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Regulation of dynamin-related protein 1-mediated mitochondrial fission by reversible phosphorylation and its contribution to neuronal survival following injurySlupe, Andrew Michael 01 May 2014 (has links)
Mitochondria are dynamic organelles that constantly undergo opposing fission and fusion events which impact many aspects of mitochondrial and cellular homeostasis including bioenergetic activity, calcium buffering and organelle transport. The large GTPase dynamin-related protein 1 (Drp1) acts as a mechanoenzyme to catalyze fission of mitochondria. Drp1 activity is regulated through a series of reversible posttranslational modifications. Phosphorylation of the conserved serine residue, S656, by cAMP dependent protein kinase A (PKA) acts as a master regulator of Drp1 activity. Two phosphatases oppose PKA by dephosporylating Drp1 S656, a mitochondrial isoform of protein phosphatase 2A and the calcium-calmodulin dependent phosphatase calcineurin (CaN). Here I report the characterization of a conserved CaN docking site on Drp1, an LxVP motif, just upstream of the Drp1 S656 site. Mutational modification of the Drp1 LxVP motif resulted in selective bidirectional modulation of formation of the CaN:Drp1 complex. Stability of the CaN:Drp1 LxVP motif mutant complexes was qualitatively described by affinity purification and quantitatively described by isothermal titration calorimetry. Stability of the CaN:Drp1 complex was found to directly correlate with Drp1 S656 dephosphorylation kinetics as demonstrated by studies conducted in vitro and in intact cells. Further, the CaN:Drp1 signaling axis was shown to shape basal mitochondrial morphology in a heterologous cell line system and in primary hippocampal neurons. Finally, disruption of the CaN:Drp1 signaling axis was found to protect neurons from oxygen-glucose deprivation, an in vitro model of ischemic injury. While these results suggest that the CaN:Drp1 signaling axis may be a potential target for neuroprotective therapeutic exploitation, the mechanism by which disruption of the CaN:Drp1 signaling axis specifically and mitochondrial elongation generally results in resistance to ischemic injury remains unknown.
Additional studies reported here demonstrate that mitochondrial fragmentation remains a prominent feature of injured neurons regardless of the fidelity of the CaN:Drp1 signaling axis. Mitochondrial fragmentation at the time of injury was found to occur in a Drp1-independent manner. Chronic mitochondrial elongation was also found to leave unaltered the ability of neurons to detoxify reactive oxygen species, buffer intracellular calcium and supply ATP for homeostatic function.
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Role of pp2a/bβ2 and pka/akap1 in brain development and function via dynamin-related protein 1 (drp1) control of mitochondria shape and bioenergeticsDickey, Audrey Sarah 01 December 2010 (has links)
Mitochondria are critical for energy production and Ca2+ homeostasis and undergo fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by Drp1 (dynamin-related protein 1), a large GTPase tightly controlled by various posttranslational modifications, including phosphorylation. Bβ2 is a neuron-specific postnatally induced protein phosphatase 2A (PP2A) regulatory subunit that mediates PP2A translocation to the outer mitochondrial membrane (OMM) to promote mitochondrial fragmentation and sensitize neurons to various injuries. Opposing PP2A/Bβ2's effect on mitochondrial morphology and cell death is protein kinase A (PKA) anchored to the OMM via A kinase anchoring protein 1 (AKAP1). This dissertation describes how reversible phosphorylation of Drp1 at a conserved Serine residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase complex (PP2A/Bβ2) affects dendrite and synapse development in hippocampal neurons and synaptic plasticity and learning and memory in vivo.
Inducing mitochondria fragmentation decreases dendritic arbor complexity, but increases spine and synapse number. Mitochondrial elongation induces opposite effects. L-carnitine increases mitochondria membrane potential and recapitulates the dendritic and synaptic effects of mitochondrial elongation. Epistasis experiments substantiate our hypothesis that PP2A/Bβ2 dephosphorylates and PKA/AKAP1 phosphorylates Drp1 to change mitochondrial shape and regulate mitochondria localization, dendrite outgrowth, and synapse development.
Bβ2 null mice are viable and fertile, without obvious abnormalities. Bβ2 null mice demonstrate significantly larger cortical and hippocampal neuronal mitochondria than in wildtype. Bβ2 deletion decreases spine number on apical and basal cortical dendrites and hippocampal dendrites. Bβ2 null mice display significantly decreased input/output relationship in the hippocampus, consistent with a decrease in synapse number. In a combined context and cued fear-conditioning protocol, the hippocampal-dependent context recall trial revealed significant deficits in Bβ2 null and heterozygous mice. This deficit is also seen in hippocampal-dependent Barnes maze performance. These results are consistent with the reduced hippocampal long-term potentiation (LTP) found in Bβ2 null mice and demonstrate the importance of Bβ2 in hippocampal synaptic plasticity and memory. In conclusion, PP2A/Bβ2 and PKA/AKAP1 have important roles in mitochondria regulation and dendritic and synaptic development as seen in our results in vitro with rat hippocampal cultures and in vivo with Bβ2 null mice.
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Understanding How O-GlcNAcylation and Phosphorylation Regulates the Mitochondrial Fission Machinery in GlioblastomaAkinbiyi, Elizabeth O. 25 January 2022 (has links)
No description available.
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