Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498-499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains. Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron-ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498-499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy.

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.

Neurodegeneration associated with defective pantothenate kinase-2 (PKAN) is an early-onset monogenic autosomal-recessive disorder. The hallmark of the disease is the massive accumulation of iron in the globus pallidus brain region of patients. PKAN is caused by mutations in the PANK2 gene encoding the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway. To date, the way in which this alteration leads to brain iron accumulation has not been elucidated. Starting from previously obtained hiPS clones, we set up a differentiation protocol able to generate inhibitory neurons. We obtained striatal-like medium spiny neurons composed of approximately 70-80% GABAergic neurons and 10-20% glial cells. Within this mixed population, we detected iron deposition in both PKAN cell types, however, the viability of PKAN GABAergic neurons was strongly affected. CoA treatment was able to reduce cell death and, notably, iron overload. Further differentiation of hiPS clones in a pure population of astrocytes showed particularly evident iron accumulation, with approximately 50% of cells positive for Perls staining. The analysis of these PKAN astrocytes indicated alterations in iron metabolism, mitochondrial morphology, respiratory activity, and oxidative status. Moreover, PKAN astrocytes showed signs of ferroptosis and were prone to developing a stellate phenotype, thus gaining neurotoxic features. This characteristic was confirmed in iPS-derived astrocyte and glutamatergic neuron cocultures, in which PKAN glutamatergic neurons were less viable in the presence of PKAN astrocytes. This newly generated astrocyte model is the first in vitro disease model recapitulating the human phenotype and can be exploited to deeply clarify the pathogenetic mechanisms underlying the disease.

Ferritin is the main intracellular protein of iron storage with a central role in the regulation of iron metabolism and detoxification. Nucleotide insertions in the last exon of the ferritin light chain cause a neurodegenerative disease known as Neuroferritinopathy, characterized by iron deposition in the brain, particularly in the cerebellum, basal ganglia and motor cortex. The disease progresses relentlessly, leading to dystonia, chorea, motor disability and neuropsychiatry features. The characterization of a good animal model is required to compare and contrast specific features with the human disease, in order to gain new insights on the consequences of chronic iron overload on brain function and behavior. To this aim we studied an animal model expressing the pathogenic human FTL mutant 498InsTC under the phosphoglycerate kinase (PGK) promoter. Transgenic (Tg) mice showed strong accumulation of the mutated protein in the brain, which increased with age, and this was accompanied by brain accumulation of ferritin/iron bodies, the main pathologic hallmark of human neuroferritinopathy. Tg-mice were tested throughout development and aging at 2-, 8- and 18-months for motor coordination and balance (Beam Walking and Footprint tests). The Tg-mice showed a significant decrease in motor coordination at 8 and 18 months of age, with a shorter latency to fall and abnormal gait. Furthermore, one group of aged naïve subjects was challenged with two herbicides (Paraquat and Maneb) known to cause oxidative damage. The treatment led to a paradoxical increase in behavioral activation in the transgenic mice, suggestive of altered functioning of the dopaminergic system. Overall, data indicate that mice carrying the pathogenic FTL498InsTC mutation show motor deficits with a developmental profile suggestive of a progressive pathology, as in the human disease. These mice could be a powerful tool to study the neurodegenerative mechanisms leading to the disease and help developing specific therapeutic targets.

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.

The novel brain-penetrant peroxisome proliferator-activated receptor gamma agonist leriglitazone, previously validated for other rare neurodegenerative diseases, is a small molecule that acts as a regulator of mitochondrial function and exerts neuroprotective, anti-oxidative and anti-inflammatory effects. Herein, we tested whether leriglitazone can be effective in ameliorating the mitochondrial defects that characterize an hiPS-derived model of Pantothenate kinase-2 associated Neurodegeneration (PKAN). PKAN is caused by a genetic alteration in the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway, and for which no effective cure is available. The PKAN hiPS-derived astrocytes are characterized by mitochondrial dysfunction, cytosolic iron deposition, oxidative stress and neurotoxicity. We monitored the effect of leriglitazone in comparison with CoA on hiPS-derived astrocytes from three healthy subjects and three PKAN patients. The treatment with leriglitazone did not affect the differentiation of the neuronal precursor cells into astrocytes, and it improved the viability of PKAN cells and their respiratory activity, while diminishing the iron accumulation similarly or even better than CoA. The data suggest that leriglitazone is well tolerated in this cellular model and could be considered a beneficial therapeutic approach in the treatment of PKAN.

Epsins are part of the internalization machinery pivotal to control clathrin-mediated endocytosis. Here, we report that epsin family members are expressed in mouse embryonic stem cells (mESCs) and that epsin1/2 knockdown alters both mESC exits from pluripotency and their differentiation. Furthermore, we show that epsin1/2 knockdown compromises the correct polarization and division of mESC-derived neural progenitors and their conversion into expandable radial glia-like neural stem cells. Finally, we provide evidence that Notch signaling is impaired following epsin1/2 knockdown and that experimental restoration of Notch signaling rescues the epsin-mediated phenotypes. We conclude that epsins contribute to control mESC exit from pluripotency and allow their neural differentiation by appropriate modulation of Notch signaling.

Neuroferritinopathy (NF) is a movement disorder caused by alterations in the L-ferritin gene that generate cytosolic free iron. NF is a unique pathophysiological model for determining the direct consequences of cell iron dysregulation. We established lines of induced pluripotent stem cells from fibroblasts from two NF patients and one isogenic control obtained by CRISPR/Cas9 technology. NF fibroblasts, neural progenitors, and neurons exhibited the presence of increased cytosolic iron, which was also detectable as: ferritin aggregates, alterations in the iron parameters, oxidative damage, and the onset of a senescence phenotype, particularly severe in the neurons. In this spontaneous senescence model, NF cells had impaired survival and died by ferroptosis. Thus, non-ferritin-bound iron is sufficient per se to cause both cell senescence and ferroptotic cell death in human fibroblasts and neurons. These results provide strong evidence supporting the primary role of iron in neuronal aging and degeneration.

PKAN disease is caused by mutations in the PANK2 gene, encoding the mitochondrial enzyme pantothenate kinase 2, catalyzing the first and key reaction in Coenzyme A (CoA) biosynthetic process. This disorder is characterized by progressive neurodegeneration and excessive iron deposition in the brain. The pathogenic mechanisms of PKAN are still unclear, and the available therapies are only symptomatic. Although iron accumulation is a hallmark of PKAN, its relationship with CoA dysfunction is not clear. We have previously developed hiPS-derived astrocytes from PKAN patients showing iron overload, thus recapitulating the human phenotype. In this work, we demonstrated that PKAN astrocytes presented an increase in transferrin uptake, a key route for cellular iron intake via transferrin receptor-mediated endocytosis of transferrin-bound iron. Investigation of constitutive exo-endocytosis and vesicular dynamics, exploiting the activity-enriching biosensor SynaptoZip, led to the finding of a general impairment in the constitutive endosomal trafficking in PKAN astrocytes. CoA and 4-phenylbutyric acid treatments were found to be effective in partially rescuing the aberrant vesicular behavior and iron intake. Our results demonstrate that the impairment of CoA biosynthesis could interfere with pivotal intracellular mechanisms involved in membrane fusions and vesicular trafficking, leading to an aberrant transferrin receptor-mediated iron uptake.

The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL-Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.

Pantothenate kinase-associated neurodegeneration (PKAN) is an early onset and severely disabling neurodegenerative disease for which no therapy is available. PKAN is caused by mutations in PANK2, which encodes for the mitochondrial enzyme pantothenate kinase 2. Its function is to catalyze the first limiting step of Coenzyme A (CoA) biosynthesis. We generated induced pluripotent stem cells from PKAN patients and showed that their derived neurons exhibited premature death, increased ROS production, mitochondrial dysfunctions-including impairment of mitochondrial iron-dependent biosynthesis-and major membrane excitability defects. CoA supplementation prevented neuronal death and ROS formation by restoring mitochondrial and neuronal functionality. Our findings provide direct evidence that PANK2 malfunctioning is responsible for abnormal phenotypes in human neuronal cells and indicate CoA treatment as a possible therapeutic intervention.

The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.

The discovery of the role of the RAS/RAF/MEK/ERK pathway in melanomagenesis and its progression have opened a new era in the treatment of this tumor. Vemurafenib was the first specific kinase inhibitor approved for therapy of advanced melanomas harboring BRAF-activating mutations, followed by dabrafenib and encorafenib. However, despite the excellent results of first-generation kinase inhibitors in terms of response rate, the average duration of the response was short, due to the onset of genetic and epigenetic resistance mechanisms. The combination therapy with MEK inhibitors is an excellent strategy to circumvent drug resistance, with the additional advantage of reducing side effects due to the paradoxical reactivation of the MAPK pathway. The recent development of RAS and extracellular signal-related kinases (ERK) inhibitors promises to add new players for the ultimate suppression of this signaling pathway and the control of pathway-related drug resistance. In this review, we analyze the pharmacological, preclinical, and clinical trial data of the various MAPK pathway inhibitors, with a keen interest for their clinical applicability in the management of advanced melanoma.