Cerebral Phospho-Tau Acts Synergistically with Soluble Aß42 Leading to Mild Cognitive Impairment in AAV-AD Rats.

BACKGROUND: Alzheimer's disease (AD) is a continuum of events beginning with an increase in brain soluble Aß42 followed by the appearance of hyperphosphorylated tau (P-tau, asymptomatic stage). Mild Cognitive Impairment (MCI) then appears (prodromal stage). However, the individual contribution of these two soluble proteins in the onset of the first cognitive symptoms remains unclear. OBJECTIVES: We sought to understand the specific impact of p-tau on the development of MCI in the AAV-AD rat model, a model of late-onset Alzheimer's disease (LOAD) predementia. METHODS: We specifically reduced the phosphorylation level of tau while leaving Aß42 levels unchanged using a DYRK1A protein kinase inhibitor, Leucettine L41, in an adeno-associated virus-based Alzheimer's disease (AAV-AD) rat model. Leucettine L41 was administered by intraperitoneal injection at 20 mg/kg per day in AAV-AD rats from 9 (late asymptomatic phase) to 10 (prodromal phase) months of age. RESULTS: Decreased soluble forms of P-tau induced by chronic administration of Leucettine L41 did not change soluble Aß42 levels but prevented MCI onset in 10-month-old AAV-AD rats. CONCLUSIONS: The present study argues that P-tau is required to induce the development of MCI. Consistent with our previous findings that soluble Aß42 is also required for MCI onset, the data obtained in the AAV-AD rat model confirm that the transition from the asymptomatic to the prodromal stage may be caused by the combined presence of both soluble brain forms of Aß42 and p-tau, suggesting that the development of MCI may be the consequence of their synergistic action.

Evaluation of Memantine in AAV-AD Rat: A Model of Late-Onset Alzheimer's Disease Predementia.

BACKGROUND: Though our understanding of Alzheimer's disease (AD) remains elusive, it is well known that the disease starts long before the first signs of dementia. This is supported by the large number of symptomatic drug failures in clinical trials and the increased trend to enroll patients at predementia stages with either mild or no cognitive symptoms. However, the design of pre-clinical studies does not follow this attitude, in particular regarding the choice of animal models, often irrelevant to mimic predementia Late Onset Alzheimer's Disease (LOAD). OBJECTIVES: We aimed to pharmacologically validate the AAV-AD rat model to evaluate preventive treatment of AD. METHODS: We evaluated an N-methyl-D-aspartate receptor antagonist, named memantine, in AAV-AD rats, an age-dependent amyloid rat model which closely mimics Alzheimer's pathology including asymptomatic and prodromal stages. Memantine was used at a clinically relevant dose (20 mg daily oral administration) from 4 (asymptomatic phase) to 10 (mild cognitive impairment phase) months of age. RESULTS: A 6-month treatment with memantine promoted a non-amyloidogenic cleavage of APP followed by a decrease in soluble Aß42. Consequently, both long-term potentiation and cognitive impairments were prevented. By contrast, the levels of hyperphosphorylated endogenous tau remained unchanged, indicating that a long-term memantine treatment is ineffective to restrain the APP processing-induced tauopathy. CONCLUSIONS: Together, our data confirm that relevant models to LOAD, such as the AAV-AD rat, can provide a framework for a better understanding of the disease and accurate assessment of preventive treatments.

Candida nivariensis: Identification strategy in mycological laboratories.

Candida nivariensis is a cryptic fungal species classified within the Candida glabrata complex. It was described for the first time in 2005 by the means of DNA sequencing. We report a rare case of C. nivariensis deep-seated infection occurring in a 77-year-old man hospitalized for cysto-prostatectomy. Phenotypic testing based on the direct examination and the macroscopic features of the in vitro culture initially suggested C. glabrata species, while MALDI-TOF mass spectrometry enables correct identification. The isolate was found resistant to fluconazole, like in almost 20% of the reported cases. Herein, we present our practical strategy to reliably characterize this rare cryptic species. To date, MALDI-TOF mass spectrometry-based analysis showed very good results for such a purpose.

Current and emerging avenues for Alzheimer's disease drug targets.

Alzheimer's disease (AD), the most frequent cause of dementia, is escalating as a global epidemic, and so far, there is neither cure nor treatment to alter its progression. The most important feature of the disease is neuronal death and loss of cognitive functions, caused probably from several pathological processes in the brain. The main neuropathological features of AD are widely described as amyloid beta (Aß) plaques and neurofibrillary tangles of the aggregated protein tau, which contribute to the disease. Nevertheless, AD brains suffer from a variety of alterations in function, such as energy metabolism, inflammation and synaptic activity. The latest decades have seen an explosion of genes and molecules that can be employed as targets aiming to improve brain physiology, which can result in preventive strategies for AD. Moreover, therapeutics using these targets can help AD brains to sustain function during the development of AD pathology. Here, we review broadly recent information for potential targets that can modify AD through diverse pharmacological and nonpharmacological approaches including gene therapy. We propose that AD could be tackled not only using combination therapies including Aß and tau, but also considering insulin and cholesterol metabolism, vascular function, synaptic plasticity, epigenetics, neurovascular junction and blood-brain barrier targets that have been studied recently. We also make a case for the role of gut microbiota in AD. Our hope is to promote the continuing research of diverse targets affecting AD and promote diverse targeting as a near-future strategy.

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.

Gene therapy strategies for X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is the most frequently seen genetic disorder involving the myelin of the central nervous system. The cerebral form affects mainly boys between five to 12 years, leading to vegetative state or death within two to four years. The adult form affects the spinal cord, leading to severe paraplegia often complicated by cerebral demyelination. The ALD gene encodes an ATP-binding cassette transporter involved in the transport of very long chain fatty acids into peroxysomes. Specific subpopulations of oligodendrocytes and microglia are particularly affected by the ALD gene mutation and thus should be the target cells of gene therapy approaches. Two different and potentially complementary therapeutic strategies are currently evaluated. The first approach aims at replacing the endogenous brain microglia from patients by autotransplantation of genetically corrected hematopoietic stem cells using a lentiviral vector. The second approach aims at targeting directly the ALD gene into brain glial cells using stereotactic injections of viral vectors.

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.

Homo- and heterodimerization of peroxisomal ATP-binding cassette half-transporters.

Mammalian peroxisomal proteins adrenoleukodystrophy protein (ALDP), adrenoleukodystrophy-related protein (ALDRP), and 70-kDa peroxisomal protein (PMP70) belong to the superfamily of ATP-binding cassette (ABC) transporters. Unlike many ABC transporters that are single functional proteins with two related halves, ALDP, ALDRP, and PMP70 have the structure of ABC half-transporters. The dysfunction of ALDP is responsible for X-linked adrenoleukodystrophy (X-ALD), a neurodegenerative disorder in which saturated very long-chain fatty acids accumulate because of their impaired peroxisomal beta-oxidation. No disease has so far been associated with mutations of adrenoleukodystrophy-related or PMP70 genes. It has been proposed that peroxisomal ABC transporters need to dimerize to exert import functions. Using the yeast two-hybrid system, we show that homo- as well as heterodimerization occur between the carboxyl-terminal halves of ALDP, ALDRP, and PMP70. Two X-ALD disease mutations located in the carboxyl-terminal half of ALDP affect both homo- and heterodimerization of ALDP. Co-immunoprecipitation demonstrated the homodimerization of ALDP, the heterodimerization of ALDP with PMP70 or ALDRP, and the heterodimerization of ALDRP with PMP70. These results provide the first evidence of both homo- and heterodimerization of mammalian ABC half-transporters and suggest that the loss of ALDP dimerization plays a role in X-ALD pathogenesis.

Retroviral-mediated adrenoleukodystrophy-related gene transfer corrects very long chain fatty acid metabolism in adrenoleukodystrophy fibroblasts: implications for therapy.

X-linked adrenoleukodystrophy is a demyelinating disorder of the central nervous system with an impaired very long chain fatty acid metabolism. The adrenoleukodystrophy gene encodes a peroxisomal membrane protein that is part of a family of related ATP-binding transporters including the adrenoleukodystrophy-related protein. The adrenoleukodystrophy protein and adrenoleukodystrophy-related protein show 66% identity and have a mirror expression in most mouse tissues. We show that retroviral-mediated adrenoleukodystrophy-related gene transfer corrects very long chain fatty acid accumulation in adrenoleukodystrophy fibroblasts, irrespective of the presence or absence of adrenoleukodystrophy protein. Pharmacological approaches aiming at overexpressing the adrenoleukodystrophy-related gene in the central nervous system of adrenoleukodystrophy patients might thus offer new therapeutic leads.

Retroviral transfer and long-term expression of the adrenoleukodystrophy gene in human CD34+ cells.

Adrenoleukodystrophy (ALD) is a demyelinating disease of the central nervous system that results from a genetic deficiency of ALDP, an ABC protein involved in the transport of very long-chain fatty acids (VLCFAs). The cloning of the ALD gene and the positive effects of allogeneic bone marrow transplantation support the feasibility of a gene therapy approach. We report the retroviral transfer of the ALD cDNA to peripheral blood and bone marrow CD34+ cells from control donors and ALD patients. Prestimulation of these cells with cytokines, followed by infection with the M48-ALD retroviral vector, resulted in 20% transduction efficiency (4-40%) and expression of the vector-encoded ALDP in 20% of CD34+ cells (7.3-50%). Long-term culture (LTC) of transduced CD34+ cells from two ALD patients showed efficient transduction (24-28%) and stable expression (25-32%) of ALDP in derived clonogenic progenitors at 3 weeks of culture. The expression of ALDP in CFU cells derived from 5 and 6 weeks of LTC confirmed the effective transduction of LTC-initiating cells. Expression of ALDP was observed in CD68+ CFU-derived cells, suggesting that monocyte-macrophages, the target bone marrow cells in ALD, were produced from transduced progenitor cells. VL-CFA content was corrected in LTC and CFU-derived cells in proportion to the percentage of transduced cells, indicating that the vector-encoded ALDP was functional. Although not efficient yet to allow a clinical perspective, these results demonstrate the feasibility of ALD gene transfer into CD34+ cells of ALD patients.

Discrete brain areas express the insulin-responsive glucose transporter GLUT4.

Whether or not glucose utilization in the brain is insulin-dependent is still a controversial issue. We looked for the presence of the insulin-sensitive glucose transporter (GLUT4) in rat brain and obtained the following results: (1) poly(A) RNAs from the hypothalamus and anterior medulla oblongata hybridize with a cDNA probe for GLUT4; (2) reverse transcription-polymerase chain reaction (RT-PCR) on RNA from various brain nuclei detects GLUT4 transcripts; (3) immunocytochemistry, using a polyclonal antibody to GLUT4; reveals a specific immunostaining pattern, whereas both electronic microscopy and double immunofluorescence staining, using a neurofilament protein marker, indicate a neuronal localization. These results are discussed in terms of a putative neuromodulator role of insulin, via glucose utilization, in brain areas involved in the regulation of fuel metabolism.

Retroviral-mediated gene transfer corrects very-long-chain fatty acid metabolism in adrenoleukodystrophy fibroblasts.

Adrenoleukodystrophy (ALD), a lethal demyelinating disease of the brain, is caused by mutations of a gene encoding an ATP-binding transporter, called ALDP, localized in the peroxisomal membrane. It is associated with a defective oxidation of very-long-chain fatty acids, leading to their accumulation in many tissues. This study reports that the retroviral-mediated transfer of the ALD cDNA restored very-long-chain fatty acid oxidation in ALD fibroblasts in vitro following abundant expression and appropriate targeting of the vector-encoded ALDP in peroxisomes. The same method may be used in hematopoietic cells as a further step of a gene therapy approach of ALD.

[The creation of diet-dependent cancer models using transgenesis in animals]. / Création de modèles de cancers dépendant du régime alimentaire par transgenèse chez l'animal.

We have created transgenic mice lines in which SV40 T and c-myc expression was controlled by the L-pyruvate kinase gene regulatory region which is responsible for hepatic and pancreatic expression specificity, and strong dependence of this expression upon the carbohydrate composition of the diet. Models of hepatoma and endocrine pancreatic tumors have been obtained. Both tumors were dependent upon the diet, since carbohydrates strongly increased frequency and precocity of both hepatic and pancreatic carcinomas.

[Medium-chain acyl-CoA dehydrogenase deficiency: contribution of molecular biology]. / Déficit en déshydrogénase des acyl-CoA à chaîne moyenne: apport de la biologie moléculaire.

BACKGROUND: Medium-chain acyl-CoA dehydrogenase deficiency is the most frequent cause of defective congenital fatty acid oxidation. Its molecular characterization is now possible. Case n. 1. A girl, 15 month-old, was admitted because she suffered from fever and vomiting, requiring the administration of aspirin. One day later, she showed signs of drowsiness and hypotonia; her blood glucose concentration was 0.3 g/l. She was given intravenous glucose and this episode rapidly passed. Case n. 2. A boy, brother of the preceding patient, was routinely investigated; he was never symptomatic. Case n. 3. A boy, sibling of the two preceding children, was admitted at the age of 18 months because he had gone into a coma during a febrile episode. His blood glucose concentration was 0.15 g/l. This episode was rapidly resolved by a glucose infusion. His fasting blood concentrations of glucose, non esterified fatty acids. beta-hydroxybutyrate, lactate and pyruvate were normal as were his blood carnitine and ammonia, but he showed elevated urinary excretion of dicarboxylic acids. METHODS: Genomic DNA was extracted from peripheral leukocytes of the three sibs and their parents. The A-->G mutation at nucleotide 985 of the MCAD gene was detected by amplification and creation of a restriction site (ACRS). The implicated segment of this gene was amplified by PCR. RESULTS: ACRS showed that the symptomatic children were homozygous for the A-->G mutation, whereas their parents were heterozygous. The third asymptomatic child did not carry the mutation. CONCLUSIONS: Molecular biology techniques are appropriate for diagnosing this potentially lethal disease and their use for screening is important for disease prevention.

Expression of the L-type pyruvate kinase gene and the hepatocyte nuclear factor 4 transcription factor in exocrine and endocrine pancreas.

The L-pyruvate kinase (L-PK) gene is slightly active in normal and tumoral endocrine pancreatic tissues while, in vivo, this gene is not transcribed in the exocrine pancreas. Nevertheless, the L-PK gene is re-expressed at a very low level in cultured 266.6 cells derived from an exocrine pancreas carcinoma. The L-PK gene is early activated in endodermal tissues, e.g. yolk sac and primitive intestine; it remains transcribed in fetal pancreas. In adult, L-PK gene expression is restricted to some endocrine cells. Hepatocyte nuclear factor (HNF) 1 and HNF4 are the main tissue-restricted transcription factors involved in tissue-specific expression of the L-PK gene. HNF1 concentration is similar in liver and all pancreatic cells. HNF4 concentration is high in liver, much lower in islets of Langerhans, endocrine pancreatic tumors, and cultured insulinoma cells, and is scarcely detectable in adult exocrine pancreas. This distribution of HNF4 parallels the expression of the L-PK gene. In vivo footprinting experiments show that the HNF1 binding site is similarly occupied in both adult liver and adult pancreas, in which this gene is practically inactive. In this latter tissue, however, the HNF4 binding site is differently occupied with respect to the liver. Since the chromatin structure remains open around the L-PK promoter in pancreas, the L-PK gene can probably be re-expressed under certain circumstances, for instance in cancerous pancreatic cells.

Glucose transporter 2 (GLUT 2): expression in specific brain nuclei.

In the brain, certain neurons appear to be sensitive to changes in local and/or plasma glucose concentration. The alterations in the electrical activity of these neurons probably depend on the existence of 'glucose sensors', which may be one of the glucose transporters described so far. Because of suitable kinetic properties, we hypothesized that the glucose transporter 2 (GLUT 2) may well constitute one of the cerebral 'glucose sensors'. In this study, it was demonstrated, using the polymerase chain reaction, that GLUT 2 mRNAs are present in a limited number of brain nuclei, including the nucleus tractus solitarius, the motor nucleus of the vagus, the paraventricular hypothalamic nucleus, the lateral hypothalamic area, the arcuate nucleus and the olfactory bulbs. These localizations were confirmed by immunocytochemistry, but the cerebral distribution of GLUT 2-like immunoreactivity was far larger than initially expected. Furthermore, electron microscopic observations showed that, within the regions examined, GLUT 2 was localized to a restricted population of astrocytes. The localization of GLUT 2 in regions previously connected with feeding behavior supports an indirect role for GLUT 2 in 'glucose sensing' in these specific cerebral structures.

[Creation of diet-dependent cancer models in transgenic animals]. / Création de modèles de cancers dépendant du régime alimentaire par transgénèse chez l'animal.

We have created transgenic mice lines in which SV40 T and c-myc expression was controlled by the L-pyruvate kinase gene regulatory region which is responsible for hepatic and pancreatic expression specificity, and strong dependence of this expression upon the carbohydrate composition of the diet. Models of hepatoma and endocrine pancreatic tumors have been obtained. Both tumors were dependent upon the diet, since carbohydrates strongly increased frequency and precocity of both hepatic and pancreatic carcinomas.

Abnormal messenger RNA expression and a missense mutation in patients with X-linked adrenoleukodystrophy.

A candidate gene for X-linked adrenoleukodystrophy (ALD) has been identified via positional cloning strategies. We now report messenger RNA expression in fibroblasts from 6 unrelated ALD patients. Four patients lacked the normal 4.2 kb transcript, three of them having deletions of the ALD gene. A fifth patient with a deletion of 1.6 kb had a smaller 4.0 kb transcript. The last patient had a normal sized transcript and a missense mutation at base 1258 leading to Glu-291-Lys substitution in a region of the candidate gene protein which is conserved in the 70 kD peroxisomal membrane protein. These results provide further evidence that this candidate gene is indeed the ALD gene.