Tricarboxylic acid cycle enzyme activities in a mouse model of methylmalonic aciduria.

Methylmalonic acidemia (MMA) is a propionate pathway disorder caused by dysfunction of the mitochondrial enzyme methylmalonyl-CoA mutase (MMUT). MMUT catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA, an anaplerotic reaction which feeds into the tricarboxylic acid (TCA) cycle. As part of the pathological mechanisms of MMA, previous studies have suggested there is decreased TCA activity due to a "toxic inhibition" of TCA cycle enzymes by MMA related metabolites, in addition to reduced anaplerosis. Here, we have utilized mitochondria isolated from livers of a mouse model of MMA (Mut-ko/ki) and their littermate controls (Ki/wt) to examine the amounts and enzyme functions of most of the TCA cycle enzymes. We have performed mRNA quantification, protein semi-quantitation, and enzyme activity quantification for TCA cycle enzymes in these samples. Expression profiling showed increased mRNA levels of fumarate hydratase in the Mut-ko/ki samples, which by contrast had reduced protein levels as detected by immunoblot, while all other mRNA levels were unaltered. Immunoblotting also revealed decreased protein levels of 2-oxoglutarate dehydrogenase and malate dehydrogenase 2. Interesting, the decreased protein amount of 2-oxoglutarate dehydrogenase was reflected in decreased activity for this enzyme while there is a trend towards decreased activity of fumarate hydratase and malate dehydrogenase 2. Citrate synthase, isocitrate dehydrogenase 2/3, succinyl-CoA synthase, and succinate dehydrogenase are not statistically different in terms of quantity of enzyme or activity. Finally, we found decreased activity when examining the function of methylmalonyl-CoA mutase in series with succinate synthase and succinate dehydrogenase in the Mut-ko/ki mice compared to their littermate controls, as expected. This study demonstrates decreased activity of certain TCA cycle enzymes and by corollary decreased TCA cycle function, but it supports decreased protein quantity rather than "toxic inhibition" as the underlying mechanism of action. SUMMARY: Methylmalonic acidemia (MMA) is an inborn metabolic disorder of propionate catabolism. In this disorder, toxic metabolites are considered to be the major pathogenic mechanism for acute and long-term complications. However, despite optimized therapies aimed at reducing metabolite levels, patients continue to suffer from late complications, including metabolic stroke and renal insufficiency. Since the propionate pathway feeds into the tricarboxylic acid (TCA) cycle, we investigated TCA cycle function in a constitutive MMA mouse model. We demonstrated decreased amounts of the TCA enzymes, Mdh2 and Ogdh as semi-quantified by immunoblot. Enzymatic activity of Ogdh is also decreased in the MMA mouse model compared to controls. Thus, when the enzyme amounts are decreased, we see the enzymatic activity also decreased to a similar extent for Ogdh. Further studies to elucidate the structural and/or functional links between the TCA cycle and propionate pathways might lead to new treatment approaches for MMA patients.

AAV gene therapy corrects OTC deficiency and prevents liver fibrosis in aged OTC-knock out heterozygous mice.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder of the urea cycle. Hemizygous males and heterozygous females may experience life-threatening elevations of ammonia in blood and brain, leading to irreversible cognitive impairment, coma, and death. Recent evidence of acute liver failure and fibrosis/cirrhosis is also emerging in OTC-deficient patients. Here, we investigated the long-term consequences of abnormal ureagenesis in female mice heterozygous (Het) for a null mutation in the OTC gene. Two-month-old Het OTC knockout (KO) mice received a single dose of self-complementary adeno-associated virus (AAV) encoding a codon-optimized human OTC gene at 1×1010, 3×1010, or 1×1011 vector genome copies per mouse. We compared liver pathology from 18-month-old treated Het OTC-KO mice, age-matched untreated Het OTC-KO mice, and WT littermates, and assessed urinary orotic acid levels and vector genome copies in liver at 4, 10, and 16months following vector administration. Het OTC-KO female mice showed evidence of liver inflammation and the eventual development of significant fibrosis. Treatment with AAV gene therapy not only corrected the underlying metabolic abnormalities, but also prevented the development of liver fibrosis. Our study demonstrates that early treatment of OTC deficiency with gene therapy may prevent clinically relevant consequences of chronic liver damage from developing.

TERC and TERT gene mutations in patients with bone marrow failure and the significance of telomere length measurements.

Dyskeratosis congenita (DC) is a rare inherited form of bone marrow failure (BMF) caused by mutations in telomere maintaining genes including TERC and TERT. Here we studied the prevalence of TERC and TERT gene mutations and of telomere shortening in an unselected population of patients with BMF at our medical center and in a selected group of patients referred from outside institutions. Less than 5% of patients with BMF had pathogenic mutations in TERC or TERT. In patients with BMF, pathogenic TERC or TERT gene mutations were invariably associated with marked telomere shortening (<< 1st percentile) in peripheral blood mononuclear cells (PBMCs). In asymptomatic family members, however, telomere length was not a reliable predictor for the presence or absence of a TERC or TERT gene mutation. Telomere shortening was not pathognomonic of DC, as approximately 30% of patients with BMF due to other causes had PBMC telomere lengths at the 1st percentile or lower. We conclude that in the setting of BMF, measurement of telomere length is a sensitive but nonspecific screening method for DC. In the absence of BMF, telomere length measurements should be interpreted with caution.

Study of the cortical representation of whisker directional deflection using voltage-sensitive dye optical imaging.

Using voltage-sensitive dye optical imaging methods, we visualized neural activity in the rat barrel cortex in response to the deflection of a single whisker in different directions. Obtained data indicates that fast movements of single whiskers in varying directions correlate with different patterns of activation in the somatosensory cortex. A functional map was created based on the voltage-sensitive dye optical signal. This supports prior research that vibrissae deflections cause responses in different cortical neurons within the barrel field according to the direction of the deflection. By analogy with the orientation columns in the visual cortex, directionally biased single-whisker responses to different directions of deflection could be a possible mechanism for the directional selectivity of this important sensory response.

Acquired monosomy 7 myelodysplastic syndrome in a child with clinical features suggestive of dyskeratosis congenita and IMAGe association.

We describe a case of acquired monosomy 7 myelodysplastic syndrome (MDS) in a boy with congenital adrenocortical insufficiency, genital anomalies, growth delay, skin hyperpigmentation, and chronic lung disease. Some of his clinical manifestations were suggestive of dyskeratosis congenita (DC), while other features resembled IMAGe association. DC has been linked to mutations in telomere maintenance genes. The genetic basis of IMAGe association is unknown, although mice harboring a mutation in a telomere maintenance gene, Tpp1, have adrenal hypoplasia congenita. We considered the possibility that this patient has a defect in telomere function resulting in features of both DC and IMAGe association.

Planar implantable sensor for in vivo measurement of cellular oxygen metabolism in brain tissue.

BACKGROUND: Brain imaging methods are continually improving. Imaging of the cerebral cortex is widely used in both animal experiments and charting human brain function in health and disease. Among the animal models, the rodent cerebral cortex has been widely used because of patterned neural representation of the whiskers on the snout and relative ease of activating cortical tissue with whisker stimulation. NEW METHOD: We tested a new planar solid-state oxygen sensor comprising a polymeric film with a phosphorescent oxygen-sensitive coating on the working side, to monitor dynamics of oxygen metabolism in the cerebral cortex following sensory stimulation. RESULTS: Sensory stimulation led to changes in oxygenation and deoxygenation processes of activated areas in the barrel cortex. We demonstrate the possibility of dynamic mapping of relative changes in oxygenation in live mouse brain tissue with such a sensor. COMPARISON WITH EXISTING METHOD: Oxygenation-based functional magnetic resonance imaging (fMRI) is very effective method for functional brain mapping but have high costs and limited spatial resolution. Optical imaging of intrinsic signal (IOS) does not provide the required sensitivity, and voltage-sensitive dye optical imaging (VSDi) has limited applicability due to significant toxicity of the voltage-sensitive dye. Our planar solid-state oxygen sensor imaging approach circumvents these limitations, providing a simple optical contrast agent with low toxicity and rapid application. CONCLUSIONS: The planar solid-state oxygen sensor described here can be used as a tool in visualization and real-time analysis of sensory-evoked neural activity in vivo. Further, this approach allows visualization of local neural activity with high temporal and spatial resolution.

Study of the cortical representation of whisker frequency selectivity using voltage-sensitive dye optical imaging.

The facial whiskers of rodents act as a high-resolution tactile apparatus that allow the animal to detect the finest details of its environment. Previously it was shown that whisker-sensitive neurons in the somatosensory cortex show frequency selectivity to small amplitude stimuli, An intravital voltage-sensitive dye optical imaging (VSDi) method in combination with the different frequency whisker stimulation was used in order to visualize neural activity in the mice somatosensory cortex in response to the stimulation of a single whisker by different frequencies. Using the intravital voltage-sensitive dye optical imaging (VSDi) method in combination with the different frequency whisker stimulation we visualized neural activity in the mice somatosensory cortex in response to the stimulation of a single whisker by different frequencies. We found that whisker stimuli with different frequencies led to different optical signals in the barrel field. Our results provide evidence that different neurons of the barrel cortex have different frequency preferences. This supports prior research that whisker deflections cause responses in cortical neurons within the barrel field according to the frequency of the stimulation. Many studies of the whisker frequency selectivity were performed using unit recording but to map spatial organization, imaging methods are essential. In the work described in the present paper, we take a serious step toward detailed functional mapping of the somatosensory cortex using VSDi. To our knowledge, this is the first demonstration of whisker frequency sensitivity and selectivity of barrel cortex neurons with optical imaging methods.

In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe.

Several approaches have been adopted for real-time imaging of neural activity in vivo. We tested a new cell-penetrating phosphorescent oxygen-sensitive probe, NanO2-IR, to monitor temporal and spatial dynamics of oxygen metabolism in the neocortex following peripheral sensory stimulation. Probe solution was applied to the surface of anesthetized mouse brain; optical imaging was performed using a MiCAM-02 system. Trains of whisker stimuli were delivered and associated changes in phosphorescent signal were recorded in the contralateral somatosensory ("barrel") cortex. Sensory stimulation led to changes in oxygenation of activated areas of the barrel cortex. The oxygen imaging results were compared to those produced by the voltage-sensitive dye RH-1691. While the signals emitted by the two probes differed in shape and amplitude, they both faithfully indicated specific whisker evoked cortical activity. Thus, NanO2-IR probe can be used as a tool in visualization and real-time analysis of sensory-evoked neural activity in vivo.

Intrinsic optical imaging of directional selectivity in rat barrel cortex: application of a multidirectional magnetic whisker stimulator.

Using intrinsic optical imaging methods, somatosensory-evoked neural activity was visualized in the rat barrel cortex at high spatial resolution in order to obtain cortical maps of the directional selectivity of whisker deflection. For this task, a special multidirectional mechanical stimulator was developed in our laboratory. The vibrissae were covered with magnetic paint and deflection was performed using a custom-built deflecting device with four small crosswise electromagnets; the whisker was placed at the intersection of the magnet axes. The results show that differences between responses to directional deflection were indicated by a change in the intrinsic optical signal of incident light at 546 nm corresponding to an increase in blood volume. Our results provide a preliminary indication that the different regions of the barrel have different directional preference, supporting recent electrophysiological studies suggesting that the barrel might have a fine, directionally-sensitive anatomical structure.

Optical imaging of interaural time difference representation in rat auditory cortex.

We used in vivo voltage-sensitive dye optical imaging to examine the cortical representation of interaural time difference (ITD), which is believed to be involved in sound source localization. We found that acoustic stimuli with dissimilar ITD activate various localized domains in the auditory cortex. The main loci of the activation pattern shift up to 1 mm during the first 40 ms of the response period. We suppose that some of the neurons in each pool are sensitive to the definite ITD and involved in the transduction of information about sound source localization, based on the ITD. This assumption gives a reasonable fit to the Jeffress model in which the neural network calculates the ITD to define the direction of the sound source. Such calculation forms the basis for the cortex's ability to detect the azimuth of the sound source.

Complex inheritance pattern of dyskeratosis congenita in two families with 2 different mutations in the telomerase reverse transcriptase gene.

Heterozygous mutations in the telomerase components TERT, the reverse transcriptase, and TERC, the RNA template, cause autosomal dominant dyskeratosis congenita due to telomere shortening. Anticipation, whereby the disease severity increases in succeeding generations due to inheritance of shorter telomeres, is a feature of this condition. Here we describe 2 families in which 2 TERT mutations are segregating. Both families contain compound heterozygotes. In one case the proband is homozygous for a novel mutation causing a P704S substitution, while his father's second allele encodes an H412Y mutation. The proband in the second family has mutant alleles Y846C and H876Q. Transfection studies show codominant expression of the mutated alleles with no evidence of a dominant negative effect or of intragenic complementation. Thus in these families the expression of both TERT alleles and the inherited telomere length contribute to the clinical phenotype.

Clinical, biochemical, and cytochemical studies on a Japanese Salla disease case associated with a renal disorder.

We report the first Japanese case of Salla disease. A 5-year-old male patient developed unique proteinuria with other clinical manifestations, including coarse facies, dysostosis multiplex, mild mitral valve regurgitation, umbilical and inguinal herniation, and mild developmental delay. Pathological analysis of biopsied kidney tissues showed marked vacuolation of podocytes, mesangial cells, capillary endothelial cells, and tubular cells. Biochemical studies involving thin-layer chromatography and mass spectrometry revealed increased excretion of free sialic acid (N-acetylneuraminic acid) into the patient's urine. Immuno- and lectin staining of the patient's cells demonstrated the accumulation of sialyl and asialyl glycoconjugates in lysosomes and late endosomes. A defect in sialyl glycoconjugate metabolism is thought to have occurred in the patient's cells, besides impairment of the lysosomal transport of free sialic acid residues. A renal disorder should be considered as an important manifestation, not only in infantile free sialic acid storage disease but also in Salla disease.