Transglutaminase Type 2-MITF axis regulates phenotype switching in skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is the deadliest form of skin cancer due to its high heterogeneity that drives tumor aggressiveness. Melanoma plasticity consists of two distinct phenotypic states that co-exist in the tumor niche, the proliferative and the invasive, respectively associated with a high and low expression of MITF, the master regulator of melanocyte lineage. However, despite efforts, melanoma research is still far from exhaustively dissecting this phenomenon. Here, we discovered a key function of Transglutaminase Type-2 (TG2) in regulating melanogenesis by modulating MITF transcription factor expression and its transcriptional activity. Importantly, we demonstrated that TG2 expression affects melanoma invasiveness, highlighting its positive value in SKCM. These results suggest that TG2 may have implications in the regulation of the phenotype switching by promoting melanoma differentiation and impairing its metastatic potential. Our findings offer potential perspectives to unravel melanoma vulnerabilities via tuning intra-tumor heterogeneity.

Dysfunctional mitochondria accumulate in a skeletal muscle knockout model of Smn1, the causal gene of spinal muscular atrophy.

The approved gene therapies for spinal muscular atrophy (SMA), caused by loss of survival motor neuron 1 (SMN1), greatly ameliorate SMA natural history but are not curative. These therapies primarily target motor neurons, but SMN1 loss has detrimental effects beyond motor neurons and especially in muscle. Here we show that SMN loss in mouse skeletal muscle leads to accumulation of dysfunctional mitochondria. Expression profiling of single myofibers from a muscle specific Smn1 knockout mouse model revealed down-regulation of mitochondrial and lysosomal genes. Albeit levels of proteins that mark mitochondria for mitophagy were increased, morphologically deranged mitochondria with impaired complex I and IV activity and respiration and that produced excess reactive oxygen species accumulated in Smn1 knockout muscles, because of the lysosomal dysfunction highlighted by the transcriptional profiling. Amniotic fluid stem cells transplantation that corrects the SMN knockout mouse myopathic phenotype restored mitochondrial morphology and expression of mitochondrial genes. Thus, targeting muscle mitochondrial dysfunction in SMA may complement the current gene therapy.

Kv1.3 K+ Channel Physiology Assessed by Genetic and Pharmacological Modulation.

Potassium channels are widespread over all kingdoms and play an important role in the maintenance of cellular ionic homeostasis. Kv1.3 is a voltage-gated potassium channel of the Shaker family with a wide tissue expression and a well-defined pharmacology. In recent decades, experiments mainly based on pharmacological modulation of Kv1.3 have highlighted its crucial contribution to different fundamental processes such as regulation of proliferation, apoptosis, and metabolism. These findings link channel function to various pathologies ranging from autoimmune diseases to obesity and cancer. In the present review, we briefly summarize studies employing Kv1.3 knockout animal models to confirm such roles and discuss the findings in comparison to the results obtained by pharmacological modulation of Kv1.3 in various pathophysiological settings. We also underline how these studies contributed to our understanding of channel function in vivo and propose possible future directions.

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo.

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoKCa3.1) of various cancer cell lines. The role mitoKCa3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity KCa3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane KCa3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoKCa3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoKCa3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial KCa3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.

Dopamine signaling modulates microglial NLRP3 inflammasome activation: implications for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by the loss of nigral dopaminergic neurons leading to impaired striatal dopamine signaling, α-synuclein- (α-syn-) rich inclusions, and neuroinflammation. Degenerating neurons are surrounded by activated microglia with increased secretion of interleukin-1ß (IL-1ß), driven largely by the NLRP3 inflammasome. A critical role for microglial NLRP3 inflammasome activation in the progression of both dopaminergic neurodegeneration and α-syn pathology has been demonstrated in parkinsonism mouse models. Fibrillar α-syn activates this inflammasome in mouse and human macrophages, and we have shown previously that the same holds true for primary human microglia. Dopamine blocks microglial NLRP3 inflammasome activation in the MPTP model, but its effects in this framework, highly relevant to PD, remain unexplored in primary human microglia and in other in vivo parkinsonism models. METHODS: Biochemical techniques including quantification of IL-1ß secretion and confocal microscopy were employed to gain insight into dopamine signaling-mediated inhibition of the NLRP3 inflammasome mechanism in primary human microglia and the SYN120 transgenic mouse model. Dopamine and related metabolites were applied to human microglia together with various inflammasome activating stimuli. The involvement of the receptors through which these catecholamines were predicted to act were assessed with agonists in both species. RESULTS: We show in primary human microglia that dopamine, L-DOPA, and high extracellular K+, but not norepinephrine and epinephrine, block canonical, non-canonical, and α-syn-mediated NLRP3 inflammasome-driven IL-1ß secretion. This suggests that dopamine acts as an inflammasome inhibitor in human microglia. Accordingly, we provide evidence that dopamine exerts its inhibitory effect through dopamine receptor D1 and D2 (DRD1 and DRD2) signaling. We also show that aged mice transgenic for human C-terminally truncated (1-120) α-syn (SYN120 tg mice) display increased NLRP3 inflammasome activation in comparison to WT mice that is diminished upon DRD1 agonism. CONCLUSIONS: Dopamine inhibits canonical, non-canonical, and α-syn-mediated activation of the NLRP3 inflammasome in primary human microglia, as does high extracellular K+. We suggest that dopamine serves as an endogenous repressor of the K+ efflux-dependent microglial NLRP3 inflammasome activation that contributes to dopaminergic neurodegeneration in PD, and that this reciprocation may account for the specific vulnerability of these neurons to disease pathology.

Pharmacological modulation of Kv1.3 potassium channel selectively triggers pathological B lymphocyte apoptosis in vivo in a genetic CLL model.

BACKGROUND: Ion channels are emerging as promising oncological targets. The potassium channels Kv1.3 and IKCa are highly expressed in the plasma membrane and mitochondria of human chronic lymphocytic leukemia (CLL) cells, compared to healthy lymphocytes. In vitro, inhibition of mitoKv1.3 by PAPTP was shown to kill ex vivo primary human CLL cells, while targeting IKCa with TRAM-34 decreased CLL cell proliferation. METHODS: Here we evaluated the effect of the above drugs in CLL cells from ibrutinib-resistant patients and in combination with Venetoclax, two drugs used in the clinical practice. The effects of the drugs were tested also in the Eµ-TCL1 genetic CLL murine model, characterized by a lympho-proliferative disease reminiscent of aggressive human CLL. Eµ-TCL1 mice showing overt disease state were treated with intraperitoneal injections of non-toxic 5 nmol/g PAPTP or 10 nmol/g TRAM-34 once a day and the number and percentage of pathological B cells (CD19+CD5+) in different, pathologically relevant body districts were determined. RESULTS: We show that Kv1.3 expression correlates with sensitivity of the human and mouse neoplastic cells to PAPTP. Primary CLL cells from ibrutinib-resistant patients could be killed with PAPTP and this drug enhanced the effect of Venetoclax, by acting on mitoKv1.3 of the inner mitochondrial membrane and triggering rapid mitochondrial changes and cytochrome c release. In vivo, after 2 week- therapy of Eµ-TCL1 mice harboring distinct CLL clones, leukemia burden was reduced by more than 85%: the number and percentage of CLL B cells fall in the spleen and peritoneal cavity and in the peripheral blood, without signs of toxicity. Notably, CLL infiltration into liver and spleen and splenomegaly were also drastically reduced upon PAPTP treatment. In contrast, TRAM-34 did not exert any beneficial effect when administered in vivo to Eµ-TCL1 mice at non-toxic concentration. CONCLUSION: Altogether, by comparing vehicle versus compound effect in different Eµ-TCL1 animals bearing unique clones similarly to CLL patients, we conclude that PAPTP significantly reduced leukemia burden in CLL-relevant districts, even in animals with advanced stage of the disease. Our results thus identify PAPTP as a very promising drug for CLL treatment, even for the chemoresistant forms of the disease.

The mitochondrial protein Opa1 promotes adipocyte browning that is dependent on urea cycle metabolites.

White to brown/beige adipocytes conversion is a possible therapeutic strategy to tackle the current obesity epidemics. While mitochondria are key for energy dissipation in brown fat, it is unknown if they can drive adipocyte browning. Here, we show that the mitochondrial cristae biogenesis protein optic atrophy 1 (Opa1) facilitates cell-autonomous adipocyte browning. In two cohorts of patients with obesity, including weight discordant monozygotic twin pairs, adipose tissue OPA1 levels are reduced. In the mouse, Opa1 overexpression favours white adipose tissue expandability as well as browning, ultimately improving glucose tolerance and insulin sensitivity. Transcriptomics and metabolomics analyses identify the Jumanji family chromatin remodelling protein Kdm3a and urea cycle metabolites, including fumarate, as effectors of Opa1-dependent browning. Mechanistically, the higher cyclic adenosine monophosphate (cAMP) levels in Opa1 pre-adipocytes activate cAMP-responsive element binding protein (CREB), which transcribes urea cycle enzymes. Flux analyses in pre-adipocytes indicate that Opa1-dependent fumarate accumulation depends on the urea cycle. Conversely, adipocyte-specific Opa1 deletion curtails urea cycle and beige differentiation of pre-adipocytes, and is rescued by fumarate supplementation. Thus, the urea cycle links the mitochondrial dynamics protein Opa1 to white adipocyte browning.

Exploiting pyocyanin to treat mitochondrial disease due to respiratory complex III dysfunction.

Mitochondrial diseases impair oxidative phosphorylation and ATP production, while effective treatment is still lacking. Defective complex III is associated with a highly variable clinical spectrum. We show that pyocyanin, a bacterial redox cycler, can replace the redox functions of complex III, acting as an electron shunt. Sub-µM pyocyanin was harmless, restored respiration and increased ATP production in fibroblasts from five patients harboring pathogenic mutations in TTC19, BCS1L or LYRM7, involved in assembly/stabilization of complex III. Pyocyanin normalized the mitochondrial membrane potential, and mildly increased ROS production and biogenesis. These in vitro effects were confirmed in both DrosophilaTTC19KO and in Danio rerioTTC19KD, as administration of low concentrations of pyocyanin significantly ameliorated movement proficiency. Importantly, daily administration of pyocyanin for two months was not toxic in control mice. Our results point to utilization of redox cyclers for therapy of complex III disorders.

An Angiopep2-PAPTP Construct Overcomes the Blood-Brain Barrier. New Perspectives against Brain Tumors.

A developing family of chemotherapeutics-derived from 5-(4-phenoxybutoxy)psoralen (PAP-1)-target mitochondrial potassium channel mtKv1.3 to selectively induce oxidative stress and death of diseased cells. The key to their effectiveness is the presence of a positively charged triphenylphosphonium group which drives their accumulation in the organelles. These compounds have proven their preclinical worth in murine models of cancers such as melanoma and pancreatic adenocarcinoma. In in vitro experiments they also efficiently killed glioblastoma cells, but in vivo they were powerless against orthotopic glioma because they were completely unable to overcome the blood-brain barrier. In an effort to improve brain delivery we have now coupled one of these promising compounds, PAPTP, to well-known cell-penetrating and brain-targeting peptides TAT48-61 and Angiopep-2. Coupling has been obtained by linking one of the phenyl groups of the triphenylphosphonium to the first amino acid of the peptide via a reversible carbamate ester bond. Both TAT48-61 and Angiopep-2 allowed the delivery of 0.3-0.4 nmoles of construct per gram of brain tissue upon intravenous (i.v.) injection of 5 µmoles/kg bw to mice. This is the first evidence of PAPTP delivery to the brain; the chemical strategy described here opens the possibility to conjugate PAPTP to small peptides in order to fine-tune tissue distribution of this interesting compound.

α-Synuclein evokes NLRP3 inflammasome-mediated IL-1ß secretion from primary human microglia.

Synucleinopathies such as Parkinson's disease (PD) are hallmarked by α-synuclein (α-syn) pathology and neuroinflammation. This neuroinflammation involves activated microglia with increased secretion of interleukin-1ß (IL-1ß). The main driver of IL-1ß secretion from microglia is the NLRP3 inflammasome. A critical link between microglial NLRP3 inflammasome activation and the progression of both α-syn pathology and dopaminergic neurodegeneration has been identified in various PD models in vivo. α-Syn is known to activate the microglial NLRP3 inflammasome in murine models, but its relationship to this inflammasome in human microglia has not been established. In this study, IL-1ß secretion from primary mouse microglia induced by α-syn fibrils was dependent on NLRP3 inflammasome assembly and caspase-1 activity, as previously reported. We show that exposure of primary human microglia to α-syn fibrils also resulted in significant IL-1ß secretion that was dependent on inflammasome assembly and involved the recruitment of caspase-1 protein to inflammasome scaffolds as visualized with superresolution microscopy. While canonical IL-1ß secretion was clearly dependent on caspase-1 enzymatic activity, this activity was less clearly involved for α-syn-induced IL-1ß secretion from human microglia. This work presents similarities between primary human and mouse microglia in the mechanisms of activation of the NLRP3 inflammasome by α-syn, but also highlights evidence to suggest that there may be a difference in the requirement for caspase-1 activity in IL-1ß output. The data represent a novel characterization of PD-related NLRP3 inflammasome activation in primary human microglia and further implicate this mechanism in the pathology underlying PD.

DRP1-mediated mitochondrial shape controls calcium homeostasis and muscle mass.

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca2+ handling.

Transcriptome analysis of LRRK2 knock-out microglia cells reveals alterations of inflammatory- and oxidative stress-related pathways upon treatment with α-synuclein fibrils.

Several previous studies have linked the Parkinson's disease (PD) gene LRRK2 to the biology of microglia cells. However, the precise ways in which LRRK2 affects microglial function have not been fully resolved. Here, we used the RNA-Sequencing to obtain transcriptomic profiles of LRRK2 wild-type (WT) and knock-out (KO) microglia cells treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS) as a general inflammatory insult. We observed that, although α-synuclein PFFs and LPS mediate overlapping gene expression profiles in microglia, there are also distinct responses to each stimulus. α-Synuclein PFFs trigger alterations of oxidative stress-related pathways with the mitochondrial dismutase Sod2 as a strongly differentially regulated gene. We validated SOD2 at mRNA and protein levels. Furthermore, we found that LRRK2 KO microglia cells reported attenuated induction of mitochondrial SOD2 in response to α-synuclein PFFs, indicating a potential contribution of LRRK2 to oxidative stress-related pathways. We validate several genes in vivo using single-cell RNA-Seq from acutely isolated microglia after striatal injection of LPS into the mouse brain. Overall, these results suggest that microglial LRRK2 may contribute to the pathogenesis of PD via altered oxidative stress signaling.

Synapsin III deficiency hampers α-synuclein aggregation, striatal synaptic damage and nigral cell loss in an AAV-based mouse model of Parkinson's disease.

Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by the progressive loss of nigral dopamine neurons. The deposition of fibrillary aggregated α-synuclein in Lewy bodies (LB), that is considered to play a causative role in the disease, constitutes another key neuropathological hallmark of PD. We have recently described that synapsin III (Syn III), a synaptic phosphoprotein that regulates dopamine release in cooperation with α-synuclein, is present in the α-synuclein insoluble fibrils composing the LB of patients affected by PD. Moreover, we observed that silencing of Syn III gene could prevent α-synuclein fibrillary aggregation in vitro. This evidence suggests that Syn III might be crucially involved in α-synuclein pathological deposition. To test this hypothesis, we studied whether mice knock-out (ko) for Syn III might be protected from α-synuclein aggregation and nigrostriatal neuron degeneration resulting from the unilateral injection of adeno-associated viral vectors (AAV)-mediating human wild-type (wt) α-synuclein overexpression (AAV-hαsyn). We found that Syn III ko mice injected with AAV-hαsyn did not develop fibrillary insoluble α-synuclein aggregates, showed reduced amount of α-synuclein oligomers detected by in situ proximity ligation assay (PLA) and lower levels of Ser129-phosphorylated α-synuclein. Moreover, the nigrostriatal neurons of Syn III ko mice were protected from both synaptic damage and degeneration triggered by the AAV-hαsyn injection. Our observations indicate that Syn III constitutes a crucial mediator of α-synuclein aggregation and toxicity and identify Syn III as a novel therapeutic target for PD.

Synapsin III is a key component of α-synuclein fibrils in Lewy bodies of PD brains.

Lewy bodies (LB) and Lewy neurites (LN), which are primarily composed of α-synuclein (α-syn), are neuropathological hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We recently found that the neuronal phosphoprotein synapsin III (syn III) controls dopamine release via cooperation with α-syn and modulates α-syn aggregation. Here, we observed that LB and LN, in the substantia nigra of PD patients and hippocampus of one subject with DLB, displayed a marked immunopositivity for syn III. The in situ proximity ligation assay revealed the accumulation of numerous proteinase K-resistant neuropathological inclusions that contained both α-syn and syn III in tight association in the brain of affected subjects. Most strikingly, syn III was identified as a component of α-syn-positive fibrils in LB-enriched protein extracts from PD brains. Finally, a positive correlation between syn III and α-syn levels was detected in the caudate putamen of PD subjects. Collectively, these findings indicate that syn III is a crucial α-syn interactant and a key component of LB fibrils in the brain of patients affected by PD.

Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum-mitochondria tether.

The discovery of the multiple roles of mitochondria-endoplasmic reticulum (ER) juxtaposition in cell biology often relied upon the exploitation of Mitofusin (Mfn) 2 as an ER-mitochondria tether. However, this established Mfn2 function was recently questioned, calling for a critical re-evaluation of Mfn2's role in ER-mitochondria cross-talk. Electron microscopy and fluorescence-based probes of organelle proximity confirmed that ER-mitochondria juxtaposition was reduced by constitutive or acute Mfn2 deletion. Functionally, mitochondrial uptake of Ca2+ released from the ER was reduced following acute Mfn2 ablation, as well as in Mfn2-/- cells overexpressing the mitochondrial calcium uniporter. Mitochondrial Ca2+ uptake rate and extent were normal in isolated Mfn2-/- liver mitochondria, consistent with the finding that acute or chronic Mfn2 ablation or overexpression did not alter mitochondrial calcium uniporter complex component levels. Hence, Mfn2 stands as a bona fide ER-mitochondria tether whose ablation decreases interorganellar juxtaposition and communication.

Opa1 overexpression ameliorates the phenotype of two mitochondrial disease mouse models.

Increased levels of the mitochondria-shaping protein Opa1 improve respiratory chain efficiency and protect from tissue damage, suggesting that it could be an attractive target to counteract mitochondrial dysfunction. Here we show that Opa1 overexpression ameliorates two mouse models of defective mitochondrial bioenergetics. The offspring from crosses of a constitutive knockout for the structural complex I component Ndufs4 (Ndufs4(-/-)), and of a muscle-specific conditional knockout for the complex IV assembly factor Cox15 (Cox15(sm/sm)), with Opa1 transgenic (Opa1(tg)) mice showed improved motor skills and respiratory chain activities compared to the naive, non-Opa1-overexpressing, models. While the amelioration was modest in Ndufs4(-/-)::Opa1(tg) mice, correction of cristae ultrastructure and mitochondrial respiration, improvement of motor performance and prolongation of lifespan were remarkable in Cox15(sm/sm)::Opa1(tg) mice. Mechanistically, respiratory chain supercomplexes were increased in Cox15(sm/sm)::Opa1(tg) mice, and residual monomeric complex IV was stabilized. In conclusion, cristae shape amelioration by controlled Opa1 overexpression improves two mouse models of mitochondrial disease.

The OPA1-dependent mitochondrial cristae remodeling pathway controls atrophic, apoptotic, and ischemic tissue damage.

Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo.

Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency.

Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function.