Biochemical Defect Research Articles

Simultaneous Activation of Nrf2, Elevation of Antioxidants and Reduction in Glutamate Level: An Essential Strategy for Prevention and Improved Management of Neurodegenerative Diseases

Despite extensive research on the biochemical and genetic defects in Alzhemier’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and post-traumatic stress disorders (PTSDs), there are no effective preventive strategies; and the treatment methods remain unsatisfactory. The reviews of these studies suggested that enhanced production of free radicals, persistence inflammation were one of the earliest events in the development and progression of these diseases. Excess release of glutamate occurred in HD and PTSD earlier than in AD and PD. Glutamate together with excess free radicals and pro-inflammatory cytokines participate in the progression of these diseases. Thus, reducing simultaneously these biochemical defects may prevent, and together with standard therapy, enhance the care of neurodegenerative diseases. Previous studies using primarily individual antioxidants produced variable outcomes ranging from transient benefits in the early phase of the disease to no effect. In order to optimally attenuate oxidative stress, persistence inflammation and glutamate, it is necessary to simultaneously increase the cellular levels of cytoprotective enzymes including antioxidant enzymes, antioxidant compounds that are derived from the diet and made in the body and reduce glutamate level. Enhancement of antioxidant compounds and attenuation of glutamate level are achieved by supplementation with antioxidants and B-vitamins; however, increasing the cellular levels of antioxidant enzymes needs an activation of Nr2 that is ROS-dependent and ROSindependent. In neurodegenerative diseases, Nrf2 is not activated by ROS; however, antioxidants activate ROSindependent Nrf2. This commentary briefly describes the genetic and epigenetic factors that regulate the activation of Nrf2, and proposes a micronutrient mixture that may simultaneously activate ROS-independent Nrf2, increase the cellular levels of antioxidants, and decrease the release and toxicity of glutamate. This micronutrient mixture may simultaneously and optimally reduce oxidative, chronic inflammation and glutamate, and thus, may prevent and together with standard therapy, enhance the care of these neurodegenerative diseases.

Inhibition of Early Biochemical Defects in Prodromal Huntington's Disease by Simultaneous Activation of Nrf2 and Elevation of Multiple Micronutrients.

Huntington's disease (HD) is a progressive fatal dominant hereditary neurodegenerative disease of the brain, which primarily affects the cortex and the striatum. The disorder is typified by an expansion of more than 35 repeats of the nucleotide triplet cytosine- adenine-guanosine (CAG) which codes for the amino acid glutamine in the huntingtin gene. Despite studies of several decades, there are no effective means to block or postpone the appearance of symptoms of HD. Analysis of these studies led us to propose that increased oxidative stress and chronic inflammation are earliest events in the pathogenesis of HD, and together with excessive glutamate release, participate in the progression of the disease. This review briefly describes evidence for the involvement of oxidative stress, chronic inflammation and glutamate in the pathogenesis of HD. It is proposed that attenuation of these biochemical abnormalities together, may delay the appearance of symptoms of HD. In order to achieve this goal, the simultaneous activation of the nuclear transcriptional factor-2/antioxidant response elements (Nrf2/ARE) pathway that would enhance the transcription of target genes coding for antioxidant enzymes and phase-2-detoxifying enzymes, and an elevation of the levels of antioxidant compounds by supplementation may be needed. Normal mechanisms of activation of Nrf2 requiring reactive oxygen species (ROS) may be impaired in HD, but certain antioxidant compounds can activate Nrf2 without ROS. Use of a combination of micronutrients that can activate the Nrf2/ARE pathway and enhance the levels of antioxidant compounds is suggested.

Platelet Pitp-Alpha Promotes Thrombin Generation and the Dissemination of Tumor Metastasis, but Has Minimal Effect on Vascular Plug Formation

Phosphatidylinositol (PtdIns) is a relatively rare phospholipid in cell membranes. However, because of its unique ability to be transiently phosphorylated, it is critical for platelet signaling and vascular plug formation. Phosphatidylinositol transfer proteins (PITPs) facilitate the transfer of aqueous insoluble phosphatidylinositol in vitro from one cell membrane to another. Although murine platelets contain multiple PITP variants, PITPα is by far the most abundant isoform. Platelets are best known for their contribution to hemostasis, however, several lines of evidence indicate that they also contribute to tumor metastasis formation. To test the hypothesis that platelet phosphoinositide signaling contributes to tumor dissemination, we asked whether deletion of platelet PITPα impacts tumor metastasis formation by engineering PITPαfl/fl PF4Cre+ mice that lack PITPα only within their platelets and megakaryocytes. We found that loss of PITPα in platelets decreased PtdIns(4)P and PtdIns(4,5)P2 production by 30-40%. Even more striking was the 80% reduction in IP3 formation following thrombin-stimulation. However, we found no significant defect in the platelet ex vivo aggregation in response to typical doses of most of the platelet agonists, and only a small defect in response to low doses of thrombin. We investigated whether platelets lacking PITPα normally formed thrombi in vivo by using three well-established murine models. First, we observed an extremely minor (but statistically real) prolongation of the tail bleeding time in the PITPαfl/fl PF4Cre+ mice. Second, we found that mice lacking PITPα in their platelets had no defect in forming intravascular clots in response to a chemical induced carotid injury. Finally, we observed that thrombosis and platelet a-granule secretion in response to a laser-induced injury were completely normal in the knockout mice. Therefore, despite the biochemical defect in phosphoinositide signaling induced by the loss of PITPα, there was essentially no hemostastic defect. To determine whether PITPα-mediated phosphoinositide metabolism in platelets is required for tumor dissemination, we utilized a well-characterized B16F10 melanoma model of tumor metastasis. We observed that lung metastasis formation was reduced by 47%±18% in mice lacking PITPα in their platelets. We also found that during the first 3 hours after tumor injection, the control mice, but not the PITPαfl/fl PF4Cre+ mice, developed a rapid and transient thrombocytopenia. Histology analysis of the lung tissue at this time point revealed the presence of 30% more clots in the lung tissue of control mice. Further analysis showed that these thrombi were actually heterogenous complexes composed of tumor cells surrounded by platelets and fibrin. To understand why platelet PITPα influences tumor-induced fibrin formation, we investigated the ability of tumor cells to cause thrombin generation in platelet rich plasma derived from PITPαfl/fl PF4Cre+ and PITPαfl/fl PF4Cre- mice. We observed that the loss of PITPα in platelets resulted in an 88% reduction of thrombin generation compared to the controls. Furthermore, we found that PITPαfl/fl PF4Cre+ platelets also have impaired Annexin V binding suggesting that the defective fibrin formation seen in PITPαfl/fl PF4Cre+ mice is likely due to a role of PITPα in the exposure of phosphatidylserine on the platelet surface that is required for thrombin generation. Finally, we observed a mucosal immune response composed of NK-cells, T-cells, and neutrophils that was strikingly hyperplastic in mice lacking platelet PITPα at 48 hours after tumor injection, but was essentially absent in the control mice. Together, these findings demonstrate that PITPα-mediated phosphoinositide metabolism within platelets is not essential for platelet plug formation in vivo, but is required for the dissemination of tumors in vivo. Our work further demonstrates that platelet PITPα is required for tumor-induced phosphatidylserine exposure and thrombin generation. This process induces a shroud of platelets and fibrin that surround the surface of tumor cells, and thereby protects the tumors from elimination by the mucosal immune system. These results demonstrate that it is possible to clearly distinguish the platelet signaling processes required for platelet plug formation from those processes that augment metastasis formation. DisclosuresNo relevant conflicts of interest to declare.

Diagnostic Power of Laser Assisted Optical Rotational Cell Analyzer (LoRRca MaxSis) Evaluated in 118 Patients Affected By Hereditary Hemolytic Anemias

▪Introduction: Hereditary hemolytic anemias (HAs) is a group of heterogeneous disorders mainly due to defects of red cell (RBC) membrane and metabolism. HAs of RBC membrane are classified according to the morphological appearance on a blood smear: the more common are hereditary spherocytosis (HS), elliptocytosis, followed by stomatocytosis (dehydrated, DHSt and overhydrated, OHSt). Among RBC enzyme defects associated with chronic hemolysis, pyruvate kinase (PK) deficiency is the most common, followed by glucose-6-phosphate isomerase (GPI), pyrimidin 5' nucleotidase (P5'N), and more rarely, other enzymes deficiencies. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a battery of laboratory tests. Ektacytometry, and more recently, laser-assisted optical rotational cell analyser (LoRRca MaxSis, Mechatronics Instruments, NL), able to measure RBC deformability in osmotic gradient conditions (Osmoscan), is reported to be a useful tool in the detection of RBC membrane disorders and in particular for the differential diagnosis of HSt. Few data are available in other haemolytic anemias.Methods: We evaluated the diagnostic power of laser-assisted optical rotational cell analyser in 84 cases with RBC membrane disorders (69 HS, 7 HE, 7 DHSt, 1 OHSt) and 26 enzymopathies: 19 PK deficiency (14 not splenectomised), 4 GPI deficiency and 3 P5N deficiency. Moreover, we analysed 8 congenital diserythropoietic anemia type II (CDA II), of whom 3 not splenectomised. A reference curve interval was obtained from 89 healthy controls. The evaluated parameters were: Omin (osmotic value at which the EI reaches its minimum, coinciding with the 50% hemolysis in osmotic fragility assays), EImax (maximal deformability obtained near the isotonic osmolality), and Ohyper (osmotic value at which the EI reaches half of its maximum value, representing cellular hydration status).Results: Considering membrane defects, all the 69 HS, regardless the biochemical defect, showed typical altered ektacytometry curves, with a decreased EImax (0.516±0.05 vs 0.595±0.008 for controls, p<0.0001) and right shifted Omin (167±11.4 vs 143.7±7.2, p<0.0001); the 7 HE cases displayed a trapezoidal feature and a decreased EImax (0.547±0.05 vs 0.595±0.008, p<0.001). Among the hereditary stomatocytosis, the 7 DHSt were characterized by a left-shifted curve (Fig 1a).Concerning RBC enzymopathies, we found that all the GPI deficient cases showed altered enlarged Osmoscan curves associated with significant increased Ohyper (539±36 vs 462±13.4, p<0.0001), whereas not splenectomised patients with PK deficiency showed an Osmoscan curve characterised by an increased EImax (0.601±0.01 vs 0.595±0.008), although not significant (Fig 1b). Osmoscan curve in not splenectomised CDAII falls in the low reference area of normal controls. However, Omin and EImax were signifificantly different from HS patients thus permitting a differential diagnosis (Omin 149.3 ±3.0 vs 167±11.4, p<0.01; EImax 0.578±0.02 vs 0.516±0.05, p<0.05) (Fig 1c).We separately analysed splenectomised and not splenectomised patients. Interestingly, for some diseases (PK, P5N, CDAII and OHSt) Osmoscan curve after splenectomy, falls in a defined atypical area, regardless the pathology (Fig 1d).Conclusion: The Osmoscan curves were diagnostic for all the HS and HE cases analysed, with Omin and EImax parameters significantly different from controls. DHSt cases always showed typical left-shifted curve. Interestingly, Ohyper was firstly described increased in GPI deficiency, offering a new diagnostic tool for this rare enzyme defect. The interpretation of Osmoscan requires caution in splenectomised cases as splenectomy may interfere with the RBC deformability. In conclusion, the Osmoscan analysis performed by LoRRca Maxsis represents a useful and feasible first step screening test in the diagnosis of RBC membrane disorders and other rare haemolytic anemias. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Shwachman-Diamond Syndrome: Energetic Stress, Calcium Homeostasis and mTOR Pathway

Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two high-energy consuming cellular processes. The reported increment in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest a defect in the energy production in SDS cells.In this study, we analyzed the energetic metabolism in SDS cells and find that the oxygen consumption is impaired when it is induced by pyruvate/malate or succinate. This induces poor ATP production and AMP accumulation with a consequent alteration in the ATP/AMP ratio. Also respiratory chain activity was impaired because of faulty function of the complex IV; this defect is not dependent from impaired protein synthesis despite ribosome biogenesis and transduction defects in SDS. In fact, COX5A and Cox2, two subunits of Complex IV encoded respectively by a nuclear and mitochondrial gene, were expressed at normal levels. Impaired function of complex IV could be due to an increment of cytoplasmic calcium concentration that inhibits complex IV activity.Energetic stress induces changes in cellular metabolism, stimulating or inhibiting a network of molecules involved in regulation of energetic balance, such as AMPK and mTOR. In SDS cells as consequence of energetic stress, AMPK is hyper activated and the glycolytic pathway stimulated. Surprisingly, we found that also the AKT/mTOR pathway is aberrantly hyper activated since both these proteins are hyper-phosphorylated. We can speculate that hyper activation of mTOR is a way through which SDS cells support the energy defect and protein synthesis. All these defects were recovered when the SDS cells were complemented with SDS gene.Finally, leucine is an essential amino acid that induces cell proliferation and protein synthesis, restored OXPHOS and ATP synthesis, reduced the cytoplasmic calcium concentration and the AMPK and AKT/mTOR activity, and improved in vitro erythropoiesis from SDS individuals pointing to leucine as potential tool helpful to sustain deranged energetic metabolism and erythropoiesis in SDS patients.In conclusion, we report for the first time that SDS cells suffer of energetic stress and severe respiratory defect that is related to faulty SBSD protein. These defects are compensated by an enhanced activation of AMPK, glycolysis and mTOR/Akt pathways, which appear to adequately support protein synthesis. A pivotal role in the maintenance of this altered metabolism could be played by altered calcium homeostasis. Noteworthy biochemical defects might be largely corrected by leucine which also favourably affects in vitro erythropoiesis thus pointing to biochemical defects as important determinant for impaired hematopoiesis od SDS. DisclosuresDufour:Pfizer: Consultancy.

Mechanisms Governing Disruption of Telomere Maintenance in Dyskeratosis Congenita

Dyskeratosis congenita (DC) is a bone marrow failure syndrome characterized by widespread defects in diverse tissues and a strong predisposition to cancer. DC is caused by germline mutations in genes controlling maintenance of telomeres, nucleoprotein caps that protect chromosome ends. DC is part of a larger collection of telomere disorders whose manifestations include pulmonary fibrosis, liver cirrhosis and defects in many other tissues. Mutations in components of the telomerase enzyme comprise a large share of cases, including in TERT, TERC, dyskerin, TCAB1, NOP10 and NHP2. These mutations compromise telomerase function leading to telomere shortening, which in turn impairs stem cell function. The telomerase holoenzyme operates according to a complex cycle of assembly, nuclear trafficking, recruitment to telomeres and catalytic action to elongate telomeres. In patients with telomere disease states, each of these steps can be impaired leading to a common set of disease phenotypes. We previously created patient-derived iPS cells from patients with mutations in TERT, dyskerin or TCAB1 and analyzed these cells to understand the biochemical defects in the telomerase pathway. Molecular defects in these patients operate according to diverse mechanisms including: reduced catalytic function, impaired telomerase assembly, mislocalization of the enzyme to nucleoli and defective recruitment to telomeres. The development of therapeutics to treat these disease states will require approaches targeted to the underlying molecular defects in each form of the disease. DisclosuresNo relevant conflicts of interest to declare.

Abstract B1-58: Computational modeling provides new insights into KRAS mutant-specific responses to targeted therapy

Abstract Emerging data suggest different activating Ras mutants may have different biological behaviors. The most striking example may be in colon cancer, where activating KRAS mutations generally indicate a lack of benefit to treatment with cetuximab, with the activating KRAS G13D mutation an apparent exception. It is unclear why patients with KRAS G13D would respond to cetuximab; KRAS G13D shares the same general biochemical defects as the other oncogenic KRAS mutants. Here, a previously developed mathematical model of the biochemical reactions that regulate Ras signaling is used to investigate this problem. The purpose of the analysis is to determine whether known mechanisms of Ras signaling allow similar, but different, Ras mutants to respond differently to the same inhibitor, and to determine if the known biochemical properties of the G13D Ras mutant are consistent with increased sensitivity to upstream inhibition. Computational analysis of the mathematical model finds that differential response to upstream inhibition between different Ras mutants is indeed consistent with known mechanisms of Ras biology. Moreover, modeling demonstrates that the subtle biochemical differences between the responsive G13D mutant and the non-responsive G12D and G12V mutants are sufficient to explain the different responses to cetuximab. Simulations further identify differences in wild-type Ras signaling as the key difference between the mutants. The analysis suggests the elevated Km for the Ras GAP reaction on the G13D mutant is the biochemical property that most contributes to differences in wild-type Ras signaling. The identification of a single parameter that influences sensitivity to a targeted therapy is significant in that it suggests an approach to evaluate other, less common, Ras mutations for their anticipated response to cetuximab. Note: This abstract was not presented at the conference. Citation Format: Edward C. Stites. Computational modeling provides new insights into KRAS mutant-specific responses to targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-58.

Monocyte-derived extracellular Nampt-dependent biosynthesis of NAD(+) protects the heart against pressure overload.

Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD+ by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD+ concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD+, were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD+ levels and NAD+-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD+ against heart failure.

Mitochondrial leukoencephalopathy and complex II deficiency associated with a recessive SDHB mutation with reduced penetrance

Mitochondrial disease involving complex II is rare among respiratory chain deficiencies and its genetic cause remains often unknown. Two main clinical presentations are associated with this biochemical defect: mitochondrial encephalomyopathy and susceptibility to tumors. Only one homozygous SDHB mutation has been described in a patient with mitochondrial disorder. We report here two sisters, who presented highly different phenotypes (neurological impairment with leukoencephalopathy vs. asymptomatic status) and harbored the same homozygous SDHB mutation, suggesting reduced penetrance.

Molecular, biochemical, and structural analysis of a novel mutation in patients with methylmalonyl-CoA mutase deficiency

BackgroundMethylmalonic aciduria (MMA) is an inborn error of metabolism resulting from genetic defects in methylmalonyl-CoA mutase (MCM). This enzyme is encoded by the MUT gene and is required for the degradation of odd-chain fatty acids, the amino acids valine, isoleucine, methionine, and threonine, and cholesterol. MethodThree unrelated affected patients with isolated MMA and their parents were studied. The MUT gene was analyzed by PCR and sequencing of its entire coding region and the highly conserved exon-intron splice junctions. The homology modeling of the novel mutation found in the MUT gene was performed using the online Swiss-Prot server for automated modeling and then analyzed with special bioinformatics software to better study the structural effects caused by the mutation. ResultWe found one homozygous nucleotide change in intron 12 of the MUT gene (c.2125-3 C>G). The variant is located near the highly conserved acceptor splice site of intron 12. A region at the C-terminus of the protein from ASP709 to GLN748 has been deleted by the alteration of c.2125-3 C>G in intron 12 of the MUT gene. Further studies of the novel mutation in the MUT gene by means of homology modeling revealed abnormalities in the protein's structure, which causes the protein to act malfunctioning and also the mRNA expression analysis of MUT gene confirmed these results. ConclusionWe report this novel mutation, including its clinical and biochemical features and genetic defects, in the MUT gene of three patients affected with isolated MMA. Structural analyses of the mutated protein identified changes in the energy and stereochemical features of the protein that unfortunately altered the protein's functionalities. Therefore, we demonstrate that a novel splice site mutation in intron 12 of the MUT gene is a potential highly pathogenic allele via inhibition of alternative splicing.

Nutrition and nutritional signaling as therapy targets for mitochondrial disorders

Mitochondrial disorders are degenerative diseases known to be responsible for a number of heterogenous clinical presentations with multi-systemic dysfunctions that often involves the nervous, endocrine, renal and cardiac system. Impaired oxidative phosphorylation leading to a decrease in cellular energy (ATP) production is the most important cause underlying these conditions. Despite significant progress made in the field of mitochondrial medicine during the last two decades, defining the specific biochemical defects and underlying molecular mechanisms, the avaliable information about effective treatment approaches is limited. The goal of nutritional treatment of mitochondrial dysfunction in various neurological syndromes are based primarily in improving theefficiency of the processes of oxidative phosphorylation at the system level. Accumulation of toxic metabolites and reduction of electron transfer activity have prompted the use of new therapeutic agents, such as antioxidants, radical scavangers and cofactors. A literature review of the use of these supplements in mitochondrial disorders is presented.

Clinicopathological findings in mitochondrial disorders

Mitochondrial encephalomyopathies have shifted from being regarded just a few years ago as a category of rare metabolic diseases to a current recognition that they are much more common than previously thought, are a large and heterogeneous group of distinct diseases, and that they may occur not only as primary biochemical and genetic defects, but also as secondary defects in a wide spectrum of both genetic and acquired diseases of the nervous system ranging from Pompe disease (glycogenosis II, acid maltase deficiency), spinal muscular atrophy, septo-optic-pituitary dysplasia, some inflammatory myopathies, and secondary to a broad range of drugs that include immunosuppressive and chemotherapeutic agents, statins and valproic acid. Most of the research on mitochondrial diseases has been generated, not surprisingly, in the countries with the greatest funding and biochemical laboratory resources for investigating them, including detailed biochemical and genetic studies, neuroimaging and neuropathological studies, mainly from western Europe and North America. It is therefore encouraging to see studies of mitochondrial diseases eminating also from countries with fewer resources, such as the paper by Selim et al. [1] from Egypt, in this current issue of the JPN.

Prevalence of Mitochondrial ND4 Mutations in 1281 Han Chinese Subjects With Leber's Hereditary Optic Neuropathy.

To investigate the prevalence and spectrum of mitochondrial ND4 mutations in subjects with Leber's hereditary optic neuropathy (LHON). A cohort of 1281 Chinese Han probands and 478 control subjects underwent clinical and genetic evaluation, and sequence analysis of mitochondrial (mt) DNA, as well as enzymatic assay of NADH:ubiquinone oxidoreductase. In this cohort, 503 probands had a family history of optic neuropathy and 778 subjects were sporadic cases. Mutational analysis of ND4 gene identified 149 (102 known and 47 novel) variants. The prevalence of known m.11778G>A mutation was 35.36%. Furthermore, we identified the known m.11696G>A and m.11253T>C mutations and five novel putative LHON-associated mutations. These mutations accounted for 2.74% of cases of LHON subjects. By enzymatic assay, we showed a mild decrease in the activity of NADH:ubiquinone oxidoreductase in mutant cell lines carrying only one putative mtDNA mutation. The low penetrance of optic neuropathy and mild biochemical defects in these pedigrees carrying only m.11696G>A mutation and one putative LHON-associated mutation suggested that the mutation(s) is(are) necessary but is(are) itself(themselves) insufficient to produce a visual failure. Moreover, mtDNAs in 169 probands carrying the LHON-associated mutation(s) were widely dispersed among 13 Eastern Asian haplogroups. In particular, the frequencies of haplogroups D, M8, M10, M11, and H in probands carrying the LHON-associated mtDNA mutation(s) were higher than those in Chinese controls. These results suggested that the ND4 gene is the hot spot for mutations associated with LHON. Thus, these findings may provide valuable information for the further understanding of pathogenic mechanism of LHON.

Detection of glutaric acidemia type 1 in infants through tandem mass spectrometry

Glutaric acidemia type 1 (GA1) is a rare inherited metabolic disorder which goes underdiagnosed due to its latency period and subtle presentation. A pilot clinical study was conducted to assess the usefulness, specificity and sensitivity of the tandem mass (MS/MS) spectrometer, specifically the Abbott (AB) Sciex 3200, in the screening for GA1 using dried blood spots. A total of 17,100 specimens, comprising pediatric patients and healthy newborns, were screened from June 2012 to June 2014. A selection criterion was applied to increase the range of samples tested. 14 of the total specimens tested presumptive positive for GA1, of whom all were symptomatic. The diagnosis was confirmed in 4 of the 14 cases and they were started on treatment. 4 cases expired before confirmation. The remaining cases were empirically started on treatment. Most of the patients responded favorably to the dietary management. One important observation was that the older symptomatic children diagnosed with GA1 had poorer outcomes in terms of recovery of delayed milestones and mental deterioration, further emphasizing the need for early diagnosis of organic acidemias along with the other biochemical defects. Tandem mass spectrometry was found to be more than 93.33% sensitive and more than 99.42% specific. The screening test proved to be very simple and economical.

PP12.15 – 2538: Therapeutic interventions for movement disorders in children with inborn errors of metabolism: What is the evidence?

Objective Movement disorders (MD) are common symptoms in patients with inborn errors of metabolism (IEM). Based on a recent study, these symptoms seem to have serious impact on quality of life and daily functioning. Therefore knowledge about the optimal treatment is crucial. However, up till now a clear overview of the evidence for the efficacy and safety of therapeutic interventions for MD in IEM is lacking, in particular for the pediatric population. Methods A systematic literature search in Pubmed and Embase was performed to identify all articles reporting the effect of therapeutic interventions for MD in children with IEM. Results 288 papers were included, describing 63 different IEM. The most common observed MD were dystonia, ataxia and myoclonus. The effect on MD of disease specific treatments, primary aimed at the underlying condition (e.g. dietary measures), was mostly restricted to prevention with only mixed success. In the case of already existing MD the role of disease specific treatment is minimal. An exception is formed by neurotransmitter disorders, in this group resolving the biochemical defect usually leads to a dramatic improvement of the MD. When disease specific treatment lacks or fails, symptomatic therapies can be applied. In particular for dystonia, without excluding other MD, there are several effective therapeutic options available, of which trihexyfenidyl (Artane) and botulin toxin injections are amongst the most successful. In severe cases neurosurgical intervention in the form of deep brain stimulation is an option, although experiences in patients with IEM are still limited. Conclusion This is the first study reviewing the evidence for MD therapies in children with IEM. Most evidence consists of relatively small case reports. However, putting all data together gives valuable insight in which treatment options might work, and which not. Our overview provides a more rational basis for treatment choices in clinical practice.

Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions

The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end, the ssa1-45 mutant has proved invaluable. However, the biochemical defects associated with the corresponding Ssa1-45 protein (P417L) are unknown. Consequently, we characterized Ssa1 P417L, as well as a P417S variant, which corresponds to a mutation in the gene encoding the yeast mitochondrial Hsp70. We discovered that the P417L and P417S proteins exhibit accelerated ATPase activity that was similar to the Hsp40-stimulated rate of ATP hydrolysis of wild-type Ssa1. We also found that the mutant proteins were compromised for peptide binding. These data are consistent with defects in peptide-stimulated ATPase activity and with results from limited proteolysis experiments, which indicated that the mutants' substrate binding domains were highly vulnerable to digestion. Defects in the reactivation of heat-denatured luciferase were also evident. Correspondingly, yeast expressing P417L or P417S as the only copy of Ssa were temperature sensitive and exhibited defects in Ssa1-dependent protein translocation and misfolded protein degradation. Together, our studies suggest that the structure of the substrate binding domain is altered and that coupling between this domain and the nucleotide binding domain is disabled when the conserved P417 residue is mutated. Our data also provide new insights into the nature of the many cellular defects associated with the ssa1-45 allele.

A review of gene therapy in canine and feline models of lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are inherited diseases that result from the intracellular accumulation of incompletely degraded macromolecules. The majority of LSDs affect both the peripheral and central nervous systems and are not effectively treated by enzyme replacement therapy, substrate reduction therapy, or bone marrow transplantation. Advances in adeno-associated virus and retroviral vector development over the past decade have resurged gene therapy as a promising therapeutic intervention for these monogenic diseases. Animal models of LSDs provide a necessary intermediate to optimize gene therapy protocols and assess the safety and efficacy of treatment prior to initiating human clinical trials. Numerous LSDs are naturally occurring in large animal models and closely reiterate the lesions, biochemical defect, and clinical phenotype observed in human patients, and whose lifetime is sufficiently long to assess the effect on symptoms that develop later in life. Herein, we review that gene therapy in large animal models (dogs and cats) of LSDs improved many manifestations of disease, and may be used in patients in the near future.

The Phosphorylation State of GSK3β Serine 9 Correlated to the Development of Valproic Acid-Associated Fetal Cardiac Teratogenicity, Fetal VPA Syndrome, Rescued by Folic Acid Administration.

The effects of the phosphorylation state of the glycogen synthase kinase 3β involved in the cardiac myocytes (jelly-like cells) epithelial-mesenchymal transition-associated migration during heart-valve formation were examined through the valproic acid-induced cardiac teratogenicity of transgenic line A34 of Tg in a the Brachydanio rerio embryo model. Valproic acid is an effective anti-epileptic drug; however, when taken by pregnant women to treat epilepsy, it can produce cardiac developmental defects in fetuses. In this study, the role of glycogen synthase kinase 3β in valproic acid-induced cardiac teratogenicity was investigated. Transgenic line A34 of zebrafish embryos was used at 3 days postfertilization. The results show that 78% (18/23) of the embryos treated with 0.10 mM valproic acid (group A) had incomplete chamber formation with normal looping and 22 % (5/23) had abnormal looping. Bradycardia was also found in comparison with control embryos (P < 0.001). For the embryos treated with 0.25 mM valproic acid (group B), 92% (22/24) demonstrated chamber formation failure and looping abnormality. Pericardial effusion, noncontracting ventricles, and enlarged, slowly beating atriums were observed at 6 days postfertilization. Valproic acid inhibited phosphorylation of serine 9 in glycogen synthase kinase 3β in a dose-dependent manner. According to immunochemical staining results, valproic acid was shown to inhibit the mass migration and proliferation of cardiomyocytes in the development of the heart-valve region through inhibition of the GSK3β Ser 9 phosphorylation. Folic acid rescued the GSK3β Ser 9 phosphorylation and reversed the valproic acid-induced cardiac morphological, functional, and biochemical defects.

A review of gene therapy in canine and feline models of lysosomal storage disorders

Lysosomal storage disorders (LSDs) are inherited diseases that result from the intracellular accumulation of incompletely degraded macromolecules. The majority of LSDs affect both the peripheral and central nervous systems and are not effectively treated by enzyme replacement therapy, substrate reduction therapy, or bone marrow transplantation. Advances in adeno-associated virus and retroviral vector development over the past decade have resurged gene therapy as a promising therapeutic intervention for these monogenic diseases. Animal models of LSDs provide a necessary intermediate to optimize gene therapy protocols and assess the safety and efficacy of treatment prior to initiating human clinical trials. Numerous LSDs are naturally occurring in large animal models and closely reiterate the lesions, biochemical defect, and clinical phenotype observed in human patients, and whose lifetime is sufficiently long to assess the effect on symptoms that develop later in life. Herein, we review that gene therapy in large animal models (dogs and cats) of LSDs improved many manifestations of disease, and may be used in patients in the near future.

PDE-4 inhibition rescues aberrant synaptic plasticity in Drosophila and mouse models of fragile X syndrome.

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS.