Parkinsonism Sac domain mutation in Synaptojanin-1 affects ciliary properties in iPSC-derived dopaminergic neurons.

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4, 5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting a defect in the clearing of ubiquitinated proteins at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.

ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport.

AIMS: Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in patients with pulmonary arterial hypertension (PAH). ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. In this study, we sought to determine the biological function of ATP13A3 in vascular endothelial cells (ECs) and how PAH-associated variants may contribute to disease pathogenesis. METHODS AND RESULTS: We studied the impact of ATP13A3 deficiency and overexpression in EC models [human pulmonary ECs, blood outgrowth ECs (BOECs), and human microvascular EC 1], including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. ATP13A3 localized to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation, and increased monolayer permeability to thrombin. The assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, and L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germline Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling, and muscularization of pulmonary vessels. CONCLUSION: We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, a deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH.

The lipid flippase ATP10B enables cellular lipid uptake under stress conditions.

Pathogenic ATP10B variants have been described in patients with Parkinson's disease and dementia with Lewy body disease, and we previously established ATP10B as a late endo-/lysosomal lipid flippase transporting both phosphatidylcholine (PC) and glucosylceramide (GluCer) from the lysosomal exoplasmic to cytoplasmic membrane leaflet. Since several other lipid flippases regulate cellular lipid uptake, we here examined whether also ATP10B impacts cellular lipid uptake. Transient co-expression of ATP10B with its obligatory subunit CDC50A stimulated the uptake of fluorescently (NBD-) labeled PC in HeLa cells. This uptake is dependent on the transport function of ATP10B, is impaired by disease-associated variants and appears specific for NBD-PC. Uptake of non-ATP10B substrates, such as NBD-sphingomyelin or NBD-phosphatidylethanolamine is not increased. Remarkably, in stable cell lines co-expressing ATP10B/CDC50A we only observed increased NBD-PC uptake following treatment with rotenone, a mitochondrial complex I inhibitor that induces transport-dependent ATP10B phenotypes. Conversely, Im95m and WM-115 cells with endogenous ATP10B expression, present a decreased NBD-PC uptake following ATP10B knockdown, an effect that is exacerbated under rotenone stress. Our data show that the endo-/lysosomal lipid flippase ATP10B contributes to cellular PC uptake under specific cell stress conditions.

Parkinsonism Sac domain mutation in Synaptojanin-1 affects ciliary properties in iPSC-derived dopaminergic neurons.

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4,5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting an impaired clearing of proteins from cilia which may result from an endocytic defect at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.

Enhancing the Value of Surgical Entrustable Professional Activities through Integrative Learning Analytics.

OBJECTIVE: To demonstrate the value of integrating surgical resident Entrustable Professional Activity (EPA) data into a learning analytics platform that provides meaningful feedback for formative and summative decision-making. DESIGN: Description of the Surgical Entrustable Professional Activities (SEPA) analytics dashboard, and examples of summary analytics and intuitive display features. SETTING: Department of Surgery, University of Wisconsin Hospital and Clinics. PARTICIPANTS: Surgery residents, faculty, and residency program administrators. RESULTS: We outline the major functionalities of the SEPA dashboard and offer concrete examples of how these features are utilized by various stakeholders to support progressive entrustment decisions for surgical residents. CONCLUSIONS: Our intuitive analytics platform allows for seamless integration of SEPA microassessment data to support Clinical Competency Committee (CCC) decisions for resident evaluation and provides point of training feedback to faculty and trainees in support of progressive autonomy.

ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization.

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson's disease and Kufor-Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

Polyamine Transport Assay Using Reconstituted Yeast Membranes.

ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020 ) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.

ATP13A3 is a major component of the enigmatic mammalian polyamine transport system.

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.

ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress.

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6 These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Necroptosis in Immuno-Oncology and Cancer Immunotherapy.

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.

Mutated ATP10B increases Parkinson's disease risk by compromising lysosomal glucosylceramide export.

Parkinson's disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of α-synuclein-positive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD patients and identified 3 carriers of compound heterozygous mutations in the ATP10B P4-type ATPase gene. Genetic screening of a Belgian PD and dementia with Lewy bodies (DLB) cohort identified 4 additional compound heterozygous mutation carriers (6/617 PD patients, 0.97%; 1/226 DLB patients, 0.44%). We established that ATP10B encodes a late endo-lysosomal lipid flippase that translocates the lipids glucosylceramide (GluCer) and phosphatidylcholine (PC) towards the cytosolic membrane leaflet. The PD associated ATP10B mutants are catalytically inactive and fail to provide cellular protection against the environmental PD risk factors rotenone and manganese. In isolated cortical neurons, loss of ATP10B leads to general lysosomal dysfunction and cell death. Impaired lysosomal functionality and integrity is well known to be implicated in PD pathology and linked to multiple causal PD genes and genetic risk factors. Our results indicate that recessive loss of function mutations in ATP10B increase risk for PD by disturbed lysosomal export of GluCer and PC. Both ATP10B and glucocerebrosidase 1, encoded by the PD risk gene GBA1, reduce lysosomal GluCer levels, emerging lysosomal GluCer accumulation as a potential PD driver.

CHCHD2 harboring Parkinson's disease-linked T61I mutation precipitates inside mitochondria and induces precipitation of wild-type CHCHD2.

The T61I mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a protein residing in the mitochondrial intermembrane space (IMS), causes an autosomal dominant form of Parkinson's disease (PD), but the underlying pathogenic mechanisms are not well understood. Here, we compared the subcellular localization and solubility of wild-type (WT) and T61I mutant CHCHD2 in human cells. We found that mitochondrial targeting of both WT and T61I CHCHD2 depended on the four cysteine residues in the C-terminal coiled-coil-helix-coiled-coil-helix (CHCH) domain but not on the N-terminal predicted mitochondrial targeting sequence. The T61I mutation did not interfere with mitochondrial targeting of the mutant protein but induced its precipitation in the IMS. Moreover, T61I CHCHD2 induced increased mitochondrial production of reactive oxygen species and apoptosis, which was prevented by treatment with anti-oxidants. Retention of T61I CHCHD2 in the cytosol through mutation of the cysteine residues in the CHCH domain prevented its precipitation as well as its apoptosis-inducing effect. Importantly, T61I CHCHD2 potently impaired the solubility of WT CHCHD2. In conclusion, our data show that the T61I mutation renders mutant CHCHD2 insoluble inside mitochondria, suggesting loss of function of the mutant protein. In addition, T61I CHCHD2 exerts a dominant-negative effect on the solubility of WT CHCHD2, explaining the dominant inheritance of this form of PD.

ATP13A2 deficiency disrupts lysosomal polyamine export.

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.

LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10.

Parkinson disease (PD) is a disabling, incurable disorder with increasing prevalence in the western world. In rare cases PD is caused by mutations in the genes for PINK1 (PTEN induced kinase 1) or PRKN (parkin RBR E3 ubiquitin protein ligase), which impair the selective autophagic elimination of damaged mitochondria (mitophagy). Mutations in the gene encoding LRRK2 (leucine rich repeat kinase 2) are the most common monogenic cause of PD. Here, we report that the LRRK2 kinase substrate RAB10 accumulates on depolarized mitochondria in a PINK1- and PRKN-dependent manner. RAB10 binds the autophagy receptor OPTN (optineurin), promotes OPTN accumulation on depolarized mitochondria and facilitates mitophagy. In PD patients with the two most common LRRK2 mutations (G2019S and R1441C), RAB10 phosphorylation at threonine 73 is enhanced, while RAB10 interaction with OPTN, accumulation of RAB10 and OPTN on depolarized mitochondria, depolarization-induced mitophagy and mitochondrial function are all impaired. These defects in LRRK2 mutant patient cells are rescued by LRRK2 knockdown and LRRK2 kinase inhibition. A phosphomimetic RAB10 mutant showed less OPTN interaction and less translocation to depolarized mitochondria than wild-type RAB10, and failed to rescue mitophagy in LRRK2 mutant cells. These data connect LRRK2 with PINK1- and PRKN-mediated mitophagy via its substrate RAB10, and indicate that the pathogenic effects of mutations in LRRK2, PINK1 and PRKN may converge on a common pathway.Abbreviations : ACTB: actin beta; ATP5F1B: ATP synthase F1 subunit beta; CALCOCO2: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; Co-IP: co-immunoprecipitation; EBSS: Earle's balanced salt solution; GFP: green fluorescent protein; HSPD1: heat shock protein family D (Hsp60) member 1; LAMP1: lysosomal associated membrane protein 1; LRRK2: leucine rich repeat kinase 2; IF: immunofluorescence; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; OMM: outer mitochondrial membrane; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT1: ras homolog family member T1; ROS: reactive oxygen species; TBK1: TANK binding kinase 1; WB: western blot.

Parkinson disease related ATP13A2 evolved early in animal evolution.

Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. ATP13A1 complemented a deletion of the yeast P5A ATPase SPF1, while none of ATP13A2-5 could complement either the loss of SPF1 or that of the single P5B ATPase YPK9 in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.

Clickable Polyamine Derivatives as Chemical Probes for the Polyamine Transport System.

Polyamines are essential for cell growth and differentiation, but their trafficking by the polyamine transport system is not fully understood. Herein, the synthesis of several azido-derivatized polyamines for easy conjugation by click chemistry is described. Attachment of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye gave fluorescent polyamine probes, which were tested in cell culture. The linear probe series showed superior cellular uptake compared with that of probes in which the dye was attached to a branch on one of the central amines. Interestingly, the linear probes accumulated rapidly in cancer cells (MCF-7), but not in nontumorigenic cells (MCF-10A). The fluorescent polyamine probes are therefore applicable to the study of polyamine trafficking, whereas the azido polyamines may be further utilized to transport cargo into cancer cells by exploiting the polyamine transport system.

An autophagy-driven pathway of ATP secretion supports the aggressive phenotype of BRAFV600E inhibitor-resistant metastatic melanoma cells.

The ingrained capacity of melanoma cells to rapidly evolve toward an aggressive phenotype is manifested by their increased ability to develop drug-resistance, evident in the case of vemurafenib, a therapeutic-agent targeting BRAFV600E. Previous studies indicated a tight correlation between heightened melanoma-associated macroautophagy/autophagy and acquired Vemurafenib resistance. However, how this vesicular trafficking pathway supports Vemurafenib resistance remains unclear. Here, using isogenic human and murine melanoma cell lines of Vemurafenib-resistant and patient-derived melanoma cells with primary resistance to the BRAFV600E inhibitor, we found that the enhanced migration and invasion of the resistant melanoma cells correlated with an enhanced autophagic capacity and autophagosome-mediated secretion of ATP. Extracellular ATP (eATP) was instrumental for the invasive phenotype and the expansion of a subset of Vemurafenib-resistant melanoma cells. Compromising the heightened autophagy in these BRAFV600E inhibitor-resistant melanoma cells through the knockdown of different autophagy genes (ATG5, ATG7, ULK1), reduced their invasive and eATP-secreting capacity. Furthermore, eATP promoted the aggressive nature of the BRAFV600E inhibitor-resistant melanoma cells by signaling through the purinergic receptor P2RX7. This autophagy-propelled eATP-dependent autocrine-paracrine pathway supported the maintenance and expansion of a drug-resistant melanoma phenotype. In conclusion, we have identified an autophagy-driven response that relies on the secretion of ATP to drive P2RX7-based migration and expansion of the Vemurafenib-resistant phenotype. This emphasizes the potential of targeting autophagy in the treatment and management of metastatic melanoma.

Loss-of-function mutations in the ATP13A2/PARK9 gene cause complicated hereditary spastic paraplegia (SPG78).

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C > T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with fronto-temporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement.

A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.