TNF-α triggers rapid membrane insertion of Ca2 + permeable AMPA receptors into adult motor neurons and enhances their susceptibility to slow excitotoxic injury
Highlights
► First direct demonstration of functional Ca2 + permeable AMPA receptors in adult MNs. ► TNF-α rapidly increases numbers of these receptors in the plasma membrane. ► This TNF-α effect occurs via a PI3K and PKA dependent mechanism. ► TNF-α also increases MN Ca2 + permeable AMPA receptors in slice cultures. ► Low level TNF-α exposure enhances slow excitotoxic MN injury in slice cultures.
Introduction
Amyotrophic lateral sclerosis (ALS) is a catastrophic disease characterized by relatively selective degeneration of upper and lower (spinal) motor neurons (MNs), for which there is as yet no good treatment. Observations of deficiencies in spinal cord glutamate uptake in human ALS patients, resulting from selective loss of the astrocytic glutamate transporter, GLT-1, suggested an excitotoxic contribution (Rothstein et al., 1992, Rothstein et al., 1995). It is progressively evident that cell death in ALS depends critically upon interactions between MNs and neighboring cells. Activation of both microglia and astrocytes occurs prominently in both human disease and animal models of ALS (Philips and Robberecht, 2011, Wegorzewska et al., 2009), likely contributing to MN injury via a number of mechanisms, a key one of which may be release of factors including the cytokine tumor necrosis factor-alpha (TNF-α) (Mhatre et al., 2004, Tweedie et al., 2007).
Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are tetramers, composed of combinations of subunits (GluA1-4). Most AMPA receptors are Ca2 + impermeable due to the presence of a GluA2 subunit; GluA2 lacking receptors are Ca2 + permeable. TNF-α has complex effects, which could be either beneficial or injury promoting (Cheng et al., 1994, Ghezzi and Mennini, 2001, Hermann et al., 2001, Pickering et al., 2005). One injury promoting effect is the rapid insertion of AMPA type glutamate receptors into the plasma membrane (Beattie et al., 2002, Hermann et al., 2001, Stellwagen and Malenka, 2006, Yu et al., 2002). Interestingly, the majority of these AMPA receptors contain GluA1 (Beattie et al., 2002, Leonoudakis et al., 2008, Ogoshi et al., 2005, Stellwagen et al., 2005). Furthermore, although some studies showed membrane increases in GluA2 as well, TNF-α preferentially caused the rapid insertion of GluA2 lacking, Ca2 +-permeable AMPA receptors (Ca-perm AMPAr) in hippocampal pyramidal neurons (which normally have relatively low levels of these receptors) (Ogoshi et al., 2005, Stellwagen et al., 2005) and spinal dorsal horn neurons (Choi et al., 2010), resulting in increased susceptibility to excitotoxic injury (Leonoudakis et al., 2008).
Embryonic spinal MNs possess significant numbers of Ca-perm AMPAr under basal conditions (Carriedo et al., 1995, Carriedo et al., 1996, Van Den Bosch et al., 2000, Vandenberghe et al., 2000), there is indirect evidence that adult MNs possess them as well (Corona and Tapia, 2007, Darman et al., 2004, Tateno et al., 2004, Williams et al., 1997, Yin et al., 2007), and it is likely that their presence plays a role in the high susceptibility of MNs to excitotoxic injury. Past studies concerning effects of TNF-α on MNs, and consequences for disease have mixed results. One recent study found spinal cord trauma to cause a rapid appearance of Ca-perm AMPAr on neurons via a TNF-α dependent mechanism, which contributed to the resultant injury (Ferguson et al., 2008). However, another study reported TNF-α to reduce numbers of Ca-perm AMPAr on cultured MNs (Rainey-Smith et al., 2010). Regarding possible roles in models of ALS, TNF-α appeared to contribute to MN loss in the wobbler mouse model (Bigini et al., 2008), but knock out of the TNF-α gene had no effect on disease progression in superoxide dismutase type 1 (SOD1) mutant mouse ALS models (Gowing et al., 2006).
The present study employs a histochemical stain based upon kainate-stimulated uptake of Co2 + ions (“Co2 + labeling”) to confirm the presence of functional Ca-perm AMPAr on adult ventral horn MNs in lumbar spinal cord slices, and examine effects of TNF-α on their numbers. We further make use of organotypic spinal cord slice cultures to determine effects of low levels of TNF-α on slow excitotoxic MN degeneration caused by subtoxic exposures to the glutamate uptake blocker, trans-pyrrolidine-2,4-dicarboxylic acid (PDC). Present results may be relevant to ALS or other conditions in which slow excitotoxicity and inflammation contribute to MN damage.
Section snippets
Animal tissue collection and handling
All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animal and approved by the University of California Irvine Institutional Care and Use committee.
Ca-perm AMPAr are present in spinal MNs from developing and adult rats
As discussed above, numerous studies have used techniques including kainate stimulated Co2 + uptake labeling (“Co2 + labeling”) or electrophysiological recording to demonstrate the presence of Ca-perm AMPAr on embryonic and neonatal MNs. Although it has been widely assumed that adult MNs are likely to possess these receptors as well, the evidence to date for their presence in adults is largely indirect, based upon measurements of relative AMPA subunit mRNA or protein levels (Shaw and Ince, 1997,
Summary of principal findings
In the present study, we use a well-established histochemical technique to directly demonstrate, for the first time, the presence of functional Ca-perm AMPAr on adult MNs, and find that acute TNF-α exposure induces a rapid increase in the numbers of these receptors in the neuronal membrane, via a signal transduction pathway that includes both PI3K and PKA. We further find that MNs in organotypic spinal cord slice cultures possess these Ca-perm AMPAr under basal conditions (after 10 days in
Conclusions
It is clear that the survival of MNs depends critically upon interrelationships with neighboring cells, and neuroinflammation, with astrocyte dysfunction, likely contributes to MN damage in a number of conditions including ALS. TNF-α is an important cytokine that accumulates in diseased spinal cord, and has complex effects that could well modulate MN survival. However, as discussed above, it has differing effects in different in vitro and animal models, and its net effects on MN survival in ALS
Acknowledgments
This work was supported by NIH grants NS36548 (JHW) and NS065219 (LSS), and a grant from the Muscular Dystrophy Association (JHW).
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