Efficient CNS targeting in adult mice by intrathecal infusion of single-stranded AAV9-GFP for gene therapy of neurological disorders.

Adeno-associated virus (AAV) gene therapy constitutes a powerful tool for the treatment of neurodegenerative diseases. While AAVs are generally administered systemically to newborns in preclinical studies of neurological disorders, in adults the maturity of the blood-brain barrier (BBB) must be considered when selecting the route of administration. Delivery of AAVs into the cerebrospinal fluid (CSF) represents an attractive approach to target the central nervous system (CNS) and bypass the BBB. In this study, we investigated the efficacy of intra-CSF delivery of a single-stranded (ss) AAV9-CAG-GFP vector in adult mice via intracisternal (iCist) or intralumbar (it-Lumb) administration. It-Lumb ssAAV9 delivery resulted in greater diffusion throughout the entire spinal cord and green fluorescent protein (GFP) expression mainly in the cerebellum, cortex and olfactory bulb. By contrast, iCist delivery led to strong GFP expression throughout the entire brain. Comparison of the transduction efficiency of ssAAV9-CAG-GFP versus ssAAV9-SYN1-GFP following it-Lumb administration revealed widespread and specific GFP expression in neurons and motoneurons of the spinal cord and brain when the neuron-specific synapsin 1 (SYN1) promoter was used. Our findings demonstrate that it-Lumb ssAAV9 delivery is a safe and highly efficient means of targeting the CNS in adult mice.

Toward a better understanding of brain lesions during metachromatic leukodystrophy evolution.

BACKGROUND AND PURPOSE: The prospect of new therapies in MLD stresses the need to refine the indications for treatment. The aim of this study was, therefore, to perform a detailed analysis of MRI brain lesions at diagnosis and follow-up, to better understand the natural history of MLD. MATERIAL AND METHODS: This retrospective case-control study (2005-2010) looked at 13 patients with MLD (2-5 years of age) with 28 MRIs (mean follow-up, 2 years), compared with 39 age- and sex-matched controls. All MRIs were evaluated qualitatively and semiquantitatively. The Student t test, Wilcoxon signed rank test, and Pearson correlation were used for statistical analysis (P < .05). RESULTS: In addition to diffuse symmetric supratentorial WM T2 hyperintensities with a tigroid pattern (70%) and T2 hyperintensities in the CC (100%) and internal capsules (46%), we found significant GM abnormalities such as thalamic T2 hypointensity (92%), thalamic (23%, P < .05, EJ) and caudate nuclei (23%, P < .05, EJ) atrophy, and cerebellar atrophy without WM involvement (15%). The pattern of splenium involvement progression was misleading, with initially diffuse high signal intensity, which later became curvilinear before finally progressing to atrophy (23%, P < .05; EJ). This should not be mistaken for a disease regression. Spectroscopy confirmed a decrease in the NAA/Cr ratio, an increase in the Cho/Cr ratio and in myo-inositol, and a lactate resonance. CONCLUSIONS: Thalamic changes may be a common finding in MLD, raising the prospect of primary GM lesions. This may prove important when evaluating the efficacy of new treatments.

Invariant NKT cells in adrenoleukodystrophy patients and mice.

X-linked adrenoleukodystrophy (X-ALD) is a severe neurological disease characterized by progressive demyelination within the CNS, adrenal insufficiency, and is associated with an accumulation of saturated very long chain fatty acids in plasma and tissues of patients. iNKT cells, a distinct lineage of T cells recognizing glycolipid antigens through CD1d molecules, exert immunoregulatory functions and can prevent various immune mediated-pathologies. In ALD patients, but not in ALD deficient mice, iNKT cell frequency and CD1d expression on the surface of B cells are slightly decreased. However, such minor differences might not influence the pathogenesis of the disease.

Gene therapy in metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by deficiency of the lysosomal enzyme arylsulfatase A. Deficiency of this enzyme results in intralysosomal storage of sphingolipid cerebroside 3-sulfates (sulfatides), which are abundant in myelin and neurons. A pathological hallmark of MLD is demyelination and neurodegeneration, causing various and ultimately lethal neurological symptoms. This review discusses the potential therapeutic application of hematopoietic stem cell gene therapy and intracerebral gene transfer (brain gene therapy) in patients with MLD.

[Subtle brain abnormalities in adrenomyeloneuropathy]. / Anomalies cérébrales subtiles de l'Adrénomyéloneuropathie.

PURPOSE: To describe subtle brain abnormalities detected on MRI in adult patients with adrenomyeloneuropathy (AMN). Materials and methods. Retrospective evaluation of data acquired prospectively as part of a clinical trial (Riluzole) in 66 adult patients with AMN without obvious brain lesion on MR. All patients underwent brain MR including T1W, T2W, FLAIR and spectroscopy. After a review had been validated by three different reviewers, review of MR images was performed by consensus using a semi-quantitative scale. RESULTS: Preliminary analysis of MR images confirmed the presence of signal abnormalities involving the corticospinal tracts in 36 patients (54.6%). Additional subtle abnormalities were also detected: white matter palor, mainly parieto-occipital in location, with patchy hyperintensity in 36 patients (54.6%), hyperintense pontocerebellar fibers on T2W and FLAIR in 25 patients (41.7%). The presence of elevated Cho/Cr and mI/Cr ratios, described in the literature, were confirmed. CONCLUSION: This retrospective study allows the description of an AMN pattern on MRI in patients without white matter or callosal abnormalities.

[X-linked adrenoleukodystrophy]. / Adrénoleucodystrophie liée à l'X.

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder. ALD is characterized by progressive demyelination within the central and peripheral nervous system, adrenal insufficiency (Addison's disease) and accumulation of very-long-chain fatty acids (VLCFA) in plasma, fibroblasts and tissues. The overall incidence of ALD is 1:17,000 including hemizygotes and heterozygotes who are frequently symptomatic. There are two main ALD phenotypes: 1) a cerebral demyelinating form which affects boys between 5-12 years, but also 35% of adult males; 2) a form that mainly involves the spinal cord (adrenomyeloneuropathy, AMN) in adult males between 20-50 years and 50% of heterozygous women after the age of 40 years. AMN presents with progressive spastic paraparesis. Addison's disease may be the first symptom of ALD in boys and adult males. These patients are at risk to develop cerebral ALD or AMN for life. ALD results from mutations in the ABCD1 gene without correlation between genotype and phenotype. The diagnosis of ALD relies upon the measurement of plasma VLCFA levels that allows the identification of 100% affected males and of 80-95% heterozygous women. Because of these false-negative, it is therefore mandatory to search for a mutation in the ABCD1 gene in all women at risk to be heterozygous for ALD. The ABCD1 gene encodes a peroxisomal transmembrane protein (ALD protein) with the structure of an half ATP-binding cassette transporter. It is possible that ALD protein imports VLCFA or VLCFA-CoA into peroxisomes in which they are degraded by a peroxisomal beta-oxidation system. Elongation of VLCFAs is enhanced in fibroblasts from ALD patients and likely contributes to the load of VLCFA in tissues. The underlying mechanisms that lead to cerebral demyelination, axonal degeneration in spinal cord and adrenal insufficiency are unknown. The "toxic" role of VLCFA accumulation remains to be demonstrated. The mechanisms that lead to the inflammatory reaction in cerebral ALD might involve abnormal acylation of gangliosides and phospholipids by VLCFA that would result in immune reaction of brain macrophages and astrocytes bearing CD1 molecules that recognize lipid antigens. De novo mutation of ABCD1 occurs in less than 8% of ALD patients. The genetic counseling aims to identify: 1) women who are at risk to be heterozygous; 2) neurologically asymptomatic boys. It is only at this stage that allogeneic bone marrow transplantation has clinical benefit; 3) ALD patients who have Addison's disease that can lead to sudden death. Prenatal diagnosis (chorionic villus samples, cultured amniotic fluid cells) relies upon DNA based mutation detection techniques, expression of ALD protein and measurement of VLCFA levels. Allogeneic bone marrow transplantation is the only treatment that provides a permanent cure when the procedure is performed at an early stage of cerebral demyelination, i.e when the patients are asymptomatic despite abnormal brain MRI. Treatment of Addison's disease is mandatory but does not modify the course of neurological symptoms. Dietary therapy failed to halt the neurologic progression in cerebral ALD and AMN. It might have a partial preventive effect in boys treated before 6 years of age.

Enzyme, cell and gene-based therapies for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a demyelinating storage disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). Lack of ARSA activity leads to the accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the central and peripheral nervous systems. Based on the age at onset, the disease is usually classified into three forms: the late-infantile form, which manifests in the second year of life; the juvenile variants (onset between 4 and 12 years), which are subdivided into early-juvenile (EJ, onset before 6 years) and late-juvenile (LJ, onset after 6 years); and the adult form (onset after 12 years of age). Currently, there is no efficient therapy for the late-infantile form of MLD (50% of the patients), death occurring within a few years after onset of neurological symptoms. Allogeneic haematopoietic cell transplantation (HCT), when performed at a very early stage of the disease, may improve selected patients with juvenile or adult forms of MLD. As with other lysosomal storage diseases, the physiopathology of MLD is poorly understood. Demyelination is the main pathological finding, but substantial storage of sulfatides in neurons also occurs, and may contribute to the clinical phenotype. The physiopathological process leading to neuronal and glial cell degeneration and apoptosis involves accumulation of undegraded sulfatides but also secondary abnormalities (storage/mislocalization of unrelated lipids, inflammatory processes). This review summarizes the recent advances in the understanding of the physiopathology of MLD and the new therapeutic perspectives currently under preclinical investigation, including enzyme replacement therapy, gene therapy and cell therapy.

Partial cure of established disease in an animal model of metachromatic leukodystrophy after intracerebral adeno-associated virus-mediated gene transfer.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by genetic deficiency of arylsulfatase A (ARSA) enzyme. Failure in catalyzing the degradation of its major substrate, sulfatide (Sulf), in oligodendrocytes and Schwann cells leads to severe demyelination in the peripheral (PNS) and central nervous system (CNS), and early death of MLD patients. The ARSA knockout mice develop a disease that resembles MLD but is milder, without significant demyelination in the PNS and CNS. We showed that adeno-associated virus serotype 5-mediated gene transfer in the brain of ARSA knockout mice reverses Sulf storage and prevents neuropathological abnormalities and neuromotor disabilities when vector injections are performed at a pre-symptomatic stage of disease. Direct injection of viral particles into the brain of ARSA knockout mice at a symptomatic stage results in sustained expression of ARSA, prevention of Sulf storage and neuropathological abnormalities. Despite these significant corrections, the treated mice continue to develop neuromotor disability. We show that more subtle biochemical abnormalities involving gangliosides and galactocerebroside are in fact not corrected.

[Adult onset hereditary leukoencephalopathies]. / Leucoencéphalopathies génétiques de l'adulte.

In clinical practice, the term "genetic leukoencephalopathy" refers to a group of genetic diseases whose common point is to give an aspect of diffuse leukoencephalopathy on MRI. With progress in diagnostic techniques including radiology, biochemistry or genetics, a large number of hereditary diseases causing leukoencephalopathy have been identified. Although generally beginning in childhood, these diseases often have more insidious clinical forms which can begin in adulthood. These forms remain poorly known. Some are accessible to treatment so their diagnosis appears essential. The diagnostic steps must be guided by clinical examination (neurological, ophthalmological and systemic), electromyography and MRI. The purpose of this review is to propose a classification of the genetic leukoencephalopathies and to give a progress report applicable in neurological practice.

[X-linked adrenoleukodystrophy in a female proband: clinical presentation, biological diagnosis and family consequences]. / Adrénoleucodystrophie liée à l'X chez une proposante symptomatique: présentation clinique, diagnostic biologique et conséquences familiales.

INTRODUCTION: X-linked adrenoleukodystrophy (ALD) is the most frequent type of leukodystrophy (1/17 000 males). The phenotypic range in male patients varies from the severe cerebral presentations in children to the milder myeloneuropathy and to isolate adrenal insufficiency. More than a half of the carrier females display clinical symptoms over the age of 40 years. OBSERVATION: Diagnosis of ALD was raised in a 40 year-old female who presented with spastic paraparesis and sphincterian dysfunction, occurring after the delivery of her first child. There was no family history of ALD. Very long-chain fatty acids (VLFCA) were assayed in her one-year-old son in order to propose appropriate hormonal and neurological survey. His dosage was abnormal and an adrenal insufficiency was subsequently found. A brain MRI will be proposed biannually when he reaches to age of for years. The proband's mother had an increased level of VLCFA, showing that she was a carrier. Family screening was extended to the proband's sisters and maternal aunt who already had children, but also to her brother, who may express a mild form of the disease later on, and to her maternal uncles who might be asymptomatic carriers. A frameshift mutation was found in the ABCD1 gene and will allow accurate carrier identification and prenatal diagnosis in the family. CONCLUSION: ALD diagnosis should be evoked in a woman affected by myelopathy despite the lack of family history. Such a diagnosis has severe consequences since some of the related males may carry the mutation although they do not display any symptom at time of diagnosis, and because carrier females have a risk to both have a clinical expression of the disease and give birth to an affected boy.

Microalbuminuria and markers of the atherosclerotic process: the D.E. S.I.R. study.

The relationship between microalbuminuria and tissue-type plasminogen activator antigen (tPA-ag) and fibrinogen was evaluated in non-diabetic subjects. Subjects were participants of the D.E.S.I. R. (Data from an Epidemiological Study on the Insulin Resistance syndrome) Study. Analyses were carried out on 2248 women and 2402 men for fibrinogen and on 272 women and 284 men for tPA-ag. Microalbuminuria was defined as urinary albumin concentration greater than 20 mg/l. Men with microalbuminuria had a 6% higher fibrinogen concentration than those without (3.07 g/l (95% confidence interval: 2.99,3.15) vs. 2.89 g/l (2.87,2.91), adjusted for age and smoking). This relationship existed in hypertensive as well as non-hypertensive subjects. The association between microalbuminuria and tPA-ag existed only in hypertensive men, those with microalbuminuria having a 21% higher tPA-ag than those without (4.39 ng/ml (3.70,5.08) vs. 3.63 ng/ml (3.32,3.94), adjusted for age and smoking). Adjustment for other risk markers for cardiovascular disease did not change the results. There was no relationship between microalbuminuria and these haemostatic factors in women. The results of this study suggest that in non-diabetic men, microalbuminuria is associated with fibrinogen, but with tPA-ag only when concomitant with hypertension.

Apoptosis in the central nervous system of cerebral adrenoleukodystrophy patients.

The childhood cerebral form of adrenoleukodystrophy (ALD) is a fatal demyelinating disease, yet mice deficient in the ALD gene do not show such clinicopathological phenotype. We have therefore investigated in human autopsy tissues whether the ALD gene mutation results in apoptosis of CNS cells. Specimens from telencephalic and brainstem regions of four patients, and three controls were examined for internucleosomal DNA fragmentation, in situ detection of DNA breaks by the TUNEL method, and caspase-3 immunostaining. None of the controls showed significant apoptosis in white matter, while apoptotic nuclei with chromatin alterations were detected in areas of active demyelination in three ALD patients. A large proportion of apoptotic cells were oligodendrocytes and some express activated caspase-3. TUNEL-positive nuclei and/or caspase-3 staining were also detected in perivascular infiltrates and, occasionally, in neurons. We conclude that apoptosis of oligodendrocytes may account, at least in part, for the demyelinating process in the ALD brain.

Long-term effect of bone-marrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy.

BACKGROUND: The childhood-onset cerebral form of X-linked adrenoleukodystrophy, a demyelinating disorder of the central nervous system, leads to a vegetative state and death within 3-5 years once clinical symptoms are detectable. The hypothesis to be tested was whether bone-marrow transplantation can over an extended period of time halt the inexorable progressive demyelination and neurological deterioration. METHODS: 12 patients with childhood onset of cerebral X-linked adrenoleukodystrophy have been followed for 5-10 years after bone-marrow transplantation. Magnetic resonance imaging (MRI), neurological, neuropsychological, electrophysiological, and plasma very-long-chain fatty acid (VLCFA) measurements were used to evaluate the effect of this treatment. FINDINGS: MRI showed complete reversal of abnormalities in two patients and improvement in one. One patient showed no change from baseline to last follow-up. All eight patients who showed an initial period of continued demyelination stabilised and remained unchanged thereafter. Motor function remained normal or improved after bone-marrow transplantation in ten patients. Verbal intelligence remained within the normal range for 11 patients. Performance (non-verbal) abilities were improved or were stable in seven patients. Decline in performance abilities followed by stability occurred in five patients. Plasma VLCFA concentrations decreased by 55% and remained slightly above the upper limits of normal. INTERPRETATION: 5-10-year follow-up of 12 patients with childhood-onset cerebral X-linked adrenoleukodystrophy shows the long-term beneficial effect of bone marrow transplantation when the procedure is done at an early stage of the disease.

MR spectroscopic imaging of normal-appearing white matter in adrenoleukodystrophy.

BACKGROUND: Adrenoleukodystrophy (ALD) is characterised by wide phenotypic variation, and there is no marker to predict the onset of cerebral demyelination. The indications for therapeutic approaches depend largely on the onset of cerebral demyelination. OBJECTIVE: To evaluate the brain spectroscopic pattern in normal-appearing white matter (NAWM) in patients with various phenotypes of ALD to determine if these abnormalities could be of useful prognostic value. MATERIALS AND METHODS: Spectroscopic imaging acquisition mode (MRSI, 16 x 16 voxels) was performed in 20 patients with ALD, including 7 neurologically asymptomatic patients without detectable demyelination on MRI, 3 patients with early signs of cerebral demyelination, 8 patients with adrenomyeloneuropathy (AMN) and 2 patients with cerebral ALD who had previously undergone bone marrow transplantation. Controls were 22 healthy subjects. In all patients, four voxels entirely located in the juxtaventricular NAWM were studied. The ratios NAA/Cho, NAA/ CPC and Cho/CPC for the four ROIs were measured in the patient population and compared with control values. Results. In spite of a large distribution of ratios, the statistical tests did not show any significant difference between the ratios within NAWM in the patient population compared with control values. Means of ratios in the left posterior (LP) voxel compared normal subjects were (a) in neurologically asymptomatic ALD patients (n = 7) 2.27 +/- 0.63 for NAA/CPC, 2.21 +/- 0.75 for NAA/ Cho, 1.06 +/- 0.28 for Cho/CPC, (b) in patients with early signs of demyelination (n = 3) 3.43 +/- 0.85 for NAA/ CPC, 2.47 +/- 0.32 for NAA/Cho, 1.37 +/- 0.16 for Cho/CPC and (c) in AMN patients (n = 8) 1.47 +/- 0.53 for NAA/CPC, 2.17 +/- 1.58 for NAA/ Cho, 0.83 +/- 0.32 for Cho/CPC. Conclusions. The study did not show significant differences in metabolite ratios between patients and controls. The large distribution of results precludes the possibility of detecting small variations. Part of this distribution can be due to the CSI method. Longitudinal spectroscopic studies, preferentially using monovoxel spectroscopy, are clearly needed.

Simvastatin does not normalize very long chain fatty acids in adrenoleukodystrophy mice.

X-linked adrenoleukodystrophy (ALD) is a genetic demyelinating disorder characterized by accumulation of very long chain fatty acid (VLCFA) in tissues. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, normalizes VLCFA in fibroblasts and plasma from ALD patients. We dietary treated ALD mice with simvastatin, an analog of lovastatin with similar pharmacokinetics and effects on plasma VLCFA in ALD patients at 20 or 60 mg/kg/day for 6-12 weeks. No decrease of VLCFA content was observed in mouse tissues, including the brain. A significant increase of VLCFA was rather observed in the brain of ALD mice at 60 mg/kg/day.

Molecular mechanism of detectable catalase-containing particles, peroxisomes, in fibroblasts from a PEX2-defective patient.

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.