholinergic and other adult CNS neurons. The actions of NGF on adult mammalian CNS cholinergic neurons provide the first direct evidence of the more general roles that neuronotrophic factors are likely to play in maintenance and repair of central neurons and the opportunities that these factors may represent for new therapeutic approaches to human CNS degeneration disorders and for human brain and spinal cord lesions.