In Alzheimer's disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/ tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-␤ (A␤) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or A␤ peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.

Cultured neurons tolerate low H(2)O(2) concentrations (< or =50 microM) through the activity of constitutive antioxidant response elements (ARE). At H(2)O(2) levels (> or =100 microM), neurons increase expression of the gene encoding for inducible hemoxygenase-1 while superoxide dismutase-2 and catalase remain unchanged. Despite this adaptive response, the endogenous antioxidant systems are overwhelmed, leading to decreased viability. Elevating the neuronal cell content of human neuroglobin (Ngb) prior to insult with 100 or 200 microM H(2)O(2) enhanced cell viability and this resulted in a significant decrease in oxidative stress and an increase in the intracellular ATP concentration, whereas in parental cells exposed to the same H(2)O(2)-insult, oxidative stress and ATP increased and decreased, respectively. The mechanism for this increase in ATP involves sustained activation of the mito-K(ATP) channel and an increase in phosphoinositide-3 kinase (PI3K)-mediated phosphorylation of Akt. Pharmacological inhibitors directed toward PI3K (wortmannin and LY294002), or the mito-K(ATP) channel (glybenclamide) inhibited the H(2)O(2)-mediated increase in ATP in cells overexpressing human Ngb and consequently cell viability decreased. Neuroglobin's ability to bolster the intracellular pool of ATP in response to added H(2)O(2) is central to the preservation of cytoskeletal integrity and cell viability.

1These authors contributed equally to this study.Abbreviations used: GAP-43, growth-associated protein-43; H/R, hypoxia-reoxygenation; Nb, neuroglobin; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TPA, 12-o-tetradecanoylphorbol-13-acetate; XRF, X-ray fluorescence. AbstractOxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. We have cloned a human neuroglobin (Nb) construct and over-expressed this protein in cultured human neuronal cells to assess whether Nb ameliorates the cellular response to experimental hypoxia-reoxygenation (H/R) injury. Parental cells transfected with a blank (pDEST40) vector responded to H/R injury with a significant decrease in cellular ATP at 5 and 24 h after insult. This was coupled with increases in the cytosolic Ca 2+, and the transition metals iron (Fe), copper (Cu), and zinc (Zn) within the cell body, as monitored simultaneously using X-ray fluorescence microprobe imaging. Parental cell viability decreased over the same time period with a 4 to 5-fold increase in cell death (maximum 25%) matched by an increase in caspase 3/7 activation (peaking at a 15-fold increase after 24 h) and condensation of b-actin along axonal processes. Over-expression of Nb inhibited ATP loss and except for significant decreases in the sulfur (S), chlorine (Cl), potassium (K) and Ca 2+ contents, maintained cellular ion homeostasis after H/R insult. This resulted in increased cell viability, significantly diminished caspase activation and maintenance of the b-actin cytoskeletal structure and receptor-mediated endocytosis. These data indicate that bolstering the cellular content of Nb inhibits neuronal cell dysfunction promoted by H/R insult through multiple protective actions including: (i) maintenance of cellular bioenergetics; (ii) inhibition of Ca 2+ influx; (iii) a reduction in cellular uptake of Fe, Cu and Zn at the expense of S, Cl and K; and (iv) an enhancement of cell viability through inhibiting necrosis and apoptosis.