Atlastin-1 regulates endosomal tubulation and lysosomal proteolysis in human cortical neurons.

Mutation of the ATL1 gene is one of the most common causes of hereditary spastic paraplegia (HSP), a group of genetic neurodegenerative conditions characterised by distal axonal degeneration of the corticospinal tract axons. Atlastin-1, the protein encoded by ATL1, is one of three mammalian atlastins, which are homologous dynamin-like GTPases that control endoplasmic reticulum (ER) morphology by fusing tubules to form the three-way junctions that characterise ER networks. However, it is not clear whether atlastin-1 is required for correct ER morphology in human neurons and if so what the functional consequences of lack of atlastin-1 are. Using CRISPR-inhibition we generated human cortical neurons lacking atlastin-1. We demonstrate that ER morphology was altered in these neurons, with a reduced number of three-way junctions. Neurons lacking atlastin-1 had longer endosomal tubules, suggestive of defective tubule fission. This was accompanied by reduced lysosomal proteolytic capacity. As well as demonstrating that atlastin-1 is required for correct ER morphology in human neurons, our results indicate that lack of a classical ER-shaping protein such as atlastin-1 may cause altered endosomal tubulation and lysosomal proteolytic dysfunction. Furthermore, they strengthen the idea that defective lysosome function contributes to the pathogenesis of a broad group of HSPs, including those where the primary localisation of the protein involved is not at the endolysosomal system.

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment.

The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.

ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic.

Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER-endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic.

Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia.

Many genetic neurological disorders exhibit variable expression within affected families, often exemplified by variations in disease age at onset. Epistatic effects (i.e. effects of modifier genes on the disease gene) may underlie this variation, but the mechanistic basis for such epistatic interactions is rarely understood. Here we report a novel epistatic interaction between SPAST and the contiguous gene DPY30, which modifies age at onset in hereditary spastic paraplegia, a genetic axonopathy. We found that patients with hereditary spastic paraplegia caused by genomic deletions of SPAST that extended into DPY30 had a significantly younger age at onset. We show that, like spastin, the protein encoded by SPAST, the DPY30 protein controls endosomal tubule fission, traffic of mannose 6-phosphate receptors from endosomes to the Golgi, and lysosomal ultrastructural morphology. We propose that additive effects on this pathway explain the reduced age at onset of hereditary spastic paraplegia in patients who are haploinsufficient for both genes.

AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network.

The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis, and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function, and transport via regulating the export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome buildup are highly reminiscent of those observed in Alzheimer's disease as well as in neurons modeling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4-deficient HSP. Additionally, our results also demonstrate the utility of the human i3Neuronal model system in investigating neuronal phenotypes observed in AP-4-deficient mice and/or the human AP-4 deficiency syndrome.

HIF1α-dependent mitophagy facilitates cardiomyoblast differentiation.

Mitophagy is thought to play a key role in eliminating damaged mitochondria, with diseases such as cancer and neurodegeneration exhibiting defects in this process. Mitophagy is also involved in cell differentiation and maturation, potentially through modulating mitochondrial metabolic reprogramming. Here we examined mitophagy that is induced upon iron chelation and found that the transcriptional activity of HIF1α, in part through upregulation of BNIP3 and NIX, is an essential mediator of this pathway in SH-SY5Y cells. In contrast, HIF1α is dispensable for mitophagy occurring upon mitochondrial depolarisation. To examine the role of this pathway in a metabolic reprogramming and differentiation context, we utilised the H9c2 cell line model of cardiomyocyte maturation. During differentiation of these cardiomyoblasts, mitophagy increased and required HIF1α-dependent upregulation of NIX. Though HIF1α was essential for expression of key cardiomyocyte markers, mitophagy was not directly required. However, enhancing mitophagy through NIX overexpression, accelerated marker gene expression. Taken together, our findings provide a molecular link between mitophagy signalling and cardiomyocyte differentiation and suggest that although mitophagy may not be essential per se, it plays a critical role in maintaining mitochondrial integrity during this energy demanding process.

Mammalian mitophagy - from in vitro molecules to in vivo models.

The autophagic turnover of mitochondria, termed mitophagy, is thought to play an essential role in not only maintaining the health of the mitochondrial network but also that of the cell and organism as a whole. We have come a long way in identifying the molecular components required for mitophagy through extensive in vitro work and cell line characterisation, yet the physiological significance and context of these pathways remain largely unexplored. This is highlighted by the recent development of new mouse models that have revealed a striking level of variation in mitophagy, even under normal conditions. Here, we focus on programmed mitophagy and summarise our current understanding of why, how and where this takes place in mammals.

Cysteamine (Lynovex®), a novel mucoactive antimicrobial & antibiofilm agent for the treatment of cystic fibrosis.

BACKGROUND: There remains a critical need for more effective, safe, long-term treatments for cystic fibrosis (CF). Any successful therapeutic strategy designed to combat the respiratory pathology of this condition must address the altered lung physiology and recurrent, complex, polymicrobial infections and biofilms that affect the CF pulmonary tract. Cysteamine is a potential solution to these unmet medical needs and is described here for the first time as (Lynovex®) a single therapy with the potential to deliver mucoactive, antibiofilm and antibacterial properties; both in oral and inhaled delivery modes. Cysteamine is already established in clinical practice for an unrelated orphan condition, cystinosis, and is therefore being repurposed (in oral form) for cystic fibrosis from a platform of over twenty years of safety data and clinical experience. METHODS: The antibacterial and antibiofilm attributes of cysteamine were determined against type strain and clinical isolates of CF relevant pathogens using CLSI standard and adapted microbiological methods and a BioFlux microfluidic system. Assays were performed in standard nutrient media conditions, minimal media, to mimic the low metabolic activity of microbes/persister cells in the CF respiratory tract and in artificial sputum medium. In vivo antibacterial activity was determined in acute murine lung infection/cysteamine nebulisation models. The mucolytic potential of cysteamine was assessed against DNA and mucin in vitro by semi-quantitative macro-rheology. In all cases, the 'gold standard' therapeutic agents were employed as control/comparator compounds against which the efficacy of cysteamine was compared. RESULTS: Cysteamine demonstrated at least comparable mucolytic activity to currently available mucoactive agents. Cysteamine was rapidly bactericidal against both metabolically active and persister cells of Pseudomonas aeruginosa and also emerging CF pathogens; its activity was not sensitive to high ionic concentrations characteristic of the CF lung. Cysteamine prevented the formation of, and disrupted established P. aeruginosa biofilms. Cysteamine was synergistic with conventional CF antibiotics; reversing antibiotic resistance/insensitivity in CF bacterial pathogens. CONCLUSIONS: The novel mucolytic-antimicrobial activity of cysteamine (Lynovex®) provides potential for a much needed new therapeutic strategy in cystic fibrosis. The data we present here provides a platform for cysteamine's continued investigation as a novel treatment for this poorly served orphan disease.