Functional analysis of mammalian m-AAA proteases

The m-AAA protease, an ATP-dependent proteolytic complex in the inner mitochondrial membrane, controls mitochondrial protein quality and, acting as a processing enzyme, regulates mitochondrial protein synthesis in yeast. Mammalian m-AAA proteases assemble into several isoenzymes with variable subunit composition. In mice, the three different subunits paraplegin, Afg3l1 and Afg3l2, are expressed in a tissue-specific manner. Loss-of-function mutations in the m-AAA protease subunit paraplegin cause the neurodegenerative disease hereditary spastic paraplegia (HSP) which is mainly characterized by a cell-specific axonal degeneration. Similarly, Afg3l2 mutant mice exhibit neuropathological features with a severe defect in axonal development. The molecular basis of these neuron-specific phenotypes as well as cellular functions of mammalian m-AAA proteases in general remain unclear and are studied within this thesis. Using an HSP mouse model lacking paraplegin, a liver-specific mitochondrial translation defect was observed which is consistent with a functional conservation of the m-AAA protease-dependent control of mitochondrial protein synthesis in mammals. However, a significant impairment of mitochondrial protein synthesis and mitochondrial respiration in brain and spinal cord was not observed suggesting that axonal degeneration in HSP due to a loss of paraplegin occurs in the absence of a general respiratory dysfunction. The analysis of m-AAA isoenzymes on a cellular level using RNA interference revealed redundant functions of the subunits Afg3l1 and Afg3l2 and identified paraplegin as a new substrate which is processed by Afg3l1 and Afg3l2. Depletion of the m-AAA protease in MEFs resulted in mitochondrial fragmentation accompanied by an impaired biogenesis of OPA1, an essential component of the mitochondrial fusion machinery. Long OPA1 isoforms were destabilized and cleaved in an accelerated manner. The overexpression of a non-cleavable long OPA1 isoform in m-AAA protease-depleted cells restored the tubular mitochondrial network identifying impaired OPA1 processing as the primary cause for the mitochondrial morphology defect. Furthermore, the m-AAA protease was found to be essential for inducing mitochondrial hyperfusion, a cellular stress response which results in a highly interconnected mitochondrial network. The findings of this study indicate that the m-AAA protease has crucial functions in the regulation of mitochondrial morphology by controlling the biogenesis of OPA1 isoforms which may provide new insights into the molecular mechanisms of axonopathies caused by the loss of m-AAA protease subunits in mammals

Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1

m-AAA proteases cleave OPA1 to ensure a balance of long and short OPA1 isoforms, whereas cleavage by OMA1 causes an accumulation of the short OPA1 variants. (See also companion paper from Head et al. in this issue.

Free Fatty Acids Induce a Proinflammatory Response in Islets via the Abundantly Expressed Interleukin-1 Receptor I

Islets of patients with type 2 diabetes mellitus (T2DM) display features of an inflammatory process including elevated levels of the cytokine IL-1beta, various chemokines, and macrophages. IL-1beta is a master regulator of inflammation, and IL-1 receptor type I (IL-1RI) blockage improves glycemia and insulin secretion in humans with T2DM and in high-fat-fed mice pointing to a pivotal role of IL-1RI activity in intra-islet inflammation. Given the association of dyslipidemia and T2DM, we tested whether free fatty acids (FFA) promote the expression of proinflammatory factors in human and mouse islets and investigated a role for the IL-1RI in this response. A comparison of 22 mouse tissues revealed the highest IL-1RI expression levels in islets and MIN6 beta-cells. FFA induced IL-1beta, IL-6, and IL-8 in human islets and IL-1beta and KC in mouse islets. Elevated glucose concentrations enhanced FFA-induced proinflammatory factors in human islets. Blocking the IL-1RI with the IL-1R antagonist (IL-1Ra) strongly inhibited FFA-mediated expression of proinflammatory factors in human and mouse islets. Antibody inhibition of IL-1beta revealed that FFA stimulated IL-1RI activity via the induction of the receptor ligand. FFA-induced IL-1beta and KC expression in mouse islets was completely dependent on the IL-1R/Toll-like receptor (TLR) docking protein Myd88 and partly dependent on TLR2 and -4. Activation of TLR2 in purified human beta-cells and islets stimulated the expression of proinflammatory factors, and IL-1RI activity increased the TLR2 response in human islets. We conclude that FFA and TLR stimulation induce proinflammatory factors in islets and that IL-1RI engagement results in signal amplification

SLP-2 is required for stress-induced mitochondrial hyperfusion

Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin-related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress-induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L-OPA1, MFN1, and the mitochondrial inner membrane protein SLP-2. In the absence of SLP-2, L-OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro-survival response against stress

Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits

Proceedings of the OHBM Brainhack 2022

International audienceOHBM Brainhack 2022 took place in June 2022. The first hybrid OHBM hackathon, it had an in-person component taking place in Glasgow and three hubs around the globe to improve inclusivity and fit as many timezones as possible. In the buzzing setting of the Queen Margaret Union and of the virtual platform, 23 projects were presented for development. Following are the reports of 14 of those, as well as a recapitulation of the organisation of the event