Animal models are essential tools in axonal injury research. There are several ways that animal models can help overcome the hurdles that arise during neuropathological studies in humans. Time course analyses can be performed, which are critical for examining mechanisms of disease. In most human studies, brain pathology investigations are limited to the end stage, because the tissue is inaccessible to direct observation. Second, the neurological diseases can take several years to develop and involve complex interactions between resident brain cells, limiting the ability of researchers to model these mechanisms in culture. Furthermore, trans-genic and knock-out mice can be used to directly determine factors that contribute to pathology and neurological deficits in various models of disease. Finally, animal models allow the researcher to assess therapeutic interventions.
Axonal injury has been observed in various animal models of trauma, ischemia autoimmune diseases, viral infections, toxic injury, and neurodegenerative disorders, and many of these studies have been mentioned in the various sections of this chapter. However, research using animal models of two neurological diseases, ALS and MS, merit special attention.
Transgenic mice with alterations in either the SOD1 or neurofilament (NF) genes display motor neuron pathology and deficits in motor function and are the most commonly used animal models to study the mechanisms of ALS neurodegeneration. Studies using these models have shown that damage to axons, in particular axonal transport defects, are one of the earliest known abnormalities, occurring months before clinical changes can be detected. Additional support for the view that axonal transport defects may play a critical role in the pathogenesis of ALS came from inhibition of axonal transport machinery in mice. Transgenic mice over-expressing dynamitin develop a late-onset and progressive motor neuron disease resembling ALS, with neurofilamen-tous swellings in motor axons. Transgenic mice overexpress-ing the shortest tau isoform exhibit axonal degeneration of spinal neurons and motor weakness (reviewed in Robertson et al., 2002).
Approximately 15% to 20% of autosomal dominant familial ALS cases (FALS; ~ 2% of all cases) have mutations in the SOD1 gene. However, the mechanisms of how these ubiquitously expressed mutant proteins lead to selective vulnerability of motor neurons are not understood. Several lines of transgenic mouse models that express several of the mutant forms of the SOD1 gene have been established. These transgenic mice develop phenotypes similar to human FALS and show motor dysfunction at 3 to 9 months of age (Warita et al., 1999).
Defects in axonal transport in these mice have become a focus for several groups. Axonal transport can be divided into two rate components: fast (100- to 400-mm/day) and slow (0.1-3-mm/day). Warita and colleagues (1999) have investigated whether fast axonal components are modified in ligated sciatic nerves of SOD1 transgenic mice and how they are altered developmentally from an early preclinical stage. Defects in fast anterograde transport machinery (decreased accumulation of the microtubule activated ATPase kinesin after ligation of the sciatic nerve) were observed, and led the authors to hypothesize that this impairment may contribute to selective motor neuron death.
Other groups have also shown that slowing of axonal transport is an early event in the toxicity of the ALS-linked SOD1 mutants to motor neurons (Williamson and Cleveland, 1999); however, this group showed some selectivity toward the slow component of axonal transport. They used transgenic mice expressing the human SOD1G37R and SOD1G85R mutants. [35S]Methionine was injected into the spinal cord of SOD1 mice and labeled proteins were quantified in consecutive segments from the spinal cord. Retardation of slow axonal transport was an early event in these transgenic mice. Tubulin transport was found to slow more dramatically at earlier stages, whereas the transport of neurofilaments and other cargoes was affected later. The authors interpreted these findings as an indication of a worsening defect, reflecting a decline in neuronal health and function with time; they highlighted that this is consistent with the slow accumulation of damage over a long period, ultimately culminating in late-onset disease in both mice and humans. Unlike the Warita study, no changes were detected in fast axonal transport, suggesting some selectivity toward slow transport. Results from this study provided evidence of a unifying mechanism of SOD1 mutant action that occurs long before the onset of clinical symptoms. Thus, compromised transport of selected cargoes may contribute to motor neuron vulnerability through chronic reduction in these key axonal components (Williamson and Cleveland, 1999).
ALS is characterized by depositions of neurofilament proteins in the perikarya and proximal axons. To investigate how disorganized NFs might cause neurodegeneration, Collard and colleagues (1995) examined axonal transport of newly synthesized proteins in mice that overexpress the human NF heavy-subunit gene. These mice show muscle weakness, a fine tremor, and a disturbance of gait and pathology similar to ALS; but these mice do not become fully paralyzed, and the spinal cords do not show significant motor neuron loss. This group observed dramatic defects of axonal transport of the neurofilament proteins, as well as other proteins such as tubulin and actin. Ultrastructural analysis revealed a dramatic lack of cytoskeletal elements, smooth endoplasmic reticu-lum, and mitochondria of the degenerating axons. They proposed that the neurofilament accumulations observed in these mice cause axonal degeneration and subsequent weakness by impeding the transport of important cellular components required for axonal maintenance.
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