Functional characterization of prohibitins by conditional inactivation in the mouse

Prohibitins comprise an evolutionary conserved and ubiquitously expressed family of membrane proteins implicated in a large variety of cellular processes. Large assemblies of PHB1 and PHB2 subunits are localized in the inner membrane of mitochondria, but various roles in other cellular compartments have also been proposed for both proteins. However, the function of prohibitins on the molecular level remains unclear. To investigate the physiological role of mammalian prohibitins, a mouse strain for the conditional, Cre/loxP-mediated inactivation of Phb2 was generated. Ubiquitous and brain-restricted deletion of Phb2 in vivo caused embryonic lethality indicating an essential role of Phb2 in mammalian development. To determine cellular functions of PHB2, a cell culture system was established allowing to define functional consequences of a Phb2 deletion. Mouse embryonic fibroblasts (MEFs) isolated from Phb2fl/fl embryos were transduced with cell-permeable Cre-recombinase to inactivate Phb2. Functional interdependence of prohibitin subunits was observed in MEFs after PHB2 depletion, illustrating the physiological relevance of the assembled prohibitin complex. The absence of prohibitins in MEFs leads to impaired cell proliferation and increased sensitivity towards apoptotic stimuli. This is accompanied by fragmentation of the mitochondrial network and defective morphogenesis of mitochondrial cristae. Complementation experiments attribute these defects to the loss of mitochondria-localized prohibitins indicating an essential requirement of the mitochondrial prohibitin complex in these processes. Loss of prohibitins affects the proteolytic cleavage of OPA1, a dynamin-like GTPase in the inner membrane essential for mitochondrial fusion, leading to the selective loss of long isoforms of OPA1. The specific expression of a long OPA1 isoform in prohibitin-deficient MEFs restores cristae morphogenesis, apoptotic resistance and partially cell proliferation, identifying impaired OPA1 processing as the primary cause for the cellular defects in the absence of prohibitins. These results identify a novel roles for mitochondrial prohibitins in cell proliferation and cristae formation by the proteolytic modulation of OPA1, suggesting a molecular interplay between cell growth and organelle morphogenesis

Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-κB (NF-κB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8 mice), which is a deubiquitinating enzyme that is integral to NF-κB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-κB proteins p100 and RelB in CYLDex7/8 B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants

Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics

TRPV1 pain receptors regulate longevity and metabolism by neuropeptide signaling.

The sensation of pain is associated with increased mortality, but it is unknown whether pain perception can directly affect aging. We find that mice lacking TRPV1 pain receptors are long-lived, displaying a youthful metabolic profile at old age. Loss of TRPV1 inactivates a calcium-signaling cascade that ends in the nuclear exclusion of the CREB-regulated transcriptional coactivator CRTC1 within pain sensory neurons originating from the spinal cord. In long-lived TRPV1 knockout mice, CRTC1 nuclear exclusion decreases production of the neuropeptide CGRP from sensory endings innervating the pancreatic islets, subsequently promoting insulin secretion and metabolic health. In contrast, CGRP homeostasis is disrupted with age in wild-type mice, resulting in metabolic decline. We show that pharmacologic inactivation of CGRP receptors in old wild-type animals can restore metabolic health. These data suggest that ablation of select pain sensory receptors or the inhibition of CGRP are associated with increased metabolic health and control longevity

TRPV1 pain receptors regulate longevity and metabolism by neuropeptide signaling.

The sensation of pain is associated with increased mortality, but it is unknown whether pain perception can directly affect aging. We find that mice lacking TRPV1 pain receptors are long-lived, displaying a youthful metabolic profile at old age. Loss of TRPV1 inactivates a calcium-signaling cascade that ends in the nuclear exclusion of the CREB-regulated transcriptional coactivator CRTC1 within pain sensory neurons originating from the spinal cord. In long-lived TRPV1 knockout mice, CRTC1 nuclear exclusion decreases production of the neuropeptide CGRP from sensory endings innervating the pancreatic islets, subsequently promoting insulin secretion and metabolic health. In contrast, CGRP homeostasis is disrupted with age in wild-type mice, resulting in metabolic decline. We show that pharmacologic inactivation of CGRP receptors in old wild-type animals can restore metabolic health. These data suggest that ablation of select pain sensory receptors or the inhibition of CGRP are associated with increased metabolic health and control longevity

Imbalanced OPA1 processing and mitochondrial fragmentation cause heart failure in mice

Mitochondrial morphology is shaped by fusion and division of their membranes. Here, we found that adult myocardial function depends on balanced mitochondrial fusion and fission, maintained by processing of the dynamin-like guanosine triphosphatase OPA1 by the mitochondrial peptidases YME1L and OMA1. Cardiac-specific ablation of Yme1l in mice activated OMA1 and accelerated OPA1 proteolysis, which triggered mitochondrial fragmentation and altered cardiac metabolism. This caused dilated cardiomyopathy and heart failure. Cardiac function and mitochondrial morphology were rescued by Oma1 deletion, which prevented OPA1 cleavage. Feeding mice a high-fat diet or ablating Yme1l in skeletal muscle restored cardiac metabolism and preserved heart function without suppressing mitochondrial fragmentation. Thus, unprocessed OPA1 is sufficient to maintain heart function, OMA1 is a critical regulator of cardiomyocyte survival, and mitochondrial morphology and cardiac metabolism are intimately linked

Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria

Prohibitins comprise an evolutionarily conserved and ubiquitously expressed family of membrane proteins with poorly described functions. Large assemblies of PHB1 and PHB2 subunits are localized in the inner membrane of mitochondria, but various roles in other cellular compartments have also been proposed for both proteins. Here, we used conditional gene targeting of murine Phb2 to define cellular activities of prohibitins. Our experiments restrict the function of prohibitins to mitochondria and identify the processing of the dynamin-like GTPase OPA1, an essential component of the mitochondrial fusion machinery, as the central cellular process controlled by prohibitins. Deletion of Phb2 leads to the selective loss of long isoforms of OPA1. This results in an aberrant cristae morphogenesis and an impaired cellular proliferation and resistance toward apoptosis. Expression of a long OPA1 isoform in PHB2-deficient cells suppresses these defects, identifying impaired OPA1 processing as the primary cellular defect in the absence of prohibitins. Our results therefore assign an essential function for the formation of mitochondrial cristae to prohibitins and suggest a coupling of cell proliferation to mitochondrial morphogenesis