BDNF heightens the sensitivity of motor neurons to excitotoxic insults through activation of TrkB

Abstract

The survival promoting and neuroprotective actions of brain-derived neurotrophic factor (BDNF) are well known but under certain circumstances this growth factor can also exacerbate excitotoxic insults to neurons. Prior exploration of the receptor through which BDNF exerts this action on motor neurons deflects attention away from p75. Here we investigated the possibility that BDNF acts through the receptor tyrosine kinase, TrkB, to confer on motor neurons sensitivity to excitotoxic challenge. We blocked BDNF activation of TrkB using a dominant negative TrkB mutant or a TrkB function blocking antibody, and found that this protected motor neurons against excitotoxic insult in cultures of mixed spinal cord neurons. Addition of a function blocking antibody to BDNF to mixed spinal cord neuron cultures is also neuroprotective indicating that endogenously produced BDNF participates in vulnerability to excitotoxicity. We next examined the intracellular signaling cascades that are engaged upon TrkB activation. Previously we found that inhibition of the phosphatidylinositide-3¢-kinase (PI3¢K) pathway blocks BDNF-induced excitotoxic sensitivity. Here we show that expression of a constitutively active catalytic subunit of PI3¢K, p110, confers excitotoxic sensitivity (ES) upon motor neurons not incubated with BDNF. Parallel studies with purified motor neurons confirm that these events are likely to be occuring specifically within motor neurons. The abrogation of BDNF's capacity to accentuate excitotoxic insults may make it a more attractive neuroprotective agent.

Protecting Motor Neurons from Toxic Insult by Antagonism of Adenosine A2a and Trk Receptors

“…Many previous in vitro investigations have identified motor neurons in mixed spinal cord cell culture by immunostaining for nonphosphorylated neurofilaments (or peripherin) and restricting analysis to cells with a soma diameter of 25 m or greater (Carriedo et al, 2000;Hu and Kalb, 2003). We wanted to confirm the validity of this approach using an independent method for determining whether a given cell is a motor neuron.…”

“…All other chemicals were obtained from Sigma (St. Louis, MO) and were of the highest possible grade. The source of tissue culture materials has been described previously (Hu and Kalb, 2003). KW6002 was a gift from Jacques Petzer (University of Witwatersrand, Johannesburg, South Africa).…”

“…Embryonic Sprague Dawley rat spinal cord neurons were grown on previously established cortical astrocyte monolayers as described previously (Hu and Kalb, 2003). Briefly, the cerebral cortex of postnatal day 1-3 (P1-P3) rat pups was dissociated and plated on tissue culture plastic (Primaria, Falcon, Becton Dickinson) or laminin/polylysine-coated glass coverslips and maintained in minimal essential media (10% horse serum, 10% fetal bovine serum) and 10 M AraC was added for 1 d to arrest additional proliferation when confluent.…”

“…The media was removed (and saved), cells were exposed to 100 M kainic acid or vehicle in Locke's buffer for 1 h, washed 3ϫ in Locke's buffer not containing kainic acid, and the original media restored to each dish (Hu and Kalb, 2003). Twenty-four hours later, dishes were fixed in 4% paraformaldehyde, washed extensively, and immunostained.…”

“…Previous work demonstrated that the vulnerability of motor neurons to excitotoxic insult in vitro is promoted by the peptide growth factor BDNF and is mediated by the receptor tyrosine kinase B (TrkB) (Fryer et al, 2000;Hu and Kalb, 2003). Although this is counter-intuitive, because BDNF-TrkB signaling is essential for survival of populations of developing neurons (Jones et al, 1994;Ernfors et al, 1995;Liu et al, 1995;Schwartz et al, 1997;Silos-Santiago et al, 1997), an additional prodeath activity of BDNF has also been observed in a variety of in vitro stressedneuron paradigms (Koh et al, 1995;Ishikawa et al, 2000;Kim et al, 2003).…”