Expression of the human neurofilament heavy gene (NF-H) in transgenic mice

Mammalian neurofilaments (NFs), the intermediate filaments (IFs) of nerve cells, are composed of three different polypeptides, commonly referred to as the low (NF-L), middle (NF-M) and heavy (NF-H) subunits. As members of the IF family and major components of large myelinated fibers, NFs have been assigned a putative role in maintaining the structural integrity of the neurons and in determining axonal caliber.To study the expression and function of the human NF-H gene, a genomic clone encompassing the human NF-H gene was isolated (Cos4NF-H) from a chromosome 22 enriched library and transgenic mice were generated. Four transgenic lines with multiple copies of the intact human NF-H gene were obtained. Northern blot analysis revealed that the human transgene was expressed specifically in neurons at level of approximately two-fold the endogenous mouse level. In addition, the human NF-H gene was expressed at the correct stage during the mouse nervous system development. Furthermore, expression of the human NF-H gene appeared to be proportional to gene copy-number and independent of the position of integration into the mouse genome. This is the first example of such a pattern of transgene expression in nerve cells. These results suggest that the regulatory signals necessary to direct the correct expression of the human NF-H gene lie within the injected clone and are recognized between the two mammalians species. Once identified, the control elements may constitute a valuable tool to direct the expression of foreign genes specifically to neurons.The human NF-H transgenic mice appear normal at birth but progressively develop tremors and pathological features reminiscent of the ones observed in patients with amyotrophic lateral sclerosis (ALS). For instance, one sees selective enlargements of motor neuron perikarya, dorsal root ganglion (DRG) neurons and proximal axons in addition to distal axonal atrophy and muscle atrophy. Electron microscopy studies showed that the swellings consisted of intracytoplasmic accumulation of NFs. Axonal transport experiments were undertaken to determine if cytoskeletal elements were abnormally transported as has been suggested for a number of neurodegenerative diseases such as ALS. The proteins synthesized in motor/sensory neuron cell bodies were labeled and their subsequent transport to distal axonal segments was analyzed by SDS gels and fluorography. The results showed an impairment of axonal transport of NF proteins, actin and tubulin. There was also retention of some rapidly transported organelles. A possible mechanism to explain these results is that overexpression of the human NF-H gene led to neurofilamentous swelling formation that disrupted axonal flow and eventually resulted in motor neuron degeneration. The human NF-H transgenic mice are therefore a valuable model of human motor neuron disorders and can be used to test therapeutic strategies aimed at reducing the neurofilamentous swellings