Impairment of brain and muscle energy metabolism detected by magnetic resonance spectroscopy in hereditary spastic paraparesis type 28 patients with DDHD1 mutations

Mutations in DDHD1 gene have been associated with the SPG28 subtype of Hereditary Spastic Paraparesis (HSP). Clinical phenotype includes axonal neuropathy, distal sensory loss, and cerebellar eye movement disturbances. We screened 96 index subjects from recessive HSP families for mutation and identified one family with two sibs carrying mutations in DDHD1 gene. Clinical, neuropsychological, and neuroimaging studies were performed, including MR spectroscopy of brain and muscle of the two mutated patients. Two novel heterozygous mutations in DDHD1 were found in the affected members of one family, with clinical features overlapping the SPG28 subtype. Of note, MR spectroscopy of brain and muscle in these patients indicated a mild deficit of brain energy metabolism in the oldest and most severely affected patient, while an impairment of energy metabolism was found in the skeletal muscle of both patients. Unlike the DDHD2 mutated patients, no evidence of lipid accumulation in the brain was found. Our data along with those previously reported suggest a dysfunction in the OXPHOS system possibly due to mitochondrial lipid content modification, which could be a central mechanism in the pathogenesis of SPG28

Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors

The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking

Late-onset spastic ataxia phenotype in a patient with a homozygous DDHD2 mutation

Autosomal recessive cerebellar ataxias and autosomal recessive hereditary spastic paraplegias (ARHSPs) are clinically and genetically heterogeneous neurological disorders. Herein we describe Japanese siblings with a midlife-onset, slowly progressive type of cerebellar ataxia and spastic paraplegia, without intellectual disability. Using whole exome sequencing, we identified a homozygous missense mutation in DDHD2, whose mutations were recently identified as the cause of early-onset ARHSP with intellectual disability. Brain MRI of the patient showed a thin corpus callosum. Cerebral proton magnetic resonance spectroscopy revealed an abnormal lipid peak in the basal ganglia, which has been reported as the hallmark of DDHD2-related ARHSP (SPG 54). The mutation caused a marked reduction of phospholipase A(1) activity, supporting that this mutation is the cause of SPG54. Our cases indicate that the possibility of SPG54 should also be considered when patients show a combination of adult-onset spastic ataxia and a thin corpus callosum. Magnetic resonance spectroscopy may be helpful in the differential diagnosis of patients with spastic ataxia phenotype.ArticleSCIENTIFIC REPORTS. 4:7132 (2014)journal articl

Gene identification in Hereditary Spastic Paraplegias and characterization of Spastic Paraplegia type 58 (SPG58)

Hereditary spastic paraplegias (HSPs) are a large group of inherited neurodegenerative disorders characterised by a progressive spasticity and weakness of the lower limbs. Additional symptoms variably occur and define so-called complicated forms of the disease. HSPs exhibit a very high genetic and clinical variability, with at least 84 loci identified and 67 known causative genes. They can be inherited in autosomal dominant, autosomal recessive, and X-linked manner. However, they all share a common trait: a progressive lengthdependent distal axonopathy of the motor neurons that form the corticospinal tracts. The use of whole exome sequencing (WES) has dramatically increased the speed of gene discovery in HSP. This technique granted the conjunct identification of the five genes described here, which are responsible for causing different forms of spastic paraplegia: SPG28, SPG46, SPG26, SPG54 and SPG58. These genes are respectively involved in mitochondrial function, different aspects of lipid metabolism and RNA metabolism. The in-depth study of SPG58 shows how mutations in KIF1C alter the cellular localization of KIF1C protein and affect endogenous protein levels if mutations locate to the ATP-binding domain. This work also elucidates that KIF1C interacts with known RNA-binding proteins (RBPs) and that it also binds RNA directly and is thus itself an RBP. Transcripts bound to KIF1C correspond to genes involved in key mechanisms of cell cycle and gene regulation and in various aspects of RNA metabolism. In addition to the enrichment of ribosomal RNAs, KIF1C also interacts with ribosomal proteins and influences cellular ribosome distribution. This suggests that KIF1C might have a role in the regulation and or transport of ribosomes

po 053 the phospholipase ddhd1 as a new target in colorectal cancer therapy

Introduction We have recently demonstrated that Citrus-limon derived nanovesicles are able to decrease colon cancer cell viability and that this effect is associated with the down-regulation of the intracellular phospholipase DDHD domain-containing protein 1 (DDHD1). While few studies are currently available on DDHD1 contribution in neurological disorders, information on its involvement in cancer is missing. Here we investigate the role of DDHD1 in colon cancer. Material and methods DDHD1 siRNAs and overexpression vector were transfected into colorectal cancer and normal cells to down-regulate or up-regulate DDHD1 expression. In vitro and in vivo assays were performed to investigate the functional role of DDHD1 in colorectal cancer cell growth. Quantitative proteomics by SWATH-MS was performed to determinate the molecular effects induced by DDHD1 silencing in colorectal cancer cells. Results and discussions Our evidences indicate that DDHD1 supports colon cancer cell proliferation and survival, since its down-regulation reduces in vitro colon cancer cell viability and increases apoptosis rate, without affecting normal cells. On the contrary, in vivo studies demonstrate that the xenograft tumours, derived from DDHD1-overexpressing cells, have a higher proliferation rate compared to control animals. Finally, a proteomic analysis of silenced cells opens up to the opportunity to define the molecular effects of DDHD1 silencing: we found that functional categories, significantly affected by DDHD1 silencing, was specifically related to cancer phenotype and for the first time associated to DDHD1 activity. Conclusion In summary, here we provide the first evidence of DDHD1 role in cancer, pointing out the possibility to define a new target to design more effective therapies for colon cancer patients. In addition, the proteomic analysis allows us to add new knowledge of DDHD1 cytoplasmic activity, highlighting its involvement in both known and previously unrecognised intracellular pathways and identifying multiple mechanisms that may explain the suppressed cancer cell growth induced by DDHD1 silencing

Functional characterization of mammalian and plant extracellular vesicles as systems for cancer treatment

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) have raised the interest of the scientific community due to their high yield and biocompatibility. This study aims to first evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. The DDHD1 gene encodes the phospholipase DDHD1 protein, which plays a vital role in lipid metabolism by hydrolyzing phospholipids into fatty acids and other lipophilic substances. Overexpression of DDHD1 has been linked to increased proliferation in colorectal cancer cells, suggesting its potential as a target for cancer therapies. We found that the DDHD1-siRNA TNV complexes were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells. More recently we tested whether the approach used with PDNVs can be applied also to mammalian vesicles, in particular to EVs purified from human embryonic kidney cells (HEK293T). We focused on loading onco-suppressor microRNAs, miR-146a and miR-199a, in EVs and to test the functionality of the complex on thyroid cancer cell lines. To increase the cell-specific targeting of the EV-miRNA complex, we decorated exosomes with Hyaluronic acid (HA), since cancer cells overexpress its receptor, CD44. Preliminary results suggest that the developed electroporation approach can be also used to load exogenous RNA in mammalian EVs; also, our findings suggest that the decoration of EV with HA may enhance the delivery efficiency of miRNAs to target cells

INFLUENCE OF DIETARY SELENIUM SUPPLEMENTATION FORM ON HEPATIC TRANSCRIPTOME PROFILES OF MATURING BEEF HEIFERS

Our objective was to know how the hepatic transcriptome expression of growing beef (Angus-cross) heifers (0.5 kg gain/day) was affected by the feeding of different sources of dietary (3 mg/day) Se supplements: inorganic Se (ISe, sodium selenite), organic (OSe, Sel-Plex®), or a blend (1.5 mg:1.5 mg) of ISe:OSe (Mix), compared to the adequate but non-Se supplemented “Control”. The biopsied hepatic tissues of these four groups heifers collected at day 168 (when liver Se assimilation had stabilized) after supplements of Se, was subjected to the microarray analysis to assess Se treatment effects. The results suggest that there were clear differences in the hepatic gene expression profile of the four Se treatment groups. 139 significantly treatment-induced differentially expressed transcripts were selected. Among them: 1) the gene expression profiles of Control and OSe appeared to be more similar than Control and ISe, 2) eight distinct gene expression patterns among treatments were identified and each of them indicates affected biofunctions and networks, 3) they were grouped as the expression profile relative to Control, there were solely and commonly affected transcripts for four Se treatments and they indicated different biofunctions, 4) of them, three microRNAs were identified and their predicated mRNA targets showed different biofunctions

Cardiolipin remodeling maintains the inner mitochondrial membrane in cells with saturated lipidomes.

Cardiolipin (CL) is a unique, four-chain phospholipid synthesized in the inner mitochondrial membrane (IMM). The acyl chain composition of CL is regulated through a remodeling pathway, whose loss causes mitochondrial dysfunction in Barth syndrome (BTHS). Yeast has been used extensively as a model system to characterize CL metabolism, but mutants lacking its two remodeling enzymes, Cld1p and Taz1p, exhibit mild structural and respiratory phenotypes compared to mammalian cells. Here, we show an essential role for CL remodeling in the structure and function of the IMM in yeast grown under reduced oxygenation. Microaerobic fermentation, which mimics natural yeast environments, caused the accumulation of saturated fatty acids and, under these conditions, remodeling mutants showed a loss of IMM ultrastructure. We extended this observation to HEK293 cells, where phospholipase A2 inhibition by Bromoenol lactone resulted in respiratory dysfunction and cristae loss upon mild treatment with exogenous saturated fatty acids. In microaerobic yeast, remodeling mutants accumulated unremodeled, saturated CL, but also displayed reduced total CL levels, highlighting the interplay between saturation and CL biosynthesis and/or breakdown. We identified the mitochondrial phospholipase A1 Ddl1p as a regulator of CL levels, and those of its precursors phosphatidylglycerol and phosphatidic acid, under these conditions. Loss of Ddl1p partially rescued IMM structure in cells unable to initiate CL remodeling and had differing lipidomic effects depending on oxygenation. These results introduce a revised yeast model for investigating CL remodeling and suggest that its structural functions are dependent on the overall lipid environment in the mitochondrion