Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased mitochondrial delivery to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1-light chain-3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modeling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an “eat-me” signal for the elimination of damaged mitochondria from neuronal cells.

This paper describes the synthesis, crystal structure, and physicomechanical properties of a biobased polyester prepared from 2,5-furandicarboxylic acid (FDCA) and 1,4-butanediol. Melt-polycondensation experiments were conducted by a two-stage polymerization using titanium tetraisopropoxide (Ti[OiPr] 4 ) as a catalyst. Polymerization conditions (catalyst concentration, reaction time and second stage reaction temperature) were varied to optimize poly(butylene-FDCA), PBF, and molecular weight. A series of PBFs with different M w were characterized by DSC, TGA, DMTA, X-ray diffraction and tensile testing. Influence of molecular weight and melting/ crystallization enthalpy on PBF material tensile properties was explored. Cold-drawing tensile tests at room temperature for PBF with M w 16K to 27K showed a brittle-to-ductile transition. When M w reaches 38K, the Young modulus of PBF remains above 900 MPa, and the elongation at break increases to above 1000%. The mechanical properties, thermal properties and crystal structures of PBF were similar to petroleum derived poly(butylenes-terephthalate), PBT. Fiber diagrams of uniaxially stretched PBF films were collected, indexed, and the unit cell was determined as triclinic (a = 4.78(3) Å, b = 6.03(5) Å, c = 12.3(1) Å, α = 110.1(2)°, β = 121.1(3)°, γ = 100.6(2)°). A crystal structure was derived from this data and final atomic coordinates are reported. We concluded that there is a close similarity of the PBF structure to PBT αand β-forms.

induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.

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The recent discovery of direct interactions between two important regulators of cell fate, the tumor suppressor p53 and glycogen synthase kinase-3␤ (GSK3␤), led us to examine the mechanism and outcomes of this interaction. Two regions of p53 were identified that regulate its binding to GSK3␤. Deletion of the p53 activation domain-1 (AD1), but not mutations that prevent MDM2 binding through the AD1 domain, enhanced GSK3␤ binding to p53, indicating that the AD1 domain interferes with p53 binding to GSK3␤. Deletion of the p53 basic domain (BD) abrogated GSK3␤ binding, and a ten amino acid region within the C-terminal BD domain was identified as necessary for binding to GSK3␤. GSK3␤ activity was not required for p53 binding, but inhibition of GSK3␤ stabilized the association, suggesting a transient interaction during which active GSK3␤ promotes actions of p53. This regulatory role of GSK3␤ was demonstrated by large reductions of p53-induced increases in the levels of MDM2, p21, and Bax when GSK3␤ was inhibited. Besides promoting p53-mediated transcription, GSK3␤ also contributed to mitochondrial p53 apoptotic signaling. After DNA damage, mitochondrial GSK3␤ co-immunoprecipitated with p53 and was activated, and inhibition of GSK3␤ blocked cytochrome c release and caspase-3 activation. Thus, GSK3␤ interacts with p53 in both the nucleus and mitochondria and promotes its actions at both sites.