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Bibliografia Scientifica


Si riporta di seguito un'ampia bibliografia riguardante lo stato d'arte scientifico in merito alla leucodistrofia di Krabbe. Sono state selezionale le pubblicazioni scientifiche più significative e recenti....

Per facilitare la lettura degli articoli scientifici selezionati ognuno di essi è seguito da un commento redatto della D.ssa Pannuzzo Giovanna. Per qualsiasi dubbio e/o approfondimento la D.ssa Pannuzzo rimane a vostra completa disposisizone email:[email protected]

Am J Hum Genet. 1990 Jul;47(1):37-44.
Krabbe disease locus mapped to chromosome 14 by genetic linkage.
Zlotogora J, Chakraborty S, Knowlton RG, Wenger DA.
Department of Biochemistry and Molecular Biology, Jefferson Medical College, Philadelphia, PA 19107.
Using genetic linkage we have localized the gene coding for galactocerebrosidase (GALC) to human chromosome 14. Patients with Krabbe disease and their family members were assayed for GALC activity in leukocytes or fibroblasts and were classified as affected, carrier, noncarrier, or unknown. Polymorphic DNA markers from chromosome 14 demonstrated a multipoint LOD score of 3.40 with GALC located 13 cM centromere distal to CRI-C70 (D14S24). This finding is consistent with the location of the mouse twitcher mutation (a model of human GALC deficiency) on chromosome 12, which has substantial homology to human chromosome 14. Our data do not support a previous report's localization of GALC to chromosome 17.

Tale pubblicazione riguarda la localizzazione del gene che codifica per la galattosilcerebrosidasi. Questa localizzazione è stata individuata sul cromosoma umano 14.

Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line.

Haq E, Giri S, Singh I, Singh AK.

Department of Pediatrics, Medical University of South Carolina, USA.

This study delineates the molecular mechanism underlying psychosine-induced oligodendroglial cell death. An immortalized human oligodendroglial cell line, MO3.13, was treated with exogenous psychosine (beta-galactosylsphingosine), a toxic metabolite that accumulates in the tissues of patients with Krabbe's disease. The mode of cell death induced by psychosine was found to be apoptotic, as revealed by different apoptotic markers viz., TUNEL, DNA fragmentation and caspase cleavage/activation. The action of psychosine was redox sensitive, as measured by changes in mitochondrial membrane potential (psidelta), and this effect of psychosine could be reversed by pre-treatment with the antioxidant molecules N-acetyl-l-cysteine or pro-cysteine. Psychosine directly affects the mitochondria as revealed by the activation of caspase 9 but not caspase 8. Up-regulation of the c-jun/c-jun N-terminal kinase pathway by psychosine leads to the induction of AP-1 and, at the same time, psychosine also down-regulates the lipopolysaccharide-induced NF-kappaB transactivation. These observations indicate that the mechanism of action of psychosine is, through the up-regulation of AP-1, a pro-apoptotic pathway as well as, through the down-regulation of the NF-kappaB pathway, an antiapoptotic pathway.

Questo studio riguarda il meccanismo molecolare attravero il quale la psicosina esercita il suo effetto tossico. L’apoptosi avviene tramite attivazione della caspasi 9, con sovraregolazione di AP-1(un meccanismo che favorisce l’apoptosi) e sottoregolazione del NF-KappaB(fattore neurotrofico), che è antiapoptotico.

Innherit Metab DIis. 1999 Apr;22(2):155-62.
Molecular heterogeneity of Krabbe disease.
Fu L, Inui K, Nishigaki T, Tatsumi N, Tsukamoto H, Kokubu C, Muramatsu T, Okada S.
Department of Pediatrics, Faculty of Medicine, Osaka University, Japan. [email protected]
Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive neurodegenerative disorder that affects both the central and peripheral nervous system due to an enzymatic defect of galactocerebrosidase (GALC). Following its cloning, many mutations in the galactocerebrosidase gene have been reported, but the correlation between phenotype and genotype was not clear in many cases. In this study we further investigated the molecular defects in another 10 patients (6 Japanese and 4 non-Japanese), using cultured skin fibroblasts, and found 10 mutations, of which 8 were novel, including a nonsense mutation (W647X) and 7 missense mutations (G43R, S52F, T262I, Y319C. W410G, R515H, T652R) in the coding region. Some phenotype-specific mutations were found but the other mutations were private. Mutations reported so far have been distributed over the whole GALC gene and it is difficult to speculate on functional domains of the GALC protein and phenotypically specific regions

In questo studio sono state studiate le mutazioni del gene della galattosilcerebrosidasi allo scopo di trovare una correlazione tra il fenotipo e il genotipo. Sono stati valutati 10 pazienti (6 giapponesi e 4 non giapponesi) e sono state trovate 10 mutazioni, delle quali 8 nuove, una non senso e 7 missenso nella regione codificante.

Mol Genet Metab. 2000 May;70(1):1-9.
Krabbe disease: genetic aspects and progress toward therapy.
Wenger DA, Rafi MA, Luzi P, Datto J, Costantino-Ceccarini E.
Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Krabbe disease or globoid cell leukodystrophy is a disorder involving the white matter of the peripheral and central nervous systems. Mutations in the gene for the lysosomal enzyme galactocerebrosidase (GALC) result in low enzymatic activity and decreased ability to degrade galactolipids found almost exclusively in myelin. The pathological changes observed, including the presence of globoid cells and decreased myelin, appear to result from the toxic nature of psychosine and accumulation of galactosylceramide that cannot be degraded due to the GALC deficiency. Over 60 mutations have been identified in this gene. The great majority are disease-causing; however, a few are considered polymorphisms. While most patients present with symptoms within the first 6 months of life, others present later in life including adulthood. Even patients with the same genotype can have very different clinical presentations and course. The reason for this is not known. Treatment at this time is limited to hematopoietic stem cell transplantation that appears to slow the progression of the disease and improve the magnetic resonance images. Studies using stem cells and viral vectors to transduce transplantable cells are under way in model systems. In culture, oligodendrocytes from the twitcher mouse model can assume a normal appearance after differentiation if GALC activity is provided via viral transduction or uptake from donor cells. Therefore continued myelination and/or remyelination in patients will require supplying GALC activity by transplanted cells or viral vectors to still functional endogenous oligodendrocytes or transplantation of normal oligodendrocytes or stem cells that can differentiate into oligodendrocytes. Using the animal models these options can be explored. Copyright 2000 Academic Press.

Tale studio riguarda lo stato d’arte delle possibilità terapeutiche di intervento nella leucodistrofia di Krabbe. In particolare, si parla del trapianto di cellule staminali e dello studio di vettori virali in vari modelli. In coltura cellulare, gli oligodentrociti assumono un aspetto normale dopo la differenziazione, se l’attività del GALC (l’enzima galattosilcerebrosidasi) viene fornita tramite traduzione virale o mediante cellule di un donatore

J Child Neurol. 2003 Sep;18(9):595-603.
Globoid cell leukodystrophy (Krabbe's disease): update.
Suzuki K.
Neuroscience Center, Department of Neurology, University of North Carolina School of Medicine, Chapel Hill, NC, USA. [email protected]
The classic globoid cell leukodystrophy (Krabbe's disease) is caused by genetic defects in a lysosomal enzyme, galactosylceramidase. It is one of the two classic genetic leukodystrophies, together with metachromatic leukodystrophy. The mode of inheritance is autosomal recessive. Typically, the disease occurs among infants and takes a rapidly fatal course, but rarer late-onset forms also exist. Clinical manifestations are exclusively neurologic with prominent white-matter signs. The pathology is unique, consisting of a rapid and nearly complete disappearance of myelin and myelin-forming cells--the oligodendrocytes in the central nervous system and the Schwann cells in the peripheral nervous system, reactive astroytic gliosis, and infiltration of the unique and often multinucleated macrophages ("globoid cells") that contain strongly periodic acid-Schiff (PAS)-positive materials. A normally insignificant but highly cytotoxic metabolite, galactosylsphingosine (psychosine), is also a substrate of galactosylceramidase and is considered to play a critical role in the pathogenesis. The galactosylceramidase gene has been cloned, and a large number of disease-causing mutations have been identified. Equivalent genetic galactosylceramidase deficiency occurs in several mammalian species, such as mouse, dog, and monkey. Recently, deficiency of one of the sphingolipid activator proteins, saposin A, was demonstrated to cause a late-onset, slowly progressive globoid cell leukodystrophy at least in the mouse, with all of the phenotypic consequences of impaired degradation of galactosylceramidase substrates. Human globoid cell leukodystrophy owing to saposin A deficiency might be anticipated and should be suspected in human patients with a late-onset leukodystrophy with normal galactosylceramidase activity when other possibilities are also excluded. The only serious attempt at treating human patients is bone marrow transplantation, which can provide significant alleviation of symptoms, particularly in those patients with later-onset, more slowly progressive globoid cell leukodystrophy.

Qui viene inquadrata in modo generale la leucodistrofia di Krabbe. Si tratta di una malattia genetica autosomica recessiva, causata dal difetto enzimatico della galattosilcerebrosidasi, che comporta la rapida scomparsa della mielina, con morte di oligodentrociti nel sistema nervoso centrale e di cellule di schwann nel sistema nervoso periferico, e infiltrazione di macrofagi multinucleati (cellule globoidi). Il metabolica tossico responsabile della patogenesi della malattia è la psicosina (galattosilsfingosina). Recentemente è stata inquadrata una leucodistrofie causata da deficienza di saposina A, con attività normale della galattosilcerebrosidasi.

Neurosci Lett. 2004 Apr 1;358(3):205-9.
Psychosine-induced apoptosis in a mouse oligodendrocyte progenitor cell line is mediated by caspase activation.
Zaka M, Wenger DA.

Department of Neurology, 1020 Locust Street, Room 394, Jefferson Medical College, Philadelphia, PA 19107, USA.

Globoid cell leukodystrophy (GLD) is an inherited neurological disorder caused by the deficiency of galactocerebrosidase (GALC) activity resulting in cell death of oligodendrocytes and subsequent demyelination. The death of oligodendrocytes is accompanied by accumulation of psychosine, which is also a substrate for the GALC enzyme. In this report, we investigated the mechanism of the toxic effect of psychosine in a mouse-derived oligodendrocyte progenitor cell line (OLP-II), a precursor to the cell type most affected in GLD. Psychosine caused cytotoxicity in a dose-dependent manner. Lower concentration of psychosine (5 microM) did not significantly reduce OLP-II cell numbers. However, 50 microM psychosine induced up to 45% cell death. These results were confirmed by the Tunel assay, which is a hallmark for the detection of apoptosis. Moreover, psychosine treatment resulted in the activation/cleavage of initiator caspase-8 and -9, and effector caspase-3. These results support a role for psychosine in OLP-II cell death via an apoptotic mechanism, and suggest the involvement of caspases.

In questa pubblicazione viene valutato il meccanismo attraverso il quale si verifica l’effetto tossico della psicosina. In particolare, 50 microM di psicosina inducono circa il 45% di morte cellulare, attraverso l’attivazione degli iniziatori caspasi 8 e 9, e dell’effettore caspasi 3.

Gene Ther. 2004 Aug;11(15):1188-94.
Transgenic rescue of Krabbe disease in the twitcher mouse.
De Gasperi R, Friedrich VL, Perez GM, Senturk E, Wen PH, Kelley K, Elder GA, Gama Sosa MA.
Department of Psychiatry, Mount Sinai School of Medicine of New York University, New York, NY, USA.

The twitcher mouse is a natural model of Krabbe disease caused by galactocerebrosidase (GALC) deficiency. Previous attempts at rescuing the twitcher mouse by bone marrow transplantion, viral transduction, or transgenesis were only partially successful. Here, we report the transgenic (tg) rescue of the twitcher mouse with a BAC clone harboring the entire GALC. The twi/twi/hGALC tg mice exhibited growth, motor function, and fertility similar to those of nonaffected animals. These animals had normal levels of GALC activity in brain and were free of the typical twitcher demyelinating pathology. Surprisingly, GALC expression in twi/twi hGALC tg kidneys was low and galactocerebroside storage was only partially cleared. Nonetheless, these mice have been maintained for over 1 year without any sign of disease. Since pathological damage associated with GALC deficiency is confined to the nervous system, our work represents the first successful rescue of the twitcher mouse and opens the possibility of developing novel therapeutic approaches.

Questo studio riguarda la trasduzione genica di un modello di topo twitcher con un clone BAC che contiene l’intero GALC. Tali animali avevano livelli normali dell’enzima e non mostravano la tipica patologia da demielinizzazione.

Ned Tijdschr Geneeskd. 2004 Apr 24;148(17):826-8.

[From gene to disease; Krabbe disease and galactosylceramidase deficiency]
Kleijer WJ, van Diggelen OP, Halley DJ, van der Ploeg AT, Mancini GM.
Afd. Klinische Genetica, Erasmus Medisch Centrum, Postbus 1738, 3000 DR Rotterdam. [email protected]

Krabbe disease is a devastating lysosomal storage disease with autosomal recessive inheritance. Early symptoms of leukodystrophy, such as irritability and hypertonicity, appear at 3 to 6 months of age, but progress rapidly to severe mental and motor deterioration and death in the second year. The disease is caused by the deficiency of the lysosomal enzyme galactosylceramidase, which is in turn caused by mutations in the GALC gene. The incidence of the infantile form of the disease in the Netherlands is estimated at 1.3 per 100,000 births; 50% of the patients' alleles show the large 30-kb deletion. Early diagnosis by enzyme assay in leukocytes or skin fibroblasts permits timely genetic counselling and prenatal diagnosis, which is reliably made by enzyme or mutation analysis in the chorionic villi.

Tale pubblicazione presenta la leucodistrofia di Krabbe come una malattia lisosomiale , ereditaria autosomica recessiva. I sintomi iniziali sono irritabilità e ipertonia, in genere tra i 3 e i 6 mesi di vita. Progredisce rapidamente con severo deterioramento mentale e motorio fino alla morte nel secondo anno di vita. L’incidenza in Olanda è stimata a 1.3 per ogni 100,000 nascite. È possibile la diagnosi prenatale.

Neurochem Res. 1999 Feb;24(2):287-93.
Transduction of cultured oligodendrocytes from normal and twitcher mice by a retroviral vector containing human galactocerebrosidase (GALC) cDNA.
Costantino-Ceccarini E, Luddi A, Volterrani M, Strazza M, Rafi MA, Wenger DA.
Centro Studio Cellule Germinali, Siena, Italy.
Krabbe disease or globoid cell leukodystropy is a lysosomal disorder caused by a deficiency of galactocerebrosidase (GALC) activity. This results in defects in myelin that lead to severe symptoms and early death in most human patients and animals with this disease. With the cloning of the GALC gene and the availability of the mouse model, called twitcher, it was important to evaluate the effects of providing GALC via a retroviral vector to oligodendrocytes in culture. After differentiation, the untransduced cells from normal mice extended highly branched processes while those from the twitcher mice did not. Oligodendrocytes in culture can be readily transduced to produce much higher than normal levels of GALC activity. Transduced normal and twitcher cells formed clusters when plated at high density. Transduction of twitcher oligodendrocytes plated at lower density, followed by differentiation, resulted in some cells having a completely normal appearance with highly branched processes. Other cells showed retraction and fragmentation. Perhaps over expression of GALC activity may be detrimental to oligodendrocytes. These studies demonstrate that the phenotype of twitcher oligodendrocytes can be corrected by providing GALC via gene transfer, and this could lead the way to future studies to treat this disease.

Qui si valutano gli effetti su colture di oligodentrociti a cui viene fornito GALC tramite un retrovirus. La trasduzione di oligodentrociti di modelli twitcher, a bassa densità, è seguita da differenziazione e risulta in cellule che hanno un aspetto completamente normale. Altre cellule mostrano frammentazione.

Neurobiol Dis. 2001 Aug;8(4):600-10.
Retrovirus-mediated gene transfer and galactocerebrosidase uptake into twitcher glial cells results in appropriate localization and phenotype correction.
Luddi A, Volterrani M, Strazza M, Smorlesi A, Rafi MA, Datto J, Wenger DA, Costantino-Ceccarini E.
Centro Studio Cellule Germinali, Consiglio Nazionale delle Ricerche, Via Pendola 62, Siena Italy, 53100.
Galactocerebrosidase (GALC) is deficient in all tissues from human patients and animal models with globoid cell leukodystrophy (GLD) or Krabbe disease. The deficiency results in decreased lysosomal catabolism of certain galactolipids including galactosylceramide and psychosine that are synthesized maximally during myelination. According to current theories, the accumulation of psychosine in humans and animals with GLD induces oligodendrocyte degeneration and myelination ceases. Transduction of oligodendrocytes from twitcher mice with a retroviral vector containing the GALC cDNA can correct the enzyme deficiency in these cells. Our data show that twitcher astrocytes and oligodendrocytes can internalize exogenous GALC, as well as donate the enzyme to the mutant glial cells. Antibodies against human GALC localized the GALC antigen in retrovirally transduced cells and cells receiving enzyme via cell to cell secretion and uptake to the lysosomal fraction. In fact immunocytochemical studies in transduced oligodendrocytes revealed that the GALC colocalizes in vesicles lysosomal-associated membrane protein-2 (LAMP2) (+). Moreover, labeling cells with anti-GALC and a marker for oligodendrocytes demonstrated that, upon differentiation, transduced, twitcher oligodendrocytes attained the normal branched process configuration, while untransduced cells show only abnormal morphology. Phenotype correction in mutant oligodendrocytes has also been observed after enzyme transfer. These studies indicate that GALC activity supplied to cultured oligodendrocytes from twitcher mice by different methods can correct the pathological phenotype of these cells. Copyright 2001 Academic Press.

Anche tale studio riguarda la trasduzione degli oligodentrociti di topo twitcher con GALC cDNA. Viene vista l’internalizzazione dell’enzima esogeno all’interno degli astrociti e degli oligodentrociti, con una correzione del fenotipo.

Purtroppo oggi poche persone sono a conoscenza della malattia denominata Leucodistrofia di Krabbe.... Una malattia terribile che degenera irremediabilmente il sistema nervoso sino a portare alla morte dell'individuo.Aiutaci a divulgare questo messaggio, tutti assieme possiamo lottare e dare una speranza in più a tanti bambini che come Grazia soffrono...
Intellisystem Technologies è coinvolta a pieno titolo nel Progetto Grazia e rende disponibile la propria tecnologia, prodotti e soluzioni al servizio di tutti coloro che ne possono trarre vantaggio contribuendo alla Ricerca Scientifica per lo studio di nuove terapie contro la Leudostrifoia di Krabbe...


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