Mechanisms of neurodegeneration: How oxidative stress reduces proNGF axonal transport in basal forebrain cholinergic neurons

Profound and early basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Alzheimer’s disease. BFCNs depend for survival and function on retrograde axonal transport of neurotrophins like nerve growth factor (NGF). We have recently shown that the retrograde axonal transport of proNGF, the form of NGF found in the brain, is reduced with age in cultured BFCNs. ProNGF loss coincides with loss of its receptor, tropomyosin‐related kinase A (TrkA), while p75NTR levels remain unchanged. We sought to determine whether mechanisms related to oxidative stress, a longstanding hypothesized contributor to Alzheimer’s disease, account for these reductions. Embryonic rat BFCNs were cultured in microfluidic chambers for 6‐8 days. Neurons were incubated in antioxidant‐poor medium to induce oxidative stress. Thioredoxin‐1 and thioredoxin reductase were added in the rescue condition, or PTP1B was inhibited using the antagonist TCS401 or siRNA. All treatments were applied exclusively to the cell bodies. TrkA and p75NTR levels were determined by immunocytochemistry. Retrograde transport was assayed using quantum dot‐labelled proNGF added to the axon terminals. Axonal uptake of proNGF via TrkA was determined using a mutant proNGF that only binds to TrkA (proNGF‐KKE). We found that elevation of intracellular oxidative stress via antioxidant deprivation significantly reduced both TrkA immunoreactivity and proNGF retrograde transport in rat BFCNs cultured in microfluidic chambers. TrkA levels are partially regulated by protein tyrosine phosphatase 1B (PTP1B), an enzyme whose activity is reduced by the oxidation of a cysteine residue within its active site. siRNA knockdown of PTP1B and pharmacological PTP1B antagonism via selective inhibitor TCS401 significantly reduced TrkA levels in BFCNs. Furthermore, TCS401 treatment reduced proNGF retrograde axonal transport in BFCNs and reduced axonal uptake of proNGF‐KKE, a mutant form of proNGF that only binds TrkA. Finally, treatment of BFCNs with thioredoxin‐1, an antioxidant responsible for the reactivation of oxidized PTP1B, significantly increased TrkA levels in BFCNs following antioxidant deprivation. Our results suggest that, during aging and in Alzheimer’s disease, increased oxidative stress reduces TrkA levels and proNGF transport through a PTP1B‐dependent mechanism. Impaired proNGF retrograde transport due to TrkA loss may contribute to BFCN degeneration in Alzheimer’s disease.