IGF-I prevents glutamate-induced motor neuron programmed cell death
Introduction
Insulin-like growth factor (IGF)-I is a potent neurotrophin with diverse effects on proliferation, differentiation, and survival. There are two major signaling pathways downstream of IGF-I receptor (IGF-IR) activation: phosphatidylinositol 3-kinase (PI-3K) and mitogen-activated protein kinase (MAPK). PI-3K leads to the downstream activation of protein kinase B/Akt, a potent mediator of neuronal survival Bui et al., 2002, Ciani et al., 2002, Kumari et al., 2001, Zhang et al., 2001. MAPK activation is involved in cell proliferation and differentiation. In neurons, MAPK signaling can also lead to neurite outgrowth, but like the PI-3K/Akt pathway, it may also promote cell survival (Feldman et al., 1997).
The ability of IGF-I to inhibit neuronal cell death is well established. IGF-I is neuroprotective in animal models of neuronal injury including brain ischemia Kawano et al., 2001, Wang et al., 2000, axotomy Kermer et al., 2000, Mathonnet et al., 2001, and age-induced hippocampal neuron death (Poe et al., 2001). In cell culture models, IGF-I prevents dopamine-induced cell death in cultured cerebellar granule cells, a model of Parkinson's disease (Offen et al., 2001). IGF-I also protects cortical neurons from apoptosis induced by serum deprivation (Yamada et al., 2001) and motor neurons from glutamate-induced death in organotypic slice culture (Bilak and Kuncl, 2001).
The neuroprotective effect of IGF-I indicates therapeutic potential against neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). ALS is characterized by progressive loss of motor neurons in the spinal cord and cerebral cortex. The motor neuron loss does not occur through necrosis but has some morphological features of apoptosis and was recently termed paraptosis (Sperandio et al., 2000). Cellular death that is regulated by innate cellular mechanisms is defined as programmed cell death (PCD) and this term is used for the current study. In some familial cases of ALS, the induction of motor neuron PCD is related to mutations of the Cu/Zn superoxide dismutase gene (Rosen et al., 1993) or the ALS2 gene (Hadano et al., 2001). However, the cause of most cases of ALS is not known. One factor that may be important is glutamate, which is elevated in the cerebrospinal fluid of approximately half of all ALS patients (Spreux-Varoquaux et al., 2002). This increase in glutamate is associated with the loss of spinal motor neurons Carriedo et al., 1996, Schubert and Piasecki, 2001, Tarabal et al., 2001, and in vitro, elevated glutamate causes motor neuron cell death (Bilak and Kuncl, 2001).
IGF-I can protect cultured motor neurons from glutamate-induced death (Bilak and Kuncl, 2001). Thus, IGF-I may be effective in the treatment of ALS. In fact, two clinical trials of recombinant human IGF-I in sporadic ALS have been completed. In a European trial, IGF-I provided no demonstrated benefit (Borasio et al., 1998), while a North American trial showed a slower disease progression in patients treated with IGF-I (Lai et al., 1997). A new North American trial is currently in progress.
Despite the interest in the use of IGF-I as a treatment for ALS, the mechanisms of IGF-I neuroprotection in ALS are unknown. The current studies involved the development of a cell culture model of ALS using glutamate-exposed purified rat motor neurons that can be utilized for high-throughput screening of toxins or neuroprotective agents. This model is used to examine glutamate-induced motor neuron death and to investigate the signaling mechanisms of IGF-I protection. We demonstrate that IGF-IR is expressed and is functional in motor neurons, with downstream signaling through both the PI-3K and MAPK pathways in response to IGF-I. Glutamate induces DNA fragmentation and caspase-3 activation in motor neurons, and these events are prevented through IGF-I treatment in a time- and dose-dependent manner. Finally, inhibitors of downstream IGF signaling increase motor neuron death in the presence or absence of glutamate. These findings provide a rationale for using IGF-I clinically to prevent motor neuron loss in ALS.
Section snippets
Primary culture of rat spinal motor neurons
Spinal cords were collected from E15 Sprague–Dawley rat embryos. Perineural membranes were removed and the tissue was chopped into 2–3 mm pieces. The cells were dissociated by incubating in 0.05% trypsin/EDTA for 15 min at 37°C followed by gentle trituration for 1 min with a serum-coated glass Pasteur pipette. The cell suspension was then layered on a 6.8% solution of Metrizamide in Leibowitz's L-15 media and centrifuged for 15 min at 1000 × g. The motor neurons were collected from the top
Primary motor neurons in culture express functional IGF-IR
To determine how IGF-I may modulate motor neuron survival, we first examined whether IGF-I may have direct effects on motor neurons. Cultured motor neurons were grown for 1, 3, or 7 days and stained using a specific IGF-IR antibody. In addition, motor neurons were counter-stained for the motor neuron-specific markers islet-1 or SMI-32, and nuclear chromatin was visualized with bis-benzimide. Representative motor neurons at 1 and 7 days staining for IGF-IR and islet-1 are illustrated in Fig. 1.
Discussion
ALS is the most common adult-onset motor neuron disease, affecting 1–5 per 100,000 people Cluskey and Ramsden, 2001, Newbery and Abbott, 2002. Ninety percent of cases are sporadic (SALS) and the remaining 10% follow a pattern of autosomal dominant inheritance (FALS) (Newbery and Abbott, 2002). In both forms of ALS, patients experience progressive weakness and spasticity due to a selective degeneration of motor neurons in the motor cortex, corticospinal tracts, brainstem, and spinal cord
Acknowledgements
The authors would like to acknowledge Ms. Carey Backus and Dr. Bhumsoo Kim for their technical help during the completion of these studies. This work was supported by the NIH (NS38849 and NS36778), the ALS Association, and the Program for Understanding Neurological Diseases (PFUND).
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