Comparative survey of mitochondrial ultrastructure in IDH1-mutant astrocytoma and IDH1-wildtype glioblastoma (GBM).

Gliomas are the most common malignant brain tumors with poor prognosis. The WHO's classification recognizes isocitrate dehydrogenase 1 (IDH1) mutant astrocytoma and IDH1-wildtype glioblastoma (GBM). The IDH1 mutation confers a survival advantage over the wildtype. There are several explanations for the metabolic advantage of the IDH1 mutation, some involve mitochondrial implications. Since an ultrastructural comparison of both tumor genotypes is still lacking, we surveyed the ultrastructural effects of the IDH1 mutation on the mitochondria of the IDH1-mutant astrocytoma (n = 15) and IDH1-wildtype glioblastoma (n = 15) tumors. Our results show that both IDH1 genotypes have degenerate and uncoupled mitochondria; this has not been reported before. The presence of ample lipid inclusions and lipid droplets in the cytoplasm of both genotypes support our conclusion of dysfunctional uncoupled mitochondria. Thus, the IDH1 mutation may have no ultrastructural consequences on the mitochondria, and the aberrant mitochondria in both genotypes may be the result of other unknown mutations. The status of the mitochondria in these genotypes portends a clinical challenge since tumor cells with uncoupled mitochondria are more primitive, aggressive, and considerably treatment resistant.

Astrocytoma and glioblastoma IDH1-wildtype cells colonize tumor vessels and deploy vascular mimicry.

Gliomas are the most prevalent type of malignant brain tumors with a very dismal prognosis. Angiogenesis in glioma has recently gotten more attention and its molecular aspects have been published; however, these were not complemented with ultrastructural evidence. Our ultrastructural examination of glioma vessels reveals several unique and critical features related to their mechanisms of progression and metastasis strategy. The detailed ultrastructural survey of 18 isocitrate dehydrogenase-wildtype (IDH1-wt) glioblastomas and 12 isocitrate dehydrogenase-mutant (IDH1-mt) High-grade gliomas indicated that tumor vessels of both types had undergone deformities such as the thickening of the vessel wall (VW) and proliferation of the basement membrane, contour distortions, abnormal and discontinuous basal lamina, tumor cells' invasion and colonization of VW, disappearance of endothelial cells (ECs), pericytes, and smooth muscle cells, as well as the formation of a continuous ring of tumor cells attached to the luminal side of VW in numerous cases. The latter feature is a clear sign of vascular mimicry (VM) that was previously suggested in gliomas but never shown by TEM. Additionally, the vascular invasion was carried out by a large number of tumor cells and was accompanied by the accumulation of tumor lipids in the vessels' lumina and VWs; these two features are distinct for gliomas and may alter the course of the clinical presentation and overall prognosis. This raises the issue of how to specifically target tumor cells involved in vascular invasion in order to optimize prognosis and overcome these mechanisms employed by the tumor cells.

Gain-of-function mutations in ALPK1 cause an NF-κB-mediated autoinflammatory disease: functional assessment, clinical phenotyping and disease course of patients with ROSAH syndrome.

OBJECTIVES: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. METHODS: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. RESULTS: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.

Retinal and choroidal capillaries contribution to age-related macular degeneration (AMD) phenotypes in murine models of the disease.

Mouse models of age-related macular degeneration (AMD) such as Ccl2-/- and Ccl2-/-/Cx3cr1-/- have not yet been fully characterized ultrastructurally. Although we have previously shown extranuclear DNA (enDNA) leakage into the cytoplasm and damaged mitochondria in the retinal pigment epithelium (RPE) of these AMD mouse models, little is known about the state of their vascular capillaries of the retina and choroid. Our ultrastructural survey shows that the aberrations were not restricted to the RPE cells, but also extended to the vasculature of the retina and choroid. Their endothelial aberrations included cytoplasmic degeneration, pyknotic DNA, hypertrophic nuclei, and loss of fenestration in addition to duplication of basement membrane and loss of density in Bruch's membrane. Moreover, the state of the vasculature in the mutant mice models suggests that the capillaries could also be active contributors to the pathological findings seen in AMD. The goal of this study is to gain insights into the early events of AMD that may lead to a better understanding of AMD's pathogenesis, improve our preventative measures, and formulate designed therapeutic regimens that are tailored to target the initial pathological events.Abbreviations: AMD: age-related macular degeneration; BM: Bruch's membrane; DPC: degenerate pericyte; EN: endothelial nucleus; enDNA: extranuclear DNA; GCL: ganglion cell layer; HEN: hypertrophic endothelial nucleus; IPL: inner plexiform layer; NFL: nerve fiber layer; OPL: outer plexiform layer; RBC: red blood cell; RPE: retinal pigment epithelium; SNPs: Single nucleotide polymorphisms.

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors.

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Mitochondrial autophagosomes as a mechanism of drug resistance in breast carcinoma.

We have previously described the process by which mitochondria donate their membranes for the formation of autophagosomes, and in this study we show that the same process could be involved in drug sequestration and exocytosis resulting in multidrug-resistant cancerous cells. We examine the implications of mitochondrial vesicle formation of mitoautophagosomes (MAPS) in response to the cytotoxic drug MKT-077, which targets mortalin, in a drug-resistant breast carcinoma cell line overexpressing P-glycoprotein (P-gp). The breast cancer cell line MCF-7Adr is derived from MCF-7, but differs from its ancestral line in tolerance of MKT-077-induced mitochondrial toxicity. Our ultrastructural observations suggest that autophagy in the MCF-7Adr cells entails regional sequestration of MKT077 in multilamellar LC3-labeled MAPS, which then separate from their mitochondria, and fuse with or engulf each other. MAPS appeared to be migrating through the cytoplasm and fusing with the plasma membrane, thus carrying out exocytotic secretion. This mechanism, which seems ineffective in the ancestral cell line, provides a resistance mechanism for MKT-077 by enhancing the efflux process of the cells. After 8 hr of MKT-077 exposure, a fraction of the resistant cells appeared viable and contained larger number of smaller sized mitochondria. Mitoautophagosomes, therefore, provide a potentially novel model for multidrug resistance in cancerous cells and may contribute to the P-gp efflux process.

Loss of endothelial planar cell polarity and cellular clearance mechanisms in age-related macular degeneration.

Apoptosis, autophagosomes, and lysosomes are lacking in the retinal pigment epithelium (RPE) of age-related macular degeneration (AMD) eyes. Necrosis, not apoptosis, appeared to be the prominent type of cell death in RPE, which led to the accumulation of cell debris within and on both sides of Bruch's membrane. The endothelium of the choriocapillaris had an altered planar cell polarity which encompassed the disappearance of fenestrations, the thickening of cytoplasm, and anterior nuclear dislocation. There were no significant differences in RPE and choroidal aberrations between macular and temporal regions. Loss of endothelial polarity could be at the crux of AMD initiation and progression.

Adverse effects of antiretroviral therapy on liver hepatocytes and endothelium in HIV patients: An ultrastructural perspective.

Human immunodeficiency virus and antiretroviral therapy (ART) together can be far more detrimental to liver cells than either of the two unaided. However, ultrastructural aspects of the synergistic effects of HIV and ART have been understudied. In a patient cohort receiving ART, this study characterizes ultrastructurally sinusoidal degeneration, hepatocytic aberrations, mitochondrial dysfunction, accumulation of bulky lipid droplets (steatosis), and occlusion of sinusoidal lumina. Mitochondrial dysfunction causes the accumulation of acetyl-CoA which leads to insulin upregulation and resistance, lipid synthesis, and steatosis. Lipid droplets deposited in the sinusoids could be the source of the blood's lipid profile alterations in HIV patients on ART.

NLRP3 Upregulation in Retinal Pigment Epithelium in Age-Related Macular Degeneration.

Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1ß) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis.

Platelet-derived growth factor (PDGF)-C inhibits neuroretinal apoptosis in a murine model of focal retinal degeneration.

Platelet-derived growth factor (PDGF)-C is a member of the PDGF family and is critical for neuronal survival in the central nervous system. We studied the possible survival and antiapoptotic effects of PDGF-C on focal retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)] (DKO rd8) background mice, a model for progressive and focal retinal degeneration. We found no difference in transcript and protein expression of PDGF-C in the retina between DKO rd8 mice and wild type (WT, C57BL/6N). Recombinant PDGF-CC protein (500 ng/eye) was injected intravitreally into the right eye of DKO rd8 mice with phosphate-buffered saline as controls into the left eye. The retinal effects of PDGF-C were assessed by fundoscopy, ocular histopathology, A2E levels, apoptotic molecule analysis, and direct flat mount retinal vascular labeling. We found that the PDGF-CC-treated eyes showed slower progression or attenuation of the focal retinal lesions, lesser photoreceptor and retinal pigment epithelial degeneration resulting in better-preserved photoreceptor structure. Lower expression of apoptotic molecules was detected in the PDGF-CC-treated eyes than in controls. In addition, no retinal neovascularization was observed after PDGF-CC treatment. Our results demonstrate that PDGF-C potently ameliorates photoreceptor degeneration via the suppression of apoptotic pathways without inducing retinal angiogenesis. The protective effects of PDGF-C suggest a novel alternative approach for potential age-related retinal degeneration treatment.

Upregulation of hypoxia-inducible factors and autophagy in von Hippel-Lindau-associated retinal hemangioblastoma.

PURPOSE: To describe pathological and molecular changes of three patients with clinically severe von Hippel-Lindau (VHL)-associated retinal hemangioblastoma (RH) with rapid progression. METHODS: Medical records, ocular histopathology, and transmission electron microscopy from three cases of VHL-associated RHs at the National Eye Institute were retrospectively reviewed. One eye of each patient was enucleated. Hypoxia-inducible factor (HIF) 1α and HIF2α expressions were identified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. RESULTS: All three cases had rapidly growing RHs that were resistant to multiple conventional therapies and two (patients 1 and 2) were also resistant to multiple intravitreal anti-vascular endothelial growth factor (VEGF) treatments. Macroscopically, all the enucleated eyes had multiple RHs, serous retinal detachment, severe retinal disorganization and focal hemorrhages. Histopathology showed typical RHs composed of vacuolated foamy VHL cells and capillary networks. Retinal gliosis and hemorrhages were also presented. Additionally, T lymphocytes and macrophages infiltrated in the tumors of two patients resistant to anti-VEGF therapy. Immunohistochemistry, and qRT-PCR found upregulation of HIF1α in the retinal lesions of all eyes. Importantly, upregulation of HIF2α was exclusively detected in the two cases with inflammatory infiltration and resistance to anti-VEGF therapy. Ultrastructural images showed autophagy, lipid droplets, glycogen aggregations, and cytoplasmic degeneration in many VHL cells. CONCLUSIONS: Based on the histopathological and molecular pathological findings, autophagy, inflammation, and/or upregulation of HIF2α could potentially contribute to the aggressive course of RHs, resulting in the resistance to multiple anti-VEGF and radiation therapies in these patients.

Implications of DNA leakage in eyes of mutant mice.

Extranuclear DNA (enDNA) is not well studied ultrastructurally in the retinal pigment epithelium (RPE). We analyzed the retina and vastus medialis muscle of four mouse strains that are related to focal retinal degeneration by transmission electron microscopy (TEM) and EM immunolabeling. Evaluation of enDNA would imply the involvements of enDNA is either limited to the affected tissue or generalized in the whole body. Ultrastructural analysis and EM immunolabeling revealed that enDNA was present in the RPE cells but not in the muscle. These data suggest that enDNA could be unique to unhealthy RPE and a potential biomarker for cellular abnormality.

Astrocytoma and IDH1-Wildtype Glioblastoma (GBM) Cells Colonize Tumor Vessels and Deploy Vascular Mimicry.

Gliomas are the most prevalent type of malignant brain tumors with a very dismal prognosis. Angiogenesis in glioma has recently gotten more attention and its molecular aspects have been published; however, these were not complemented with ultrastructural evidence. Our ultrastructural examination of glioma vessels reveals several unique and critical features related to their mechanisms of progression and metastasis strategy. The detailed ultrastructural survey of 18 IDH1 -wildtype glioblastomas (GBM) and 12 IDH1 -mutant High-grade gliomas indicated that tumor vessels of both types had undergone deformities such as the thickening of the vessel wall (VW) and proliferation of the basement membrane, contour distortions, abnormal and discontinuous basal lamina, tumor cells' invasion and colonization of VW, disappearance of endothelial cells (ECs), pericytes, and smooth muscle cells, as well as the formation of a continuous ring of tumor cells attached to the luminal side of VW in numerous cases. The latter feature is a clear sign of vascular mimicry (VM) that was previously suggested in gliomas but never shown by TEM. Additionally, the vascular invasion was carried out by a large number of tumor cells and was accompanied by the accumulation of tumor lipids in the vessels' lumina and VWs; these two features are distinct for gliomas and may alter the course of the clinical presentation and overall prognosis. This raises the issue of how to specifically target tumor cells involved in vascular invasion in order to optimize prognosis and overcome these mechanisms employed by the tumor cells.

Autoantibody against transient receptor potential M1 cation channels of retinal ON bipolar cells in paraneoplastic vitelliform retinopathy.

BACKGROUND: Paraneoplastic retinopathy is caused by the cross-reaction of neoplasm-directed autoantibodies against retinal antigens and results in retinal damage. Paraneoplastic vitelliform retinopathy, a presumed paraneoplastic retinopathy with features of atypical melanoma-associated retinopathy, has recently been reported in patients with metastatic melanoma. Ocular ultrastructure and its autoantibody localization of paraneoplastic vitelliform retinopathy are still indefinable. This is the first report of anti-transient receptor potential M1 antibody directly against human retinal bipolar dendritic tips in a melanoma patient with paraneoplastic vitelliform retinopathy. CASE PRESENTATION: We present a pair of postmortem eyes of an 80-year-old male with metastatic cutaneous melanoma, who developed paraneoplastic vitelliform retinopathy. The autopsied eyes were examined with light microscopy, immunohistochemistry, and transmission electron microscopy. Microscopically, the inner nuclear layer and outer plexiform layer were the most affected retinal structures, with local thinning. The lesions extended to the outer nuclear layer, resulting in focal retinal degeneration, edema, and atrophy. No active inflammation or melanoma cells were observed. Immunohistochemistry showed tightly compact bipolar cell nuclei (protein kinase C alpha/calbindin positive) with blur/loss of ON bipolar cell dendritic tips (transient receptor potential M1 positive) in diffusely condensed outer plexiform layer. The metastatic melanoma cells in his lung also showed immunoreactivity against transient receptor potential M1 antibody. Transmission electron microscopy illustrated degenerated inner nuclear layer with disintegration of cells and loss of cytoplasmic organelles. These cells contained many lysosomal and autophagous bodies and damaged mitochondria. Their nuclei appeared pyknotic and fragmentary. The synapses in the outer plexiform layer were extensively degenerated and replaced with empty vacuoles and disintegrated organelles. CONCLUSION: This case provides a convincing histological evidence of melanoma-associated autoantibodies directly against transient receptor potential M1 channels that target the ON bipolar cell structures in the inner nuclear and outer plexiform layers in paraneoplastic vitelliform retinopathy.

An Ultrastructural Perspective on Cell Death.

In the field of cell death, there is still a wide gap between the molecular models and their ultrastructural phenotypes. Because only very few published works included electron microscopy (EM) images, many ultrastructural features have not yet been incorporated into the descriptions of death modes. Some of the EM features that appear in dying cells have not been incorporated in describing death modes. It includes the accumulation of lipid droplets and glycogen, the appearance of extranuclear chromatin in the cytoplasm, and the various ways mitochondria become damaged. We argue that electron microscopy should be routinely included in these studies because it exposes some new features that molecular studies do not. It has successfully recognized new modes of cell death, such as entosis, methuosis, and paraptosis. Elucidating the precise sequence of events in death modes could be the cornerstone for offering the proper therapy of many diseases by slowing down or stopping the progression of degeneration. This review presents our own experience applying ultrastructural interpretations to death modes and explaining their biochemical implications. We complement the molecular and biochemical data and point out missing features that should be considered and studied.

The UOK 257 cell line: a novel model for studies of the human Birt-Hogg-Dubé gene pathway.

The establishment, characterization, and tumorigenicity of a new epithelial cell line (UOK 257) derived from human renal carcinoma of an individual with Birt-Hogg-Dubé (BHD) syndrome are reported. Unlike other established renal tumor cell lines from sporadic renal cell carcinoma, this is the first established renal tumor cell line of BHD, an inheritable neoplastic syndrome. The isolated tumor cells display loss of contact inhibition in vitro, and produce subcutaneous tumors in mouse xenografts. Histopathologic, ultrastructural, and cytogenetic characterizations of the established tumor cells are reported. Cytogenetic analysis using spectral karyotyping on UOK 257 cells revealed 17p loss and a near-triploid and aneuploid karyotype with multiple fluorescence in situ hybridization analysis using a locus-specific gene probe for MYC. The result demonstrates that the established tumor cells consist of two cell populations, one containing four and one containing five copies of the MYC oncogene.

Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo.

PURPOSE: The development of new cancer drugs is slow and costly. HIV protease inhibitors are Food and Drug Administration approved for HIV patients. Because these drugs cause toxicities that can be associated with inhibition of Akt, an emerging target in cancer, we assessed the potential of HIV protease inhibitors as anticancer agents. EXPERIMENTAL DESIGN: HIV protease inhibitors were screened in vitro using assays that measure cellular proliferation, apoptotic and nonapoptotic cell death, endoplasmic reticulum (ER) stress, autophagy, and activation of Akt. Nelfinavir was tested in non-small cell lung carcinoma (NSCLC) xenografts with biomarker assessment. RESULTS: Three of six HIV protease inhibitors, nelfinavir, ritonavir, and saquinavir, inhibited proliferation of NSCLC cells, as well as every cell line in the NCI60 cell line panel. Nelfinavir was most potent with a mean 50% growth inhibition of 5.2 micromol/L, a concentration achievable in HIV patients. Nelfinavir caused two types of cell death, caspase-dependent apoptosis and caspase-independent death that was characterized by induction of ER stress and autophagy. Autophagy was protective because an inhibitor of autophagy increased nelfinavir-induced death. Akt was variably inhibited by HIV protease inhibitors, but nelfinavir caused the greatest inhibition of endogenous and growth factor-induced Akt activation. Nelfinavir decreased the viability of a panel of drug-resistant breast cancer cell lines and inhibited the growth of NSCLC xenografts that was associated with induction of ER stress, autophagy, and apoptosis. CONCLUSIONS: Nelfinavir is a lead HIV protease inhibitor with pleiotropic effects in cancer cells. Given its wide spectrum of activity, oral availability, and familiarity of administration, nelfinavir is a Food and Drug Administration-approved drug that could be repositioned as a cancer therapeutic.

Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3.

Purpose: Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene. Methods: Mice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy. Results: C57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris. Conclusions: Treatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.

CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease.

Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin-dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3ß, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.

Proteomic profiling of von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2 pheochromocytomas reveals different expression of chromogranin B.

Pheochromocytomas are catecholamine-producing tumors that can occur in the context of von Hippel-Lindau syndrome (VHL) and multiple endocrine neoplasia type 2 (MEN2). Pheochromocytomas in these two syndromes differ in histopathological features, catecholamine metabolism, and clinical phenotype. To further investigate the nature of these differences, we compared the global protein expressions of 8 MEN2A-associated pheochromocytomas with 11 VHL-associated pheochromocytomas by two-dimensional gel electrophoresis proteomic profiling followed by sequencing and identification of differentially expressed proteins. Although both types of pheochromocytoma shared similarities in their protein expression patterns, the expression of several proteins was distinctly different between VHL- and MEN2A-associated pheochromocytomas. We identified several of these differentially expressed proteins. One of the proteins with higher expression in MEN2-associated tumors was chromogranin B, of which the differential expression was confirmed by western blot analysis. Our results expand the evidence for proteomic differences between these two tumor entities, and suggest that VHL-associated pheochromocytomas may be deficient in fundamental machinery for catecholamine storage. In light of these new findings, as well as existing evidence for differences between both types of pheochromocytomas, we propose that these tumors may have different developmental origins.