Elena I. Rugarli

Key Points: Lipocalin-2 inhibited clustered mitochondrial homolog (CLUH) activity and caused mitochondrial dysfunction. Loss of CLUH in kidneys triggered metabolic reprogramming and impaired mitochondrial structure. CLUH expression decreased with kidney injury and correlated with kidney function decline in patients. Background: Lipocalin-2 (LCN2) is a secreted protein involved in transporting hydrophobic molecules, regulating antibacterial responses and iron homeostasis. LCN2 expression increases after kidney damage and participates in CKD onset. We previously identified LCN2 as a regulator of mitochondrial dysfunction in renal tubular cells. This study aims to better understand LCN2's role in mitochondrial dysfunction and kidney pathology. Methods: LCN2 interactome was studied using mass spectrometry. The impact of LCN2 on clustered mitochondrial homolog (CLUH) activity was explored by immunofluorescence, Western blot, quantitative PCR, and RNA-immunoprecipitation. The role of CLUH on mitochondrial function in kidney was evaluated by RNAseq, metabolomics, electron microscopy, and immunochemistry using transgenic mice. Results: We characterized LCN2 interactome and identified CLUH in a multiprotein complex with LCN2. CLUH regulates mitochondrial homeostasis by binding mRNAs of nuclear-encoded mitochondrial proteins and promoting their translation. We demonstrated that LCN2 inhibited CLUH activity, leading to perinuclear mitochondrial clustering. We showed that CLUH was expressed in renal tubular cells and that its expression and activity decreased, while LCN2 expression increased after kidney injury leading to CKD. In human CKD patients, CLUH expression was inversely correlated with kidney lesions and kidney failure. CLUH genetic deletion in kidney nephron induced mitochondrial dysfunction and a subsequent interstitial fibrosis and kidney loss of function. Conclusions: This study shows that LCN2 forms a complex with CLUH and inhibits its activity, leading to perinuclear mitochondrial clustering. CLUH loss of function in renal tubular cells is associated with mitochondrial dysfunction and fibrosis, and in humans, CLUH expression inversely correlates with kidney lesions and loss of function.

Mitochondria are dynamic and heterogeneous organelles that rewire their network and metabolic functions in response to changing cellular needs. To this end, mitochondria integrate a plethora of incoming signals to influence cell fate and survival. A crucial and highly regulated node of cell–mitochondria communication is the translation of nuclear-encoded mitochondrial mRNAs. By controlling and monitoring the spatio-temporal translation of these mRNAs, cells can rapidly adjust mitochondrial function to meet metabolic demands, optimise ATP production and regulate organelle biogenesis and turnover. In this Review, we focus on how RNA-binding proteins that recognise nuclear-encoded mitochondrial mRNAs acutely modulate the rate of translation in response to nutrient availability. We further discuss the relevance of localised translation of these mRNAs for subsets of mitochondria in polarised cells. Finally, we highlight quality control mechanisms that monitor the translation process at the mitochondrial surface and their connections to mitophagy and stress responses. We propose that these processes collectively contribute to mitochondrial specialisation and signalling function.

Neurons integrate perception, motor, and cognitive functions, relying heavily on mitochondria for ATP production to support axonal transport, synaptic activity, and maintenance of membrane potential. Mitochondrial dysfunction disrupts energy supply, increases oxidative stress, and impairs calcium buffering, contributing to axonal degeneration in neurodegenerative diseases. Mitochondria are strategically located and transported within highly polarised neurons, posing the problem of maintaining functional organelles in axonal and dendritic regions far away from the cell body. This article highlights mechanisms that ensure the quality of the mitochondrial proteome in neurons.

Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigate the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KOs in young mice show no major phenotypes; however, combined Fam134b and Fam134c deletion ( Fam134b/c ^dKO ) , but not the combination including Fam134a deletion, leads to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/c ^dKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/c ^dKO mice exhibit expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities are present in cortical ER. Our study unveils the critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.

Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.

Complexes of ERLIN1 and ERLIN2 (ER lipid raft–associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain–containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.

Complexes of ERLIN1 and ERLIN2 form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3-ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 and RNF170. We identify a luminal N-terminal conserved region in TMUB1 and RNF170 required for this interaction. Three-dimensional modelling shows that this conserved motif binds the SPFH domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia (HSP). By using omics approaches in combination with phenotypic characterisation of cells lacking both ERLINs, we demonstrate a role for ERLIN scaffolds in maintaining cholesterol levels in the ER by favouring transport to the Golgi over esterification, thereby regulating Golgi morphology and the secretory pathway.

Transport and local translation of mRNAs in distal axonal compartments are essential for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects mitochondria from damage during their long journey along the axon, however the regulatory factors involved are largely unknown. Here, we show that CLUH, a cytosolic protein that binds mRNAs encoding mitochondrial proteins, is essential for preventing axonal degeneration of spinal motoneurons and maintaining motor behavior in the mouse. We demonstrate that CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The absence of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits specifically in the growth cone. CLUH binds ribosomal subunits, translation initiation and ribosome recycling components, and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the growth cone and translation defects in CLUH-deficient motoneurons. In conclusion, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which are essential for their development and long-term integrity and function.

Hereditary spastic paraplegia is a neurological condition characterized by predominant axonal degeneration in long spinal tracts, leading to weakness and spasticity in the lower limbs. The NAD ⁺ -consuming enzyme SARM1 has emerged as a key executioner of axonal degeneration upon nerve transection and in some neuropathies. An increase in the nicotinamide mononucleotide/NAD⁺ ratio activates SARM1, causing catastrophic NAD⁺ depletion and axonal degeneration. However, the role of SARM1 in the pathogenesis of hereditary spastic paraplegia has not been investigated. Here, we report an enhanced mouse model for hereditary spastic paraplegia caused by mutations in SPG7. eSpg7 knock-out mice carry a deletion in both Spg7 and Afg3l1, a redundant homologue expressed in mice but not in humans. eSpg7 knock-out mice recapitulate the phenotypic features of human patients, showing progressive symptoms of spastic-ataxia and degeneration of axons in the spinal cord as well as the cerebellum. We show that the lack of SPG7 rewires the mitochondrial proteome in both tissues, leading to an early onset decrease in mitoribosomal subunits and a remodelling of mitochondrial solute carriers and transporters. To interrogate mechanisms leading to axonal degeneration in this mouse model, we explored the involvement of SARM1. Deletion of SARM1 delays the appearance of ataxic signs, rescues mitochondrial swelling and axonal degeneration of cerebellar granule cells and dampens neuroinflammation in the cerebellum. The loss of SARM1 also prevents endoplasmic reticulum abnormalities in long spinal cord axons, but does not halt the degeneration of these axons. Our data thus reveal a neuron-specific interplay between SARM1 and mitochondrial dysfunction caused by lack of SPG7 in hereditary spastic paraplegia.

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multiproteomic approach to demonstrate the regulation of the m‐AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca²⁺/H⁺ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m‐AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca²⁺ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m‐AAA protease. The m‐AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca²⁺ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.