J Inherit Metab Dis DOI 10.1007/s10545-013-9663-6
ORIGINAL ARTICLE
Dietary triheptanoin rescues oligodendrocyte loss, dysmyelination and motor function in the nur7 mouse model of Canavan disease Jeremy S. Francis & Vladimir Markov & Paola Leone
Received: 30 July 2013 / Revised: 24 October 2013 / Accepted: 11 November 2013 # SSIEM and Springer Science+Business Media Dordrecht 2013
Abstract The inherited pediatric leukodystrophy Canavan disease is characterized by dysmyelination and severe spongiform degeneration, and is currently refractory to treatment. A definitive understanding of core disease mechanisms is lacking, but pathology is believed to result at least in part compromised fatty acid synthesis during myelination. Recent evidence generated in an animal model suggests that the breakdown of N-acetylaspartate metabolism in CD results in a heightened coupling of fatty acid synthesis to oligodendrocyte oxidative metabolism during the early stages of myelination, thereby causing acute oxidative stress. We present here the results of a dietary intervention designed to support oxidative integrity during developmental myelination in the nur7 mouse model of Canavan disease. Provision of the odd carbon triglyceride triheptanoin to neonatal nur7 mice reduced oxidative stress, promoted long-term oligodendrocyte survival, and increased myelin in the brain. Improvements in oligodendrocyte survival and myelination were associated with a highly significant reduction in spongiform degeneration and improved motor function in triheptanoin treated mice. Initiation of triheptanoin treatment in older animals resulted in markedly more modest effects on these same pathological indices, indicating a window of therapeutic intervention that corresponds with developmental myelination. These results support the targeting of oxidative integrity at early stages of Canavan disease, and provide a foundation for the clinical
Communicated by: Jerry Vockley Electronic supplementary material The online version of this article (doi:10.1007/s10545-013-9663-6) contains supplementary material, which is available to authorized users. J. S. Francis (*) : V. Markov : P. Leone Cell and Gene Therapy Center, Department of Cell Biology, Rowan University School of Osteopathic Medicine, 40 East Laurel Rd, Stratford, NJ, USA e-mail: [email protected]
development of a non-invasive dietary triheptanoin treatment regimen.
Introduction Leukodystrophies are a class of inherited metabolic diseases that affect the production and/or maintenance of myelin in the nervous system. These single gene disorders provide valuable insight into the complexities of myelination by highlighting key metabolic processes of broad significance. Thus, the characterization of models of leukodystrophies can potentially lead to the identification of core mechanisms that provide a foundation for the development of targeted augmentation strategies for therapeutic intervention (Saher et al 2012). Canavan disease (CD), a fatal pediatric leukodystrophy, is caused by mutations to the aspartoacylase (aspa) gene that result in loss of the ability to catabolize N-acetylaspartate (NAA; Kaul et al 1993). NAA is an abundant neuronal amino acid derivative that acts as an acetyl donor for the synthesis of select myelin lipids through the catabolic activity of oligodendrocytic ASPA (Chakraborty et al 2001). ASPA is the sole known NAA-catabolizing enzyme in the brain, and chronically elevated substrate is therefore diagnostic for CD. Pathologically, CD is characterized by dysmyelination and severe white matter vacuolation and current intractability to treatment is due in large part to a lack of consensus regarding causative mechanisms. Hypotheses for CD pathology range from deficiencies in specific myelin lipids (Madhavarao et al 2005) through to osmotic damage to white matter (Baslow 1999). Experimental evidence for causative mechanisms is limited to that defined by reduced lipid synthesis (Chakraborty et al 2001; Wang et al 2009), but these deficiencies appear not to be rate-limiting for pathology (Traka et al 2008). We have recently identified oxidative stress as an early pathological marker upstream of oligodendrocyte loss and
J Inherit Metab Dis
dysmyelination in the nur7 mouse model of CD suggestive of a role for NAA in the maintenance of oligodendrocyte metabolic integrity during myelination (Francis et al 2012). These recent studies suggest that the use of NAA-derived acetyl groups during myelination may be a means by which oligodendrocytes preserve energetic resources during myelination by uncoupling fatty acid synthesis from oxidative metabolism. Therefore, if a means by which metabolic substrates of fatty acid synthesis and energy were made available during the early stages of myelination, the progressive degeneration of the CD brain may be reduced. The odd-carbon triglyceride triheptanoin is a dietary anaplerotic substrate that provides ketone bodies capable of traversing the blood brain barrier (BBB) and increasing the mass of tricarboxylic acid (TCA) cycle intermediates to support the brains synthetic needs. Triheptanoin has shown efficacy in the treatment of disorders of mitochondrial oxidation and pyruvate metabolism (Roe and Mochel 2006; MarinValencia et al 2010), and the ability to provide AcCoA via the ß-oxidation of carbons 1–4 is believed to be the basis of reported anti-convulsant effects in models of epilepsy (Willis et al 2010; Thomas et al 2012). Because AcCoA is a key intermediate for both fatty acid synthesis and the TCA cycle, we hypothesized that triheptanoin may compensate for the loss of the ASPA function by uncoupling a portion of fatty acid synthesis from energetic metabolism through the provision of AcCoA while simultaneously providing anaplerotic support for the TCA cycle via the propionyl moiety of heptanoate.
Materials and methods Animals Animals used in this study were drawn from an in house colony. This colony was established from founder animals available commercially (Jackson Laboratories, Bar Harbor, ME). Nur7 mutants were generated from the breeding of heterozygous carrier animals and genotyped using a customized SNP assay (available upon request). All procedures performed were done so under an approved Rowan-SOM IACUC protocol, and adhered to national and institutional guidelines for animal care and use.
Table 1 Composition of control and triheptanoin chow Component
Casein Corn starch Maltodextrin (lo-dex) Sucrose Cellulose Mineral mix, Ca-P deficient Vitamin mix Calcium phosphate dibasic Calcium carbonate DL-methionine Magnesium oxide TBHQ Choline bitartrate Pantothenic acid Hydrogenated veg. oil Coconut oil Triheptanoin Corn oil Total (g) Kcal/g Protein Carbohydrates Natural fat Triheptanoin
Control diet (g/kg) 200.0 389.1 100.0 150.0 50.0 13.4 10.0 7.5 6.9 3.0 0.20 0.07
35 % triheptanoin diet (g/kg) 215.0 350.0 89.8 102.6 14.3 10.7 8.0 7.4 3.2 0.22 0.08 2.50 1.93
50.0 20.0
1000.0 3.8 18.9 63.7 17.4
170.5 23.5 1000.0 4.0 18.9 40.2 5.9 35.0
chow was produced commercially (Bio Serv, Frenchtown, NJ) and provided to animals ad libitum. For experiments involving neonatal animals, nursing dams were supplied with ad libitum triheptanoin containing chow. After weaning, all triheptanoin and control animals were housed individually to enable monitoring of food intake. All animals on triheptanoin chow were monitored for hypoglycemia from 6 weeks of age, and weighed weekly from 8 weeks of age Tail blood was gently massaged from a microincision made in the tail vein of an isoflurane anesthetized mouse. Blood was taken in 30– 50 μl sample size, applied to a test strip and glucose measured using a blood glucose monitor (Bayer COUNTOUR). Blood was collected at 6, 8, and 12 weeks of age at a prescribed time of day.
Triheptanoin chow HPLC Triheptanoin was administered to animals in dietary form (composition in Table 1). Rodent chow containing 35 % triheptanoin. Triheptanoin was synthesized to 98 % purity in liquid form (Shanghaihuating Chemical Co. Ltd., Shanghai, People’s Republic of China) and incorporated into chow to replace 35 % of calories therein. Triheptanoin containing
The analysis of N-acetylaspartate (NAA), reduced glutathione (GSH), oxidized glutathione (GSSG), adenosine tri-phosphate (ATP), malondialdehyde (MDA), ascorbic acid, propionyl coenzyme A (PropCoA), methylmalonyl coenzyme A (MeMalCoA), and acetyl coenzyme A (AcCoA) was
J Inherit Metab Dis
performed using an ion-paired high performance liquid chormoatography method. Samples were prepared from whole brains rapidly removed from deeply anesthetized animals and flash frozen in liquid nitrogen. Frozen tissue was weighed and dispersed in 2× volume/wet weight of a freshly prepared ice-cold precipitation solution consisting of 75 % acetonitrile and 25 % KH2PO4 (pH 7.4). Dispersed brain tissue was centrifuged at 12 500×g for 10 min at 4 °C and the supernatant retained. Of fresh precipitating solution 0.5 ml was added to the pellet, which was sonicated, centrifuged a second time, and the resulting supernatant added to that from the first spin. Supernatant was then extracted twice with HPLC grade chloroform, and stored at −80 °C until analyzed. Samples were run on a Thermo Scientific HPLC autosampler fitted with a 50 μl loop and a Hypersil BDS-C18 column (5 μm particle size; 25 cm×4.9 mm) attached to a Suerveyor Plus UV detector. Data was analyzed with ChromQuest software (Thermos Scientific), and target metabolites quantified against standard curves generated from purified compunds. Run conditions were as follows: 100 % buffer A 25 min, 80 % buffer A 10 min, 55 % buffer A 11 min, 40 % buffer A 11 min, 35 % buffer A 10 min, 25 % buffer A 15 min, 100 % buffer B 35 min, 100 % buffer A 25 min (buffer A: 12 mmol/L Bu4NOH, 10 mmol/L K2HPO4, 0.125 % CH3OH pH 7.00; buffer B: 2.8 mmol/L Bu4NOH, 100 mmol/L K2HPO4, 30 % CH3OH pH 5.50). Quantities of target metabolites in samples are expressed as molarity per wet weight of tissue. For the quantification of NAA, samples were diluted 4× to ensure elevated nur7 NAA signals fell within the linear limits of detection. For all other target signals, samples were run undiluted. Immunohistochemistry and stereology Immunohistochemistry was performed on paraformaldehydefixed tissue. Animals were deeply anesthetized and transcardially perfused with ice-cold 0.9 % saline followed by 4 % buffered paraformaldehyde. Perfused brains were removed and post fixed in 4 % paraformaldehyde overnight, then cryoprotected in an ascending sucrose gradient (10 %, 20 %, and 30 %). Cryoprotected brains were flash frozen and stored at −80 °C until processed. The entire forebrain was serially sectioned (40 μ m) and maintained in order for sterological sampling. Staining for adenomatous polyposis coli (APC), myelin basic protein (MBP), and MHC-II was performed using commercially available antibodies (Millipore, Billerica, MA, USA). Free-floating sections were permeabilized in 0.1 % triton-X in phosphate-buffered saline for 20 min, treated with 1 % H2O2, then washed 3×5 min in 0.1 % trition-X. Primary antibodies were diluted in immunobuffer (0.1 % trition-X with 1 % normal goat serum) and incubated with sections overnight at room temperature with gentle agitation. Following primary antibody and
washes, sections were incubated for 2 h at room temperature with biotinylated secondary antibody followed Avidinperoxidase for 1 h at room temperature. Positive cells were visualized with metal-enhanced DAB substrate (Thermo Scientific Pierce Protein Products, Rockford, IL, USA), and slide mounted in series. Mounted sections were dehydrated in an ascending ethanol series, cleared with xylene, and coverslipped. The optical fractionator was used to sample processed sections for APC, MBP, MHC-II, and vacuole volume fraction. The optical dissector was used to generate estimates of N for APC, “space balls” used to generate estimates of L for MBP-positive fibers, and an object area fraction probe used to generate estimates of vacuolation in the thalamus. MBPpositive cortical fibers were expressed as a length density (LD) calculated by dividing the total MBP-positive fiber length within the region of interest by the volume of the region of interest. Vacuole volume fraction was calculated using the formula, ∑P obj /∑P ref =A obj /A ref =Vobj /V ref , where P obj = region points interacting with vacuoles, Vobj = region points interacting with the reference space, A = area, and V = volume. Scoring for objects of interest was performed using Stereologer software (Stereology Resource Center, Chester, MD, USA). True biological variance between samples (BV) was distinguished from within-sample varaiation (CE) using the equation, (BV =√CV 2 −CE 2), where CV = total variance. Statistical significance between treatment groups was calculated using a two-tailed studen’s t-test. Electron microscopy Deeply anesthetized animals were transcardially perfused with a freshly prepared buffered solution of Kanovsky’s fixative. Brains were removed, post-fixed overnight and cryoprotected in an ascending sucrose gradient then flashfrozen and stored at −80 °C for preparation. Frozen brains were thawed in ice-cold PBS and the thalamus dissected. Dissected thalami were postfixed in 1 % osmium tetroxide for 1 h at room temp. Serial semithin sections of thalami were generated and imaged by transmission electron using a JEOL 1200EX electron microscope. Rotarod testing and open filed activity Animals were assessed for motor function using an accelerating rotarod (MedAssociates Inc.). The testing period involved three consequetive days, with the first 2 days being pre trial training. Each training day involved 3× runs with a 30 s interval between runs for each animal. On the third day, three testing runs were performed, and the average latency to fall from the accelerating rotarod (seconds) was calculated. Open field activity was assessed using automated activity chambers (MedAssociates Inc.). Animals were brought into the testing
J Inherit Metab Dis
room an hour prior to testing, and open field sessions of 30 min were conducted at 8 and 12 weeks of age. Statistical methods For all HPLC analyses, one way ANOVA with a Bonferroni correction was used to compare metabolite concentrations between groups. For the analysis of coenzyme A derivatives and redox metabolites in 2 week-old animals, a sample size of 6 animals per group was used. For the HPLC analysis of metabolites in 12 week-old animals, a sample size of 6 animals per group was used. All stereological analyses used a sample size of 6 animals per group, and statistically significant differences between groups were determined using a twotailed t-test. Statistical significance in rotarod data at individual time points was assessed using one-way ANOVA with a Bonferroni correction. For all graphs presented, the mean +/− SEM for each group is presented. Statistical significance is indicated for each graph as follows; p
ORIGINAL ARTICLE
Dietary triheptanoin rescues oligodendrocyte loss, dysmyelination and motor function in the nur7 mouse model of Canavan disease Jeremy S. Francis & Vladimir Markov & Paola Leone
Received: 30 July 2013 / Revised: 24 October 2013 / Accepted: 11 November 2013 # SSIEM and Springer Science+Business Media Dordrecht 2013
Abstract The inherited pediatric leukodystrophy Canavan disease is characterized by dysmyelination and severe spongiform degeneration, and is currently refractory to treatment. A definitive understanding of core disease mechanisms is lacking, but pathology is believed to result at least in part compromised fatty acid synthesis during myelination. Recent evidence generated in an animal model suggests that the breakdown of N-acetylaspartate metabolism in CD results in a heightened coupling of fatty acid synthesis to oligodendrocyte oxidative metabolism during the early stages of myelination, thereby causing acute oxidative stress. We present here the results of a dietary intervention designed to support oxidative integrity during developmental myelination in the nur7 mouse model of Canavan disease. Provision of the odd carbon triglyceride triheptanoin to neonatal nur7 mice reduced oxidative stress, promoted long-term oligodendrocyte survival, and increased myelin in the brain. Improvements in oligodendrocyte survival and myelination were associated with a highly significant reduction in spongiform degeneration and improved motor function in triheptanoin treated mice. Initiation of triheptanoin treatment in older animals resulted in markedly more modest effects on these same pathological indices, indicating a window of therapeutic intervention that corresponds with developmental myelination. These results support the targeting of oxidative integrity at early stages of Canavan disease, and provide a foundation for the clinical
Communicated by: Jerry Vockley Electronic supplementary material The online version of this article (doi:10.1007/s10545-013-9663-6) contains supplementary material, which is available to authorized users. J. S. Francis (*) : V. Markov : P. Leone Cell and Gene Therapy Center, Department of Cell Biology, Rowan University School of Osteopathic Medicine, 40 East Laurel Rd, Stratford, NJ, USA e-mail: [email protected]
development of a non-invasive dietary triheptanoin treatment regimen.
Introduction Leukodystrophies are a class of inherited metabolic diseases that affect the production and/or maintenance of myelin in the nervous system. These single gene disorders provide valuable insight into the complexities of myelination by highlighting key metabolic processes of broad significance. Thus, the characterization of models of leukodystrophies can potentially lead to the identification of core mechanisms that provide a foundation for the development of targeted augmentation strategies for therapeutic intervention (Saher et al 2012). Canavan disease (CD), a fatal pediatric leukodystrophy, is caused by mutations to the aspartoacylase (aspa) gene that result in loss of the ability to catabolize N-acetylaspartate (NAA; Kaul et al 1993). NAA is an abundant neuronal amino acid derivative that acts as an acetyl donor for the synthesis of select myelin lipids through the catabolic activity of oligodendrocytic ASPA (Chakraborty et al 2001). ASPA is the sole known NAA-catabolizing enzyme in the brain, and chronically elevated substrate is therefore diagnostic for CD. Pathologically, CD is characterized by dysmyelination and severe white matter vacuolation and current intractability to treatment is due in large part to a lack of consensus regarding causative mechanisms. Hypotheses for CD pathology range from deficiencies in specific myelin lipids (Madhavarao et al 2005) through to osmotic damage to white matter (Baslow 1999). Experimental evidence for causative mechanisms is limited to that defined by reduced lipid synthesis (Chakraborty et al 2001; Wang et al 2009), but these deficiencies appear not to be rate-limiting for pathology (Traka et al 2008). We have recently identified oxidative stress as an early pathological marker upstream of oligodendrocyte loss and
J Inherit Metab Dis
dysmyelination in the nur7 mouse model of CD suggestive of a role for NAA in the maintenance of oligodendrocyte metabolic integrity during myelination (Francis et al 2012). These recent studies suggest that the use of NAA-derived acetyl groups during myelination may be a means by which oligodendrocytes preserve energetic resources during myelination by uncoupling fatty acid synthesis from oxidative metabolism. Therefore, if a means by which metabolic substrates of fatty acid synthesis and energy were made available during the early stages of myelination, the progressive degeneration of the CD brain may be reduced. The odd-carbon triglyceride triheptanoin is a dietary anaplerotic substrate that provides ketone bodies capable of traversing the blood brain barrier (BBB) and increasing the mass of tricarboxylic acid (TCA) cycle intermediates to support the brains synthetic needs. Triheptanoin has shown efficacy in the treatment of disorders of mitochondrial oxidation and pyruvate metabolism (Roe and Mochel 2006; MarinValencia et al 2010), and the ability to provide AcCoA via the ß-oxidation of carbons 1–4 is believed to be the basis of reported anti-convulsant effects in models of epilepsy (Willis et al 2010; Thomas et al 2012). Because AcCoA is a key intermediate for both fatty acid synthesis and the TCA cycle, we hypothesized that triheptanoin may compensate for the loss of the ASPA function by uncoupling a portion of fatty acid synthesis from energetic metabolism through the provision of AcCoA while simultaneously providing anaplerotic support for the TCA cycle via the propionyl moiety of heptanoate.
Materials and methods Animals Animals used in this study were drawn from an in house colony. This colony was established from founder animals available commercially (Jackson Laboratories, Bar Harbor, ME). Nur7 mutants were generated from the breeding of heterozygous carrier animals and genotyped using a customized SNP assay (available upon request). All procedures performed were done so under an approved Rowan-SOM IACUC protocol, and adhered to national and institutional guidelines for animal care and use.
Table 1 Composition of control and triheptanoin chow Component
Casein Corn starch Maltodextrin (lo-dex) Sucrose Cellulose Mineral mix, Ca-P deficient Vitamin mix Calcium phosphate dibasic Calcium carbonate DL-methionine Magnesium oxide TBHQ Choline bitartrate Pantothenic acid Hydrogenated veg. oil Coconut oil Triheptanoin Corn oil Total (g) Kcal/g Protein Carbohydrates Natural fat Triheptanoin
Control diet (g/kg) 200.0 389.1 100.0 150.0 50.0 13.4 10.0 7.5 6.9 3.0 0.20 0.07
35 % triheptanoin diet (g/kg) 215.0 350.0 89.8 102.6 14.3 10.7 8.0 7.4 3.2 0.22 0.08 2.50 1.93
50.0 20.0
1000.0 3.8 18.9 63.7 17.4
170.5 23.5 1000.0 4.0 18.9 40.2 5.9 35.0
chow was produced commercially (Bio Serv, Frenchtown, NJ) and provided to animals ad libitum. For experiments involving neonatal animals, nursing dams were supplied with ad libitum triheptanoin containing chow. After weaning, all triheptanoin and control animals were housed individually to enable monitoring of food intake. All animals on triheptanoin chow were monitored for hypoglycemia from 6 weeks of age, and weighed weekly from 8 weeks of age Tail blood was gently massaged from a microincision made in the tail vein of an isoflurane anesthetized mouse. Blood was taken in 30– 50 μl sample size, applied to a test strip and glucose measured using a blood glucose monitor (Bayer COUNTOUR). Blood was collected at 6, 8, and 12 weeks of age at a prescribed time of day.
Triheptanoin chow HPLC Triheptanoin was administered to animals in dietary form (composition in Table 1). Rodent chow containing 35 % triheptanoin. Triheptanoin was synthesized to 98 % purity in liquid form (Shanghaihuating Chemical Co. Ltd., Shanghai, People’s Republic of China) and incorporated into chow to replace 35 % of calories therein. Triheptanoin containing
The analysis of N-acetylaspartate (NAA), reduced glutathione (GSH), oxidized glutathione (GSSG), adenosine tri-phosphate (ATP), malondialdehyde (MDA), ascorbic acid, propionyl coenzyme A (PropCoA), methylmalonyl coenzyme A (MeMalCoA), and acetyl coenzyme A (AcCoA) was
J Inherit Metab Dis
performed using an ion-paired high performance liquid chormoatography method. Samples were prepared from whole brains rapidly removed from deeply anesthetized animals and flash frozen in liquid nitrogen. Frozen tissue was weighed and dispersed in 2× volume/wet weight of a freshly prepared ice-cold precipitation solution consisting of 75 % acetonitrile and 25 % KH2PO4 (pH 7.4). Dispersed brain tissue was centrifuged at 12 500×g for 10 min at 4 °C and the supernatant retained. Of fresh precipitating solution 0.5 ml was added to the pellet, which was sonicated, centrifuged a second time, and the resulting supernatant added to that from the first spin. Supernatant was then extracted twice with HPLC grade chloroform, and stored at −80 °C until analyzed. Samples were run on a Thermo Scientific HPLC autosampler fitted with a 50 μl loop and a Hypersil BDS-C18 column (5 μm particle size; 25 cm×4.9 mm) attached to a Suerveyor Plus UV detector. Data was analyzed with ChromQuest software (Thermos Scientific), and target metabolites quantified against standard curves generated from purified compunds. Run conditions were as follows: 100 % buffer A 25 min, 80 % buffer A 10 min, 55 % buffer A 11 min, 40 % buffer A 11 min, 35 % buffer A 10 min, 25 % buffer A 15 min, 100 % buffer B 35 min, 100 % buffer A 25 min (buffer A: 12 mmol/L Bu4NOH, 10 mmol/L K2HPO4, 0.125 % CH3OH pH 7.00; buffer B: 2.8 mmol/L Bu4NOH, 100 mmol/L K2HPO4, 30 % CH3OH pH 5.50). Quantities of target metabolites in samples are expressed as molarity per wet weight of tissue. For the quantification of NAA, samples were diluted 4× to ensure elevated nur7 NAA signals fell within the linear limits of detection. For all other target signals, samples were run undiluted. Immunohistochemistry and stereology Immunohistochemistry was performed on paraformaldehydefixed tissue. Animals were deeply anesthetized and transcardially perfused with ice-cold 0.9 % saline followed by 4 % buffered paraformaldehyde. Perfused brains were removed and post fixed in 4 % paraformaldehyde overnight, then cryoprotected in an ascending sucrose gradient (10 %, 20 %, and 30 %). Cryoprotected brains were flash frozen and stored at −80 °C until processed. The entire forebrain was serially sectioned (40 μ m) and maintained in order for sterological sampling. Staining for adenomatous polyposis coli (APC), myelin basic protein (MBP), and MHC-II was performed using commercially available antibodies (Millipore, Billerica, MA, USA). Free-floating sections were permeabilized in 0.1 % triton-X in phosphate-buffered saline for 20 min, treated with 1 % H2O2, then washed 3×5 min in 0.1 % trition-X. Primary antibodies were diluted in immunobuffer (0.1 % trition-X with 1 % normal goat serum) and incubated with sections overnight at room temperature with gentle agitation. Following primary antibody and
washes, sections were incubated for 2 h at room temperature with biotinylated secondary antibody followed Avidinperoxidase for 1 h at room temperature. Positive cells were visualized with metal-enhanced DAB substrate (Thermo Scientific Pierce Protein Products, Rockford, IL, USA), and slide mounted in series. Mounted sections were dehydrated in an ascending ethanol series, cleared with xylene, and coverslipped. The optical fractionator was used to sample processed sections for APC, MBP, MHC-II, and vacuole volume fraction. The optical dissector was used to generate estimates of N for APC, “space balls” used to generate estimates of L for MBP-positive fibers, and an object area fraction probe used to generate estimates of vacuolation in the thalamus. MBPpositive cortical fibers were expressed as a length density (LD) calculated by dividing the total MBP-positive fiber length within the region of interest by the volume of the region of interest. Vacuole volume fraction was calculated using the formula, ∑P obj /∑P ref =A obj /A ref =Vobj /V ref , where P obj = region points interacting with vacuoles, Vobj = region points interacting with the reference space, A = area, and V = volume. Scoring for objects of interest was performed using Stereologer software (Stereology Resource Center, Chester, MD, USA). True biological variance between samples (BV) was distinguished from within-sample varaiation (CE) using the equation, (BV =√CV 2 −CE 2), where CV = total variance. Statistical significance between treatment groups was calculated using a two-tailed studen’s t-test. Electron microscopy Deeply anesthetized animals were transcardially perfused with a freshly prepared buffered solution of Kanovsky’s fixative. Brains were removed, post-fixed overnight and cryoprotected in an ascending sucrose gradient then flashfrozen and stored at −80 °C for preparation. Frozen brains were thawed in ice-cold PBS and the thalamus dissected. Dissected thalami were postfixed in 1 % osmium tetroxide for 1 h at room temp. Serial semithin sections of thalami were generated and imaged by transmission electron using a JEOL 1200EX electron microscope. Rotarod testing and open filed activity Animals were assessed for motor function using an accelerating rotarod (MedAssociates Inc.). The testing period involved three consequetive days, with the first 2 days being pre trial training. Each training day involved 3× runs with a 30 s interval between runs for each animal. On the third day, three testing runs were performed, and the average latency to fall from the accelerating rotarod (seconds) was calculated. Open field activity was assessed using automated activity chambers (MedAssociates Inc.). Animals were brought into the testing
J Inherit Metab Dis
room an hour prior to testing, and open field sessions of 30 min were conducted at 8 and 12 weeks of age. Statistical methods For all HPLC analyses, one way ANOVA with a Bonferroni correction was used to compare metabolite concentrations between groups. For the analysis of coenzyme A derivatives and redox metabolites in 2 week-old animals, a sample size of 6 animals per group was used. For the HPLC analysis of metabolites in 12 week-old animals, a sample size of 6 animals per group was used. All stereological analyses used a sample size of 6 animals per group, and statistically significant differences between groups were determined using a twotailed t-test. Statistical significance in rotarod data at individual time points was assessed using one-way ANOVA with a Bonferroni correction. For all graphs presented, the mean +/− SEM for each group is presented. Statistical significance is indicated for each graph as follows; p
