Naringenin Ameliorates Drosophila ReepA Hereditary Spastic Paraplegia-Linked Phenotypes.

Defects in the endoplasmic reticulum (ER) membrane shaping and interaction with other organelles seem to be a crucial mechanism underlying Hereditary Spastic Paraplegia (HSP) neurodegeneration. REEP1, a transmembrane protein belonging to TB2/HVA22 family, is implicated in SPG31, an autosomal dominant form of HSP, and its interaction with Atlastin/SPG3A and Spastin/SPG4, the other two major HSP linked proteins, has been demonstrated to play a crucial role in modifying ER architecture. In addition, the Drosophila ortholog of REEP1, named ReepA, has been found to regulate the response to ER neuronal stress. Herein we investigated the role of ReepA in ER morphology and stress response. ReepA is upregulated under stress conditions and aging. Our data show that ReepA triggers a selective activation of Ire1 and Atf6 branches of Unfolded Protein Response (UPR) and modifies ER morphology. Drosophila lacking ReepA showed Atf6 and Ire1 activation, expansion of ER sheet-like structures, locomotor dysfunction and shortened lifespan. Furthermore, we found that naringenin, a flavonoid that possesses strong antioxidant and neuroprotective activity, can rescue the cellular phenotypes, the lifespan and locomotor disability associated with ReepA loss of function. Our data highlight the importance of ER homeostasis in nervous system functionality and HSP neurodegenerative mechanisms, opening new opportunities for HSP treatment.

Functional and Structural Brain Damage in Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is a rare hereditary neurodegenerative disorder caused by a GAA repeat expansion in the FXN gene. There is still no cure or quantitative biomarkers reliaby correlating with the progression rate and disease severity. Investigation of functional and structural alterations characterizing white (WM) and gray matter (GM) in FRDA are needed prerequisite to monitor progression and response to treatment. Here we report the results of a multimodal cross-sectional MRI study of FRDA including Voxel-Based Morphometry (VBM), diffusion-tensor imaging (DTI), functional MRI (fMRI), and a correlation analysis with clinical severity scores. Twenty-one early-onset FRDA patients and 18 age-matched healthy controls (HCs) were imaged at 3T. All patients underwent a complete cognitive and clinical assessment with ataxia scales. VBM analysis showed GM volume reduction in FRDA compared to HCs bilaterally in lobules V, VI, VIII (L>R), as well as in the crus of cerebellum, posterior lobe of the vermis, in the flocculi and in the left tonsil. Voxel-wise DTI analysis showed a diffuse fractional anisotropy reduction and mean, radial, axial (AD) diffusivity increase in both infratentorial and supratentorial WM. ROI-based analysis confirmed the results showing differences of the same DTI metrics in cortico-spinal-tracts, forceps major, corpus callosum, posterior thalamic radiations, cerebellar penduncles. Additionally, we observed increased AD in superior (SCP) and middle cerebellar peduncles. The WM findings correlated with age at onset (AAO), short-allelle GAA, and disease severity. The intragroup analysis of fMRI data from right-handed 14 FRDA and 15 HCs showed similar findings in both groups, including activation in M1, insula and superior cerebellar hemisphere (lobules V-VIII). Significant differences emerged only during the non-dominant hand movement, with HCs showing a stronger activation in the left superior cerebellar hemisphere compared to FRDA. Significant correlations were found between AAO and the fMRI activation in cerebellar anterior and posterior lobes, insula and temporal lobe. Our multimodal neuroimaging protocol suggests that MRI is a useful tool to document the extension of the neurological impairment in FRDA.

Clinical and Paraclinical Indicators of Motor System Impairment in Hereditary Spastic Paraplegia: A Pilot Study.

BACKGROUND: Hereditary spastic paraplegias (HSP) are a composite and genetically heterogeneous group of conditions mainly expressed by the impairment of the central motor system ("pure" forms). The involvement of other components of the central nervous system or of other systems is described in the "complicate" forms. The definition of an investigation protocol capable, by assembling clinical and paraclinical indicators to fully represent the extent of the motor system impairment, would help both the clinical handling of these conditions and contribute to our understanding of their pathogenesis. METHODS: We applied a clinical and paraclinical protocol which included tools exploring motor and non motor functioning, neurophysiology and MRI to a composite cohort of 70 molecularly defined HSP patients aged 3 to 65, to define for each indicator its significance in detailing the presence and the severity of the pathology. RESULTS: Clinically increased deep tendon reflexes and lower limb (LL) weakness are constant findings in all patients. The "complicated" forms are characterized by peripheral motor impairment, cognitive and cerebellar involvement. The Spastic Paraplegia Rating Scale efficiently reflects the severity of functional problems and correlates with disease duration. Neurophysiology consistently documents the impairment of the central motor pathway to the LLs. Nevertheless, the upper extremities and sensory system involvement is a frequent finding. MRI diffusion tensor imaging (DTI) highlighted a significant alteration of FA and MD. Combining the sampling of the various portion of the cortico-spinal tract (CST) DTI consistently discriminated patients from controls. CONCLUSION: We propose a graded clinical and paraclinical protocol for HSP phenotype definition, indicating for each tool the discriminative and descriptive capacity. Our protocol applied to 9 different forms of HSP showed that the functional impairment often extends beyond the CST. The novel DTI approach may add significant elements in disease recognition, staging and mapping.

Oestrogens ameliorate mitochondrial dysfunction in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy, the most frequent mitochondrial disease due to mitochondrial DNA point mutations in complex I, is characterized by the selective degeneration of retinal ganglion cells, leading to optic atrophy and loss of central vision prevalently in young males. The current study investigated the reasons for the higher prevalence of Leber's hereditary optic neuropathy in males, exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Control and Leber's hereditary optic neuropathy osteosarcoma-derived cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in glucose or glucose-free, galactose-supplemented medium. After having shown the nuclear and mitochondrial localization of oestrogen receptors in cybrids, experiments were carried out by adding 100 nM of 17ß-oestradiol. In a set of experiments, cells were pre-incubated with the oestrogen receptor antagonist ICI 182780. Leber's hereditary optic neuropathy cybrids in galactose medium presented overproduction of reactive oxygen species, which led to decrease in mitochondrial membrane potential, increased apoptotic rate, loss of cell viability and hyper-fragmented mitochondrial morphology compared with control cybrids. Treatment with 17ß-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant enzyme superoxide dismutase 2. In addition, 17ß-oestradiol induced a general activation of mitochondrial biogenesis and a small although significant improvement in energetic competence. All these effects were oestrogen receptor mediated. Finally, we showed that the oestrogen receptor ß localizes to the mitochondrial network of human retinal ganglion cells. Our results strongly support a metabolic basis for the unexplained male prevalence in Leber's hereditary optic neuropathy and hold promises for a therapeutic use for oestrogen-like molecules.

In vitro hepatic conversion of the anticancer agent nemorubicin to its active metabolite PNU-159682 in mice, rats and dogs: a comparison with human liver microsomes.

We recently demonstrated that nemorubicin (MMDX), an investigational antitumor drug, is converted to an active metabolite, PNU-159682, by human liver cytochrome P450 (CYP) 3A4. The objectives of this study were: (1) to investigate MMDX metabolism by liver microsomes from laboratory animals (mice, rats, and dogs of both sexes) to ascertain whether PNU-159682 is also produced in these species, and to identify the CYP form(s) responsible for its formation; (2) to compare the animal metabolism of MMDX with that by human liver microsomes (HLMs), in order to determine which animal species is closest to human beings; (3) to explore whether differences in PNU-159682 formation are responsible for previously reported species- and sex-related differences in MMDX host toxicity. The animal metabolism of MMDX proved to be qualitatively similar to that observed with HLMs since, in all tested species, MMDX was mainly converted to PNU-159682 by a single CYP3A form. However, there were marked quantitative inter- and intra-species differences in kinetic parameters. The mouse and the male rat exhibited V(max) and intrinsic metabolic clearance (CL(int)) values closest to those of human beings, suggesting that these species are the most suitable animal models to investigate MMDX biotransformation. A close inverse correlation was found between MMDX CL(int) and previously reported values of MMDX LD(50) for animals of the species, sex and strain tested here, indicating that differences in the in vivo toxicity of MMDX are most probably due to sex- and species-related differences in the extent of PNU-159682 formation.

Antioxidants partially restore glutamate transport defect in leber hereditary optic neuropathy cybrids.

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by visual loss resulting from retinal ganglion cell degeneration. Despite the important role of respiratory chain deficiency and oxidative stress induced by mtDNA point mutations affecting complex I, excitotoxic injury has been postulated as a concurrent pathogenic factor. We used transmitochondrial cybrid cell lines constructed using enucleated fibroblasts from three LHON probands carrying the most severe 3460/ND1 mutation and three controls as mitochondria donors, and the osteosarcoma-derived mtDNA-less cells, to study the possible efficacy of two selected antioxidant compounds in preventing glutamate uptake reduction previously observed in LHON cybrids. We demonstrated that two antioxidants, Trolox and decylubiquinone, partially restore glutamate transport impairment occurring in LHON cybrids. Rotenone, a classic complex I inhibitor, did not worsen the glutamate uptake defect present in LHON cybrids under basal conditions but significantly reduced glutamate transport in control cybrids. Furthermore, we observed that LHON cybrids showed an increased protein carbonylation under basal conditions, not further affected by rotenone and partially counteracted by antioxidants. Our findings strengthen the hypothesis that the complex I defect associated with LHON causes free radical overproduction, which is responsible for glutamate transport inhibition. We suggest that selected antioxidants may be clinically tested in LHON patients and relatives to restore glutamate uptake defect caused by LHON-associated free radical overproduction.

Identification of molecular determinants of tumor sensitivity and resistance to anticancer drugs.

Resistance to drugs is a major problem in cancer chemotherapy. Various cellular mechanisms of drug resistance have been identified in cultured tumor cell lines selected for growth in the presence of sublethal concentrations of various anticancer drugs. They involve drug transport and detoxification, qualitative or quantitative alterations of the drug target, repair of drug-induced DNA lesions, and alterations in signaling or execution of apoptosis. More recently, the possibility to simultaneously analyze the expression of thousands of genes using DNA microarrays has allowed exploring the relationships between gene expression and sensitivity to several anticancer drugs. A number of studies using microarrays for identifying genes governing tumor chemosensitivity focused on tumor cell lines. Some clinical studies have also been carried out to investigate whether tumor gene expression patterns could predict clinical response to chemotherapy. Results of these studies are encouraging, indicating that individualization of drug treatment based on multigenic response-predictive markers is feasible.

Pentoxifylline and its major oxidative metabolites exhibit different pharmacological properties.

Previous investigations indicate that some of the metabolites of the hemorheological agent pentoxifylline (PTX), namely 1-(5-hydroxyhexyl)-3,7-dimethylxanthine (M1), 1-(4-carboxybutyl)-3,7-dimethylxanthine (M4) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (M5), concur to some of the biological effects of the drug. However, information on the bioactivity of the major circulating oxidative metabolites of PTX (M4 and M5) is scanty. Here, we compared the effects of M4 and M5 with that of PTX and its major reductive metabolite, M1, on TNF-alpha production and cytotoxicity, endothelial cell proliferation and on the ATPase activity related to some ATP-binding cassette (ABC) transporters. Unlike PTX and M1, M4 and M5 poorly inhibited lipopolysaccaride-stimulated tumor necrosis factor-alpha (TNF-alpha) release by RAW 264.7 murine macrophages, and did not affect at all cell proliferation and upregulation of TNF-alpha-induced vascular cell adhesion molecule-1 (VCAM-1) in H5V endothelioma cells. By contrast, M4 and M5 were more effective than PTX and M1 in protecting WC/1 murine fibrosarcoma cells from TNF-alpha cytotoxicity. Moreover, results from ATP hydrolase assays indicated that neither PTX nor its tested metabolites interacted significantly with the human multidrug resistance transporters p-glycoprotein/multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1), and breast cancer resistance protein (BCRP). Based on these results and literature data, M5, retaining some of the PTX effects but lacking in significant inhibition of TNF-alpha production, may be a promising candidate drug for certain pathologic conditions.

Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes.

PURPOSE: Nemorubicin (3'-deamino-3'-[2''(S)-methoxy-4''-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682. EXPERIMENTAL DESIGN: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively. RESULTS: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts. CONCLUSIONS: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.