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1.
Neurol Sci ; 37(3): 361-4, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26621361

RESUMO

Friedreich's ataxia is an autosomal recessive progressive degenerative disorder caused by deficiency of the protein frataxin. The most common genetic cause is a homozygotic expansion of GAA triplets within intron 1 of the frataxin gene leading to impaired transcription. Preclinical in vivo and in vitro studies have shown that interferon gamma (IFNγ) is able to up-regulate the expression of frataxin gene in multiple cell types. We designed a phase IIa clinical trial, the first in Italy, aimed at assessing both safety and tolerability of IFNγ in Friedreich's patients and ability to increase frataxin levels in peripheral blood mononuclear cells. Nine patients (6 female and 3 males aged 21-38 years) with genetically confirmed disease were given 3 subcutaneous escalating doses (100, 150 and 200 µg) of IFNγ (human recombinant interferon 1 b gamma, trade name IMUKIN(®)), over 4 weeks. The primary end-point was the assessment of the safety and tolerability of IFNγ by means of standard clinical and hematological criteria. The secondary end-point was the detection of changes of frataxin levels in peripheral blood mononuclear cells after each single escalating dose of the drug. IFNγ was generally well tolerated, the main adverse event was hyperthermia/fever. Although, increases in frataxin levels could be detected in a minority of patients, these changes were not significant. A large phase III multicenter, randomized clinical trial with IFNγ in Friedreich's ataxia patients is currently ongoing. This study is expected to conclusively address the clinical efficacy of IFNγ therapy in patients with Friedreich's ataxia.


Assuntos
Ataxia de Friedreich/tratamento farmacológico , Interferon gama/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Adulto , Análise Química do Sangue , Esquema de Medicação , Feminino , Ataxia de Friedreich/sangue , Humanos , Interferon gama/efeitos adversos , Proteínas de Ligação ao Ferro/sangue , Itália , Masculino , Fármacos Neuroprotetores/efeitos adversos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Resultado do Tratamento , Adulto Jovem , Frataxina
2.
Hum Mol Genet ; 21(13): 2855-61, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22447512

RESUMO

Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting ∼3 in 100 000 individuals in Caucasian populations. It is caused by intronic GAA repeat expansions that hinder the expression of the FXN gene, resulting in defective levels of the mitochondrial protein frataxin. Sensory neurons in dorsal root ganglia (DRG) are particularly damaged by frataxin deficiency. There is no specific therapy for FRDA. Here, we show that frataxin levels can be upregulated by interferon gamma (IFNγ) in a variety of cell types, including primary cells derived from FRDA patients. IFNγ appears to act largely through a transcriptional mechanism on the FXN gene. Importantly, in vivo treatment with IFNγ increases frataxin expression in DRG neurons, prevents their pathological changes and ameliorates the sensorimotor performance in FRDA mice. These results disclose new roles for IFNγ in cellular metabolism and have direct implications for the treatment of FRDA.


Assuntos
Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Interferon gama/farmacologia , Interferon gama/fisiologia , Proteínas de Ligação ao Ferro/biossíntese , Animais , Células Cultivadas , Modelos Animais de Doenças , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/patologia , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Células HeLa , Humanos , Interferon gama/uso terapêutico , Proteínas de Ligação ao Ferro/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Transcrição Gênica , Ativação Transcricional , Frataxina
3.
Hum Mol Genet ; 16(13): 1534-40, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17468497

RESUMO

The defective expression of frataxin causes the hereditary neurodegenerative disorder Friedreich's ataxia (FRDA). Human frataxin is synthesized as a 210 amino acid precursor protein, which needs proteolytic processing into mitochondria to be converted into the functional mature form. In vitro processing of human frataxin was previously described to yield a 155 amino acid mature form, corresponding to residues 56-210 (frataxin(56-210)). Here, we studied the maturation of frataxin by in vivo overexpression in human cells. Our data show that the main form of mature frataxin is generated by a proteolytic cleavage between Lys80 and Ser81, yielding a 130 amino acid protein (frataxin(81-210)). This maturation product corresponds to the endogenous frataxin detected in human heart, peripheral blood lymphocytes or dermal fibroblasts. Moreover, we demonstrate that frataxin(81-210) is biologically functional, as it rescues aconitase defects in frataxin-deficient cells derived from FRDA patients. Importantly, our data indicate that frataxin(56-210) can be produced in vivo when the primary 80-81 maturation site is unavailable, suggesting the existence of proteolytic mechanisms that can actively control the size of the mature product, with possible functional implications.


Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Aconitato Hidratase/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Células Cultivadas , Fibroblastos/metabolismo , Ataxia de Friedreich/genética , Deleção de Genes , Regulação da Expressão Gênica , Homozigoto , Humanos , Proteínas de Ligação ao Ferro/química , Linfócitos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Frataxina
4.
J Biol Chem ; 281(24): 16750-6, 2006 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-16608849

RESUMO

Frataxin is a mitochondrial protein involved in iron metabolism. Defective expression of frataxin causes Friedreich ataxia (FA), an inherited degenerative syndrome characterized by ataxia, cardiomyopathy, and high incidence of diabetes. Here we report that frataxin-deficient cells are more prone to undergo stress-induced mitochondrial damage and apoptosis, while the overexpression of frataxin confers protection to a variety of cell types. Moreover, we reveal the existence of an extramitochondrial pool of frataxin, which can efficiently prevent mitochondrial damage and apoptosis in different cellular systems. Remarkably, extramitochondrial frataxin can fully replace mitochondrial frataxin in promoting survival of FA cells.


Assuntos
Sobrevivência Celular , Proteínas de Ligação ao Ferro/química , Mitocôndrias/metabolismo , Antioxidantes/química , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Citocromos c/metabolismo , Ataxia de Friedreich/patologia , Células HeLa , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Células Jurkat , Estresse Oxidativo , Espécies Reativas de Oxigênio , Frataxina
5.
J Leukoc Biol ; 79(1): 166-72, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16244104

RESUMO

The regulation of dendritic cell (DC) survival is crucial for the modulation of adaptive immunity. Ceramide is a lipid mediator of the stress response, which accumulates intracellularly during DC differentiation. We found that ceramide levels are tightly regulated in human DCs and that the pharmacological inhibition of enzymes responsible for ceramide catabolism, such as ceramidases and sphingosine kinases, sensitizes DCs to ceramide-induced cell death. It is important that inhibition of sphingosine kinases, during lipopolysaccharide stimulation, causes extensive ceramide accumulation and death of DCs. These data indicate that ceramide catabolism regulates survival of human DCs and reveal novel potential targets for the pharmacological manipulation of the immune response.


Assuntos
Ceramidas/metabolismo , Células Dendríticas/enzimologia , Inibidores Enzimáticos/farmacologia , Galactosidases/antagonistas & inibidores , Fatores Imunológicos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Ceramidas/imunologia , Células Dendríticas/imunologia , Galactosidases/imunologia , Humanos , Imunidade Ativa/efeitos dos fármacos , Imunidade Ativa/imunologia , Lipopolissacarídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia
6.
FASEB J ; 18(13): 1553-5, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15319364

RESUMO

An accelerated activity of the GD3 synthase (ST8), with consequent GD3 accumulation, is part of the response to environmental stressors in different cell types. Depending on specific, yet largely undefined, cellular settings, this can be followed by adaptation or apoptosis, the latter mostly due to GD3-induced mitochondrial damage. Here we show that subcellular localization of ST8 could significantly affect the biological outcome of GD3 accumulation. Binding to the molecular chaperone calnexin causes the retention of ST8 within the endoplasmic reticulum (ER) and prevents its relocalization to the Golgi. Calnexin-dependent ER retention does not affect the activity of ST8; yet the de novo synthesized GD3 largely fails to reach the mitochondria. Accordingly, overexpression of calnexin suppresses the pro-apoptotic activity of ST8, while the loss of calnexin sensitizes the cells to ST8-induced apoptosis. Reconstitution of calnexin confers protection to deficient cells. Thus, calnexin controls the biological outcome of GD3 accumulation and reveals a novel role in the stress response.


Assuntos
Apoptose , Calnexina/metabolismo , Sialiltransferases/antagonistas & inibidores , Sialiltransferases/metabolismo , Animais , Calnexina/genética , Linhagem Celular , Galinhas , Cricetinae , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Mitocôndrias/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Blood ; 102(8): 2910-5, 2003 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-12829589

RESUMO

Bacterial endotoxin (lipopolysaccharide [LPS]) is a potent inducer of human dendritic cell (DC) maturation and survival. Here we show that immature DCs exposed to LPS trigger an early and sustained caspase-like activity, which can be blocked by zVAD (z-Val-Ala-Asp), in the absence of detectable caspase 8 and caspase 10 activation, or poly(ADP-ribose) polymerase (PARP)-cleaving activity. Preventing LPS-induced caspase-like activation in DC results in massive cell death. Importantly, triggering of the caspase-like activity is required for LPS-induced activation of extracellular signal-regulated kinases (ERKs) and for LPS-induced up-regulation of cFLIP (Fas-associating protein with death domain-like interleukin-1 beta-converting enzyme [FLICE]-like inhibitory protein). Therefore, a caspase-dependent pathway initiated by LPS controls survival of human DCs.


Assuntos
Caspases/metabolismo , Células Dendríticas/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/metabolismo , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/metabolismo , Caspase 10 , Caspase 8 , Caspase 9 , Separação Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Escherichia coli/metabolismo , Citometria de Fluxo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/química , Poli(ADP-Ribose) Polimerases/metabolismo , Biossíntese de Proteínas , Fatores de Tempo
8.
J Exp Med ; 196(12): 1535-41, 2002 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-12486096

RESUMO

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.


Assuntos
Apoptose , Gangliosídeos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Acetilação , Acetilesterase/genética , Acetilesterase/metabolismo , Caspases/metabolismo , Linhagem Celular , Ativação Enzimática , Citometria de Fluxo , Gangliosídeos/química , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Mitocôndrias/metabolismo , Ácido N-Acetilneuramínico/química , Receptores de Superfície Celular/metabolismo
9.
Biochimie ; 84(2-3): 123-9, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12022943

RESUMO

Much of the action in the mammalian apoptotic program takes place at the mitochondrial level. Physicochemical characteristics and integrity of mitochondrial membranes may play a crucial role in the recruitment and multimerization of pro-apoptotic Bcl-2 family members, opening of the permeability transition pore complex (PTPC) and the release of mitochondrial components which trigger the 'intrinsic' pathways of cellular apoptosis and activate executioner caspases. Recent evidence has accumulated pointing toward the mitochondrial membranes as the key targets for lipid and glycolipid mediators of stress-induced apoptosis. Mitochondrial membranes may thus act as 'sensors' of cellular stress by quantitating the local accumulation of specific lipids and glycolipids. Acute accumulation of ceramides, directly or indirectly, profoundly affects mitochondrial functions. GD3 ganglioside, a glycolipid which is actively synthesized and transiently accumulates in the early stages of apoptosis, relocates to the mitochondrial membranes causing the opening of the PTPC and the release of apoptogenic factors. Mitochondrial membranes appear to represent a common destination where protein and glycolipid mediators of stress converge and where crucial decisions about cellular adaptation or apoptotic cell death are taken.


Assuntos
Apoptose/fisiologia , Ceramidas/metabolismo , Gangliosídeos/metabolismo , Mitocôndrias/fisiologia , Esfingolipídeos/metabolismo , Humanos , Canais Iônicos , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Transdução de Sinais , Receptor fas/metabolismo
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