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. 2002 Feb 5;99(3):1604-9.
doi: 10.1073/pnas.032539299. Epub 2002 Jan 29.

Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS)

David S Howland  1 , Jian Liu, Yijin She, Beth Goad, Nicholas J Maragakis, Benjamin Kim, Jamie Erickson, John Kulik, Lisa DeVito, George Psaltis, Louis J DeGennaro, Don W Cleveland, Jeffrey D Rothstein
Affiliations

Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS)

David S Howland et al. Proc Natl Acad Sci U S A. .

Abstract

Transgenic overexpression of Cu(+2)/Zn(+2) superoxide dismutase 1 (SOD1) harboring an amyotrophic lateral sclerosis (ALS)-linked familial genetic mutation (SOD1(G93A)) in a Sprague-Dawley rat results in ALS-like motor neuron disease. Motor neuron disease in these rats depended on high levels of mutant SOD1 expression, increasing from 8-fold over endogenous SOD1 in the spinal cord of young presymptomatic rats to 16-fold in end-stage animals. Disease onset in these rats was early, approximately 115 days, and disease progression was very rapid thereafter with affected rats reaching end stage on average within 11 days. Pathological abnormalities included vacuoles initially in the lumbar spinal cord and subsequently in more cervical areas, along with inclusion bodies that stained for SOD1, Hsp70, neurofilaments, and ubiquitin. Vacuolization and gliosis were evident before clinical onset of disease and before motor neuron death in the spinal cord and brainstem. Focal loss of the EAAT2 glutamate transporter in the ventral horn of the spinal cord coincided with gliosis, but appeared before motor neuron/axon degeneration. At end-stage disease, gliosis increased and EAAT2 loss in the ventral horn exceeded 90%, suggesting a role for this protein in the events leading to cell death in ALS. These transgenic rats provide a valuable resource to pursue experimentation and therapeutic development, currently difficult or impossible to perform with existing ALS transgenic mice.

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Figures

Figure 1
Figure 1
Mutant SOD1 expression and disease in SOD1G93A transgenic rats. SOD1 expression in blood from transgenic founders (A) is highest in founder number 26. L26H F1 generation rats exhibit SOD1G93A expression throughout the nervous system and peripheral tissues (B). SOD1G93A expressed at ≈8-fold over endogenous in young (6 weeks) presymptomatic transgenic rat spinal cord increases to ≈16-fold by end-stage disease (16 weeks) (C). Normal age-matched littermate control animal (D) at ≈120 days compared with an end-stage transgenic rat showing signs of muscle wasting, paralysis of both hindlimbs and one forelimb (E). Kaplan–Meyer survival curve (n = 25) generated from F2 generation L26H transgenic rats depicting disease onset and survival.
Figure 2
Figure 2
Muscle atrophy and denervation in SOD1G93A rats. Leg muscle myofibers from end-stage (age >120 days) SOD1G93A rats were often seen as groups of atrophic angular fibers (b, arrows), compared with aged-matched control rats (a). Compound muscle action potential in nontransgenic control foot muscle (c; 5.48 mV) was reduced in the foot (d; 4.3 mV) in presymptomatic rats and was almost unobtainable in end-stage foot (e; 0.71 mV) after supramaximal stimulation (1 ms per division). Needle EMG of presymptomatic SOD1G93A rat (g) demonstrates a rare fibrillation potential recorded in the lumbosacral paraspinous muscles compared with age-matched wild-type control rat (f). EMG of end-stage (>125 days age) SOD1G93A rat (h) revealed continuous fibrillation potentials and positive sharp waves (20 ms per division).
Figure 3
Figure 3
Motor neuron and axon loss in SOD1G93A rats. Ventral spinal cord gray matter reveals vacuolar degeneration in the neuropil of presymptomatic SOD1G93A rats (c and d) and astrogliosis and loss of motor neurons in end-stage rats (e and f) compared with age-matched wild-type rats (a and b). Glial nodules, around remnants of degenerating motor neurons, were evident throughout the ventral gray matter (f, arrows). Ventral motor roots from an end-stage rat (i) were atrophic compared with aged-matched control roots (g). Closer inspection revealed active ongoing degeneration in end-stage SOD1G93A ventral roots (j), whereas roots from presymptomatic rats showed little degeneration (h). Magnification: ×4, g and i; ×10, a, c, and e; ×40, b, d, and f; ×100, h and j.
Figure 4
Figure 4
Aberrant accumulations of proteins in SOD1G93A rats. Accumulation in the neuropil of SOD1G93A rats (b) compared with age-matched wild-type rats (a). Hsc70 (c) and ubiquitin (Ub) (g) were abnormally accumulated in the neuropil and cytoplasm of ventral gray neurons. Similarly, neurofilament (NF) aggregates were found in the soma of large motor neurons (d) and their axons, often in spheroid structures in the neuropil and especially in the ventral root zone white matter (f) compared with dorsal white tracts (e).
Figure 5
Figure 5
Astroglial alterations in SOD1G93A rats. The usual ubiquitous astroglial expression of the glutamate transporter EAAT2 (a and b), surrounding motor neurons (arrows), was markedly altered in SOD1G93A rats with a patchy loss in the ventral horn in presymptomatic rats (c) and almost a complete loss of protein in end-stage ventral gray from SOD1G93A rats (d and e). This loss of EAAT2 (GLT) was paralleled in immunoblots from ventral gray of presymptomatic and end- stage rats (f). In parallel, astroglial expression of glial fibrillary acidic protein (GFAP) increased somewhat in presymptomatic ventral gray (h), compared with age-matched wild-type control (g), and was markedly increased in end-stage rats (i), especially around rare motor neuron profiles (j).
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