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Human Molecular Genetics Advance Access originally published online on February 4, 2008
Human Molecular Genetics 2008 17(10):1465-1481; doi:10.1093/hmg/ddn035
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Insulin receptor and lipid metabolism pathology in ataxin-2 knock-out mice

Isabel Lastres-Becker1, Susanne Brodesser2, Dieter Lütjohann3, Mekhman Azizov1, Jana Buchmann4, Edith Hintermann5, Konrad Sandhoff2, Annette Schürmann4, Joachim Nowock1 and Georg Auburger1,*

1 Section of Molecular Neurogenetics, Department of Neurology, Building 89, 3rd Floor, J.W. Goethe University Medical School, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany 2 LIMES, Program Unit Membrane Biology and Lipid Biochemistry, University of Bonn, c/o Kekulé-Institute of Organic Chemistry and Biochemistry, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany 3 Department of Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany 4 Department of Pharmacology, German Institute of Human Nutrition, Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany 5 Pharmazentrum Frankfurt, Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES), Johann Wolfgang Goethe University, Frankfurt am Main, Germany

* To whom correspondence should be addressed. Tel: +49 6963017428; Fax: +49 6963017142; Email: auburger{at}em.uni-frankfurt.de

Received December 13, 2007; Accepted January 30, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Ataxin-2 is a cytoplasmic protein, product of the SCA2 gene. Expansion of the normal polyglutamine tract in the protein leads to the neurodegenerative disorder Spino-Cerebellar Ataxia type 2 (SCA2). Although ataxin-2 has been related to polyribosomes, endocytosis and actin-cytoskeleton organization, its biological function remains unknown. In the present study, an ataxin-2 deficient mouse (Sca2–/–) was generated to investigate the functional role of this protein. Homozygous mice exhibited reduced fertility and locomotor hyperactivity. In analyses up to the age of 6 months, the absence of ataxin-2 led to abdominal obesity and hepatosteatosis. This was associated with reduced insulin receptor expression in liver and cerebellum, although the mRNA levels were increased indicating a post-transcriptional effect of ataxin-2 on the insulin receptor status. As in insulin resistance syndromes, insulin levels were increased in pancreas and blood serum. In the cerebellum, increased levels of gangliosides and sulfatides, as well as decreased cholesterol dynamics, may be relevant for cellular membrane functions, and alterations in the sphingomyelin cycle may affect second messengers. Thus, the data suggest altered signaling in ataxin-2 deficient organisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Spinocerebellar ataxia type 2 (SCA2) belongs to the growing family of neurodegenerative diseases caused by expansion of a polyglutamine (polyQ) tract. This group includes SCA1, Machado-Joseph disease (SCA3 or MJD), SCA6, SCA7, SCA17, Huntington's disease, spinal bulbar muscular atrophy and dentatorubral-pallidoluysian atrophy. SCA2 is an autosomal dominant disorder with a locomotor coordination deficit which is caused by progressive degeneration of cerebellar Purkinje cells, and selective loss of neurons within the brainstem and the spinal cord (1). The gene responsible for SCA2 was independently identified by three groups (2–4). The chromosomal localization of human SCA2 has been mapped to 12q24.1 (2). The genomic DNA of normal individuals contains up to 31 highly polymorphic CAG repeat units, while that of individuals suffering from the disease is expanded to 32 to over 200 CAG repeat units (4,5). Ataxin-2, the product of the SCA2 gene, is a 150 kDa cytoplasmic protein expressed in many regions of the brain and other tissue types (6). Ataxin-2 has little similarity with other proteins of known function (6). Furthermore, the mouse Sca2 gene has only one CAG codon at the site of the human repeat, suggesting that the normal function of ataxin-2 resides in the regions flanking the CAG repeat (6). Although the gene and protein structures from ataxin-2 are well known, the cellular function of ataxin-2 and the mechanism by which the polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. Several lines of evidence suggest that ataxin-2 is implicated in RNA metabolism: first, ataxin-2 contains an Lsm domain, which is known to function as an RNA-binding domain in proteins that mediate pre-mRNA splicing and mRNA decay (7). Ataxin-2 assembles with polysomes and interacts with the cytoplasmic poly(A)-binding protein 1 (PABP-1) that functions in translation initiation (8) as well as the cytoplasmic A2BP1 protein containing RNP domains characteristic for RNA binding (9). Secondly, ataxin-2 is a component of the stress granules, which are cytoplasmic sites into which untranslated mRNAs are stalled during environmental stresses (10). Apart from RNA metabolism, a role of ataxin-2 in vesicle endocytosis at the membrane has been suggested by protein interaction studies which demonstrated an association of ataxin-2 with endophilin-A (11).

In order to elucidate the function of ataxin-2, several ataxin-2 mutant animals have been generated. Using dsRNA-mediated interference (RNAi) ataxin-2 in Caenorhabditis elegans, a potent inhibitory effect on embryonic development has been found (12). Further functional genomic studies in C. elegans confirmed ataxin-2 protein depletion by RNAi to cause embryonic lethality (13,14) and sterility (15) as well as to prevent premature meiotic entry (15). In addition to its role in proliferation, ataxin-2 functions in sex determination to promote the sperm-oocyte switch at the L4 stage in XX animals (hermaphrodites). C. elegans ataxin-2 is thought to act at the post-transcriptional level downstream of the XX germline masculinizing gene, fog-2, either to enhance activity of a feminizing gene (e.g. tra-2) or to inhibit activity of a masculinizing gene (e.g. fem-3), thus regulating gene expression for germline proliferation and female sex determination (15). According to results in Drosophila, mutations that reduce ataxin-2 activity result in female sterility, aberrant sensory bristle morphology through regulation of actin-filament formation, loss or degeneration of tissues, and lethality (16). However, the widespread and conserved expression in adult tissues suggests that ataxin-2 has additional functions in the adult organism, with effects more subtle than in the embryo.

Previously, mice constitutively deficient in ataxin-2 were observed to have segregation distortion and increased weight gain while fed a fat-enriched diet, suggesting that ataxin-2 activity reduction may predispose to adult-onset obesity (17). Obesity is a readily observable phenotype, and mice with spontaneous mutations causing this phenotype have been extensively characterized (18). These include the agouti (19), obese (ob) (20), diabetic (db) (21), fat (22), tubby (23) and mahogany (24) mutant mouse strains (25). All severe mutations cause the loss-of-function of the encoded proteins. Interestingly, many of the genes that appear important in single-gene mutation events seem to be involved in a common pathway including leptin and insulin as signaling molecules (26). Knock-out (KO) or transgenic mice revealed additional obesity genes causing body weight dysregulation by hyperphagia or by changes in behavioral and metabolic responses (25).

In the present study, a new Sca2 KO mouse model has been developed. The aim of this work was to analyse the effect of ataxin-2 deficiency on a complex organism and in particular the brain, in order to gain insight into ataxin-2 function.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Absence of ataxin-2 protein in Sca2–exon 1 deleted mice
Exon 1 of the Sca2 gene codes for a considerable portion of the protein (16.6%) and might harbor an important function. Thus, the loss of mouse ataxin-2 was engineered by introduction of a targeting cassette with exon 1 flanked by loxP-sites (Fig. 1A, left panel). The design of the targeting vector would have permitted the generation of conditional Sca2–/– mice, in case the animals were not viable or tissue-specific effects were to be investigated (Fig. 1A, right panel). Attempting first the constitutive deletion, homologous recombination was achieved in three embryonal stem (ES) cell lines (Fig. 1B–D) and Cre-mediated recombination was performed by transient expression of Cre recombinase in ES cells. Chimeras and heterozygous mutant mice were bred to generate homozygous Sca2–/– mice and wild-type (WT) littermates. We observed the Sca2–/– mice to be viable, so all following experiments were performed in constitutive Sca2–/– mice. Germ-line transmission of the exon-1 deleted allele was confirmed both at the mRNA and protein level.


Figure 1
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  		Figure 1. Targeting strategy for constitutive and conditional Sca2 KO. (A) Graphic representation of the targeting strategy employed to generate constitutive (left panel) and conditional KO mice (right panel). (B-D) Proper targeting into ES cell lines 1C10, 2H3 and 2H6 was assessed with Southern blots documenting (B) 5'-homologous recombination with an NsiI restriction digest, (C) 3'-homologous recombination with an AvrII digest and (D) lack of non-homologous recombination with a SphI digest to generate the predicted restriction fragments for constitutive KO in comparison to wild-type (WT) alleles. (E) Above: scheme of ataxin-2 protein domain structure and Sca2 transcript exon structure with representation of the PCR fragments 1, 2 and 3 used for the validation of wild-type, heterozygous (HET) and homozygous KO mice. Below: 1% agarose gel showing representative RT–PCR results from cerebellum. Amplification product 1 is representative for exon 1, product 2 for exons 7–12, product 3 for exons 19–22. GAPDH was used as loading control. The absence of amplification products 1 to 3 in each KO lane demonstrates the absence of the respective Sca2 transcript fragments beyond the targeted exon 1 in Sca2–/– mice. The amplification products in each HET lane have similar intensity as in the WT lanes, as to be expected from saturation PCRs. (F) Western-blot analysis of protein extracts from cerebellum and cerebrum (brain) showed the expected loss of ataxin-2 immunoreactivity in KO tissue and a reduced intensity of ataxin-2 immunoreactivity in HET tissue, in contrast to similar intensities of the loading control glial fibrillary acidic protein (GFAP). Blots were stained with anti-ataxin-2 antibody from BD Bioscience, which recognizes an epitope between the LsmAD and the PABC domains shown in (B).

 
The successful deletion of ataxin-2 was confirmed at the RNA level using RT–PCRs at three different positions of the Sca2 transcript (Fig. 1E). The first amplicon demonstrated the successful deletion of exon 1, while the downstream amplicons demonstrated the lack of or the instability of the remaining Sca2 transcript through this mutation. Western-blot analysis with a monoclonal antibody against ataxin-2 (Fig. 1F) demonstrated the loss of the 150 kDa full-length ataxin-2 band in the cerebellum and rest brain of homozygous KO mice, and a reduced expression in heterozygous mice. The Sca2–/– mutants are therefore suitable to investigate the consequences of ataxin-2 loss-of-function. To maintain consistency, male mice were used in all phenotype experiments.

Obesity of Sca2–/– mice
The most evident phenotype observed in Sca2–/– mice was the appearance of obesity (Fig. 2A). On a normal diet, male Sca2–/– mice showed increased body weight in comparison to wild-type littermates. The weight gain was significantly elevated at the age of 3 months in male Sca2–/– mice (Fig. 2B) (17.75%), and obvious obesity was present at age 6 months (Fig. 2C) (33.85%). Similar results were observed for female mice, although the effect was not as large (data not shown). This elevated body weight was associated with the increased weight of the gonadal fat pads (2.4-fold increase) (Fig. 2D). Analysis of food consumption did not demonstrate Sca2–/– mice to be hyperphagic at the age of 6 months (Sca2+/+ mice: 3.64 ± 0.53 g/day; Sca2–/– mice: 3.46 ± 0.68 g/day).


Figure 2
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  		Figure 2. Body weight is increased progressively from age 3 months. A progressive relative increase in body weight of Sca2–/– in comparison to the WT animals is illustrated at 6 months in (A) and was quantified for ages 3 and 6 months (B) and (C), respectively. The weight of gonadal fat pads was increased accordingly (D). (Animals used at 3 months age: 10 WT and 10 KO; animals at 6 months age: 19 WT and 14 KO) (*P < 0.05; **P < 0.01; ***P < 0.005).

 
Behavioral alterations in Sca2–/– mice
To rule out decreased movement or impaired locomotor behavior as the cause for Sca2–/– mouse obesity, open field tests were carried out. In 3-month-old animals, around the onset of weight gain in Sca2–/– mice, analysis of spontaneous locomotor behavior (Fig. 3) showed a significant increase in horizontal activity, total distance, the amount of time in movement, the number of movements performed and the stereotypy counts. A trend of increase was observed for the number of stereotypies and stereotypy time. In contrast, a decrease was found for the time the animals spent at rest. At age 6 months, by which time the Sca2–/– mice were obese, their spontaneous locomotor behavior was decreased in relation to 3-month-old Sca2–/– mice, but still slightly increased with respect to WT mice of the same age (Supplementary Material, Fig. S1). These data indicate that young Sca2–/– mice are hyperactive and show no decrease of locomotor energy consumption which could account for the obesity observed.


Figure 3
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  		Figure 3. Motor activity of Sca2–/– mice is increased at age 3 months. Open-field test of spontaneous locomotor activity showed significant increases for the following parameters: horizontal activity, total distance, time of movement, number of movements and stereotypy counts. A trend of increase was observed for the number of stereotypies and stereotypy time. In contrast, the animals’ rest time was decreased. This indicates that the gain in body weight is not due to reduced energy expenditure by a decrease in movements. (Animals used at 3 months age: 8 WT and 5 KO; animals at 6 months age: 10 WT and 10 KO) (*P < 0.05; **P < 0.01; ***P < 0.005).

 
Levels of metabolic markers in blood
Several metabolic markers in blood are well known to be altered in mouse models of obesity and insulin resistance, such as insulin, glucose and leptin (27). In male Sca2–/– mice, determination of the insulin level in the serum by enzyme-linked immunosorbent assay (ELISA) showed a trend of increase apparent by 3 months, and a significant increase by 6 months of age. Since serum insulin levels are known to correlate with the amount of adipose tissue, this may be a reflection of the obesity. In contrast, the blood glucose level and the serum leptin level remained unaltered, in agreement with the apparently normal food consumption (Fig. 4). Female Sca2–/– mice also had increased insulin levels together with significantly decreased levels of blood glucose at 3 and 6 months age (data not shown).


Figure 4
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  		Figure 4. Insulin in the blood serum is increased at age 6 months. Sca2–/– animals presented a tendency toward elevated serum insulin levels at age 3 months and significantly elevated levels at age 6 months. In contrast, the blood levels of glucose and leptin were unchanged. (Animals used for glucose analysis at 3 months age: 10 WT and 10 KO; at 6 months age: 11 WT and 10 KO; animals used for insulin analysis at 3 months age: 7 WT and 6 KO; at 6 months: 8 WT and 5 KO; animals used for leptin analysis at 3 months age: 4 WT and 5 KO; at 6 months: 7 WT and 5 KO). (*P < 0.05; **P < 0.01; ***P < 0.005).

 
Insulin production in pancreas
The observation of obesity and increased blood insulin in Sca2–/– mice raises the question whether an insulin resistance is present. Insulin resistance is classically defined as a state of decreased responsiveness of target tissues to normal circulating levels of insulin (28). To test this hypothesis, several criteria were assessed experimentally. Insulin is secreted by the pancreatic β-cells in response to increased circulating levels of glucose and amino acids after a meal. Mouse pancreas was analysed for total tissue insulin levels by ELISA (Fig. 5). In 6-month-old Sca2–/– mice, the pancreatic insulin levels were significantly increased (Fig. 5A). This observation was confirmed by immunohistochemical analysis of the pancreas, which showed enlargement of pancreatic islets in Sca2–/– mice (Fig. 5B). Specifically, the number of insulin-positive β-cells appeared increased, while the lack of inflammatory infiltrations pointed against an autoimmune process as in some forms of diabetes mellitus.


Figure 5
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  		Figure 5. Pancreatic tissue at age 6 months shows increased insulin production without inflammatory infiltrations. (A) Measurement of total insulin at its production site in pancreatic tissue of 6 months old mice showed a significant elevation. (B) Immunohistochemical detection of insulin in pancreatic tissue of Sca2–/– mice showed hyperplasia of insulin-positive β-cells without evidence of inflammatory infiltration or destruction (*P < 0.05).

 
Insulin receptor levels in liver and cerebellum
Insulin resistance is directly related to insulin receptor status. To study the causes of insulin resistance, western blots for the insulin receptor (Insr) were performed in liver of 3- and 6-month-old mice. Sca2–/– mice showed a 40% decrease in the amount of Insr at the age of 6 months (Fig. 6A and B), a time at which insulin levels are increased in the Sca2–/– mouse (Figs 4 and 5A). Although the brain is the major site of glucose utilization in the basal state, it is not classically considered as an insulin-sensitive tissue. Nonetheless, the presence of insulin receptors has been demonstrated in a wide variety of brain regions, including the cerebellum (29). For this reason, analysis of insulin receptors was also performed in mouse cerebella, where a significant decrease of 19% was detected at age 6 months (Fig. 6C and D). It has previously been shown that neuronal insulin resistance may lead to obesity, hyperinsulinemia and dyslipidemia as well as contribute to reproductive abnormalities (30), in agreement with our findings in Sca2–/– mice. Insulin resistance may also be caused, at least in part, by increased levels of gangliosides. GM3, GM2 and GM1 inhibit the insulin receptor (31). Our data suggest that the absence of ataxin-2 results in insulin resistance, with increased production of pancreatic and serum insulin and reduced levels of insulin receptors in liver and cerebellar cell membranes.


Figure 6
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  		Figure 6. Insulin receptor protein levels are decreased at age 6 months in liver and cerebellum, while Insr mRNA levels are increased already at 3 months. Densitometry analysis of western blots detecting insulin receptor levels versus loading control (β-actin and {alpha}-tubulin) at 3 and 6 months in liver (A) and (B), respectively and cerebellum (C) and (D), respectively. Insr mRNA levels as determined by qRT–PCR at 3 and 6 months in liver (E) and (F) and cerebellum (G) and (H). (Number of animals used for each genotype and each age: n = 4) (*P < 0.05; **P < 0.01).

 
In order to investigate the causes of the reduction in the Insr protein levels, measurements of the Insr mRNA levels were performed by quantitative real-time reverse-transcriptase–PCR (qRT–PCR) in liver and in cerebellum. The mRNA expression of Insr was significantly increased in liver at 3 and 6 months (2.3- and 1.5-fold, respectively) (Fig. 6E and F), as well as in cerebellum at 3 and 6 months (1.4- and 1.6-fold, respectively) (Fig. 6G and H). These results indicate that the reduction of Insr protein levels in Sca2–/– mouse tissue is not correlated with a decrease in mRNA levels and that it is due to a post-transcriptional effect.

Lipid alterations in liver
Histological oil-o-red staining, which detects lipid droplets, demonstrated that Sca2–/– animals develop hepatosteatosis, as demonstrated by a marked lipid accumulation in livers of 3- and 6-month-old mice (Fig. 7A). PAS-staining, which detects glycogen, also demonstrated an increase in Sca2–/– animals at both ages (Fig. 7B). On the other hand, analyses of the cholesterol pathway in liver did not detect any alteration (Supplementary Material, Table S1). This indicates that the cholesterol pathway in liver is not changed due to the absence of ataxin-2.


Figure 7
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  		Figure 7. Liver tissue shows lipid droplet accumulation and enhanced glycogen deposition at ages 3 and 6 months. (A) Oil Red O staining demonstrated the accumulation of lipid droplets in Sca2–/– liver with marked progression over time. (B) PAS-staining demonstrated increased glycogen storage in Sca2–/– liver already at age 3 months.

 
Analyses of gene expression changes known to be related to obesity in liver were carried out by qRT–PCR. The expression levels of fatty acid synthase (FAS), glucokinase, malic enzyme, pyruvate kinase, SCD1 (stearoyl-CoA desaturase 1) and SCHAD (short-chain 3-hydroxyacyl-CoA dehydrogenase) were not altered consistently or progressively (Table 1). Collectively, these data indicate that the liver of Sca2–/– animals accumulates lipids without alterations in the expression of these key metabolic enzymes.


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  		Table 1. mRNA expression levels of liver proteins related to obesity were not consistently altered at ages 3 and 6 months

 
Interestingly, the mRNA levels of insulin-induced gene 1 (Insig1) are increased in the liver of Sca2–/– in comparison to WT mice (3 months: WT: 1.035 ± 0.529; KO: 3.469 ± 1.975, P < 0.05; 6 months: WT: 1.026 ± 0.213; KO: 1.967 ± 0.623, P < 0.05). Insig1 is upregulated by insulin and highly expressed in liver, so its induction is presumably a consequence of the elevated blood insulin level in Sca2–/– mice. Insig1 expression is known to restrict lipogenesis (32) and to decrease cholesterol synthesis (33), so presumably its induction represents an effort to limit the obesity.

Cholesterol metabolic markers in serum
To determine whether the cholesterol pathway is altered in Sca2–/– venous blood serum, analyses were performed by gas chromatography (34) at the age of 3 and 6 months (Table 2). Serum cholesterol is considered a biomarker of average whole body cholesterol metabolism, perhaps with the exception of brain. No alterations were observed either in the biosynthesis markers like lanosterol, lathosterol or desmosterol, or in the degradation products 24S-hydroxycholesterol (24SOH) or 27-hydroxycholesterol (27OH) of the cholesterol pathway. But the levels of cholesterol and its metabolite cholestanol were dramatically increased, indicating high circulating levels. These data indicate that Sca2–/– mice have increased serum cholesterol levels in addition to obesity, suggesting an elevated diabetic and vascular risk in these mice.


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  		Table 2. Analysis of the cholesterol pathway shows increased levels of cholesterol and cholestanol in blood serum at age 6 months

 
Selective brain lipid alterations in cerebellum
Our thin layer chromatography (TLC) analysis of brain lipids included the three major categories of lipids contained in neural membranes: cholesterol, sphingolipids and phospholipids (35). The cerebellum of 3-month-old Sca2–/– mice exhibited normal cholesterol and galactosylceramide levels (Fig. 8A). The amounts of ceramide and sulfatide, however, appeared slightly increased (~1.3- and ~1.5-fold, respectively), and the sphingomyelin level was significantly decreased by ~20%. The most notable alteration became obvious when analyzing the ganglioside content, which was consistently elevated in Sca2–/– cerebellum. Regarding the amounts of GM1 and GD1a, the increase was particularly distinct and significant (2.1- and 1.7-fold, respectively).


Figure 8
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  		Figure 8. Brain thin layer lipid chromatography analysis demonstrates specific alterations in the cerebellum regarding the sphingomyelin cycle and the ganglioside pathway. Brain lipid analyses were performed in 3-month-old cerebellum (A) and neocortex (B). (*P < 0.05).

 
In cortex (Fig. 8B), the levels of cholesterol, sphingomyelin and of the myelin lipids galactosylceramide and sulfatide did not differ significantly from the amounts found in WT animals. The content of ceramide, the common precursor of glycosphingolipids (GSL) and sphingomyelin, appeared slightly elevated (1.2-fold) compared to WT tissue. In addition, a non-significant decrease of ganglioside levels including GM1, GD1a, GD1b, and GT1b was detected. These sialic acid-containing GSL are particularly abundant on the surface of neurons in the brain grey matter, where they participate in recognition and signaling processes (10). Taken together, in Sca2–/– brain the lipid anomalies were inconspicuous in cortex but prominent in the cerebellum, resulting in increased gangliosides and decreased sphingomyelin.

Cholesterol metabolic markers in cerebellum
The central nervous system accounts for 2% of the whole body mass, but contains up to 25% of unesterified body cholesterol (36). Brain cholesterol is mostly independent from dietary uptake or hepatic synthesis and almost completely synthesized in situ (37). Although the steady-state levels of cholesterol were not changed in the cerebellum and cortex of Sca2–/– mice compared to wild-type, a more detailed analysis of the cholesterol metabolism pathway by gas chromatography (34) was also performed (Table 3). Although the cholesterol levels were found unchanged, the biosynthesis (lanosterol) and degradation (27OH) of cholesterol were significantly decreased in Sca2–/– cerebellum at the ages of 3 and 6 months, indicating a compensatory mechanism between biosynthesis and degradation to maintain normal levels of cholesterol.


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  		Table 3. Analysis of the cholesterol pathway shows decreased biosynthesis and degradation consistently in cerebellum at ages 3 and 6 months

 
Brain proteins related to sphingomyelin and lipid metabolism
To investigate the alterations of sphingomyelin, gangliosides and cholesterol metabolism in cerebellum in more detail, qRT–PCR expression analysis was carried out for the following lipid-related proteins: (i) neutral-sphingomyelinase (N-SMase) and acid-sphingomyelinase (A-SMase), two distinct phosphodiesterases that are responsible for the hydrolysis of sphingomyelin to yield ceramide and phosphocholine (38), (ii) peroxisome proliferator-activated receptor delta (PPAR{delta}), a transcriptional regulator enhancing lipid metabolism which has its highest expression in brain and is postulated to play a major role in neuronal cell function, (iii) liver X receptor (LXRβ), a nuclear receptor that functions as intracellular sensor for sterols to maintain a balanced and finely tuned regulation of cholesterol metabolism (39), (iv) fatty acid binding protein 7 (FABP7), also called brain lipid binding protein (BLBP), which belongs to those fatty acid binding proteins that reduce lipid accumulation induced by high levels of saturated fatty acids (40) and (v) PPAR{gamma} coactivator-1 (PGC-1), a transcription coactivator of nuclear receptors playing a critical role in the maintenance of glucose, lipid and energy homeostasis and likely involved in pathogenic conditions such as obesity, diabetes, neurodegeneration and cardiomyopathy (41). The mRNA levels of A-SMase and N-SMase were decreased at age 6 months (Table 4). For PPAR{delta}, there was a reduction at ages 3 and 6 months (Table 4). BLBP was increased at 3 and 6 months, both for mRNA (Table 4) and protein levels (Fig. 9A and B). LXRβ and PGC-1 levels were unchanged at both ages. All these data together indicate that the absence of ataxin-2 leads to a progressive alteration of the sphingomyelin cycle and other related lipids in the cerebellum. It can not be decided unequivocally on the basis of these data whether the cerebellar lipid alterations in Sca2–/– mice are a primary consequence of the ataxin-2 deficiency or are secondary to systemic lipid and insulin anomalies.


Figure 9
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  		Figure 9. Increased levels of BLBP in Sca2–/– cerebellum. Densitometry analysis of western blots detecting BLBP levels versus loading control (GAPDH) at 3 and 6 months in cerebellum. (Number of animals used for each genotype and each age: n = 4) (*P < 0.05; **P < 0.01).

 


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  		Table 4. Levels of brain lipid associated proteins in cerebellum indicates early changes of BLBP and PPAR{delta}, with later changes in sphingomyelinases

 
Brain morphology of Sca2–/– mice
It has been reported that insulin resistance with excessive insulin signaling can produce dyslipidemia with important consequences for the high lipid content of the brain. The nervous system characteristically contains a very high concentration of lipids, and displays remarkable lipid diversity (42). For this reason and in view of SCA2 mutations leading to a neurodegenerative process, morphological studies of the brain were performed in Sca2–/– mice. The KO mice appeared to be neurologically normal using the SHIRPA test battery (43), and did not display any handicap. No differences in brain weight and size were observed (data not shown). No pathology was visible in histological sections of the brain at age 3 months at levels of the striatum, dentate gyrus, substantia nigra and cerebellum (Supplementary Material, Fig. S2) stained with Klüver-Barrera, a stain using Luxol fast blue and cresyl violet in sequential steps to demonstrate myelin and the rough endoplasmic reticulum (Nissl substance), respectively.

Fertility abnormalities in Sca2–/– mice
Mice carrying the homozygous mutation were viable. In order to gain more insight in the fecundity of Sca2–/– mice, the numbers of litters and the number of animals per litter were analysed in WT and KO homozygous matings. In 15 pairs of WT and 15 pairs of KO matings, over a period of four and a half months, the numbers of litters were strongly reduced in the KO matings (Fig. 10A). In a further study, 25 pairs of WT and 17 pairs of KO matings were analysed. The number of animals per litter (Fig. 10B) in the KO matings was significantly reduced, but the number of males and females born was the same. These data indicate that ataxin-2 deficiency leads to a fecundity or fertility problem. This observation is in agreement with other findings that reproductive anomalies are frequently associated with insulin resistance (44).


Figure 10
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  		Figure 10. Fertility problems of Sca2–/– mice. Offspring in Sca2–/– inbreeding is reduced in comparison with WT. Sca2–/– inbreeding resulted in significantly fewer litters (A) and pups per litter (B). (*P < 0.05; **P < 0.01).

 
We also examined the genotypes of 44 litters derived from mating heterozygous Sca2+/– mice in a mixed C57BL/6 x 129/Ola background to assess whether the absence of ataxin-2 leads to increased lethality in utero. Detailed analysis of the genotypes showed that there was a remarkable distortion of the expected genotype ratios (Table 5) suggesting disturbed development of female KO mice, in agreement with previously published data (17).


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  		Table 5. Distortion of the genotype ratios

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
We have targeted the mouse Sca2 exon 1 with loxP sites and generated mice with constitutive deficiency of ataxin-2. These mice show progressive obesity and reduced fertility. Obesity is a complex trait influenced by age, gender, diet (energy intake), energy expenditure and metabolic factors, reflecting an inappropriate storage of excess energy. Previous analyses of diverse mouse mutants with obesity typically showed insulin resistance and subfertility as associated findings. Differences between various obese mouse mutants were evident for eating behavior, neuroendocrine signaling and the manifestation of carbohydrate and lipid anomalies (45–49). Reduced kinase activity, downregulation or absence of the insulin receptor (Insr), as well as defects in its intracellular signaling in spite of normal or high circulating levels of insulin, are the known causes of insulin resistance (45). Neuron-specific Insr knock-out (NIRKO) and Irs2 (insulin receptor substrate 2) KO mice, in particular, show mild insulin resistance with subsequently increased fat mass and decreased fertility (45–47). Insulin signaling stimulates hepatic glycogen accumulation through a coordinated increase in glucose transport and glycogen synthesis, promotes lipid synthesis and inhibits lipid degradation (28,50).

Our findings in male mice of up to 6 months of age show the absence of ataxin-2 to result in a significant reduction of Insr levels in liver and brain, accompanied by elevated insulin levels in pancreas and serum, accumulation of glycogen and fat together with induction of Insig1 expression in liver, and dyslipidemia. Specifically in the cerebellum, significant changes of sphingomyelin, gangliosides, cholesterol homeostasis, expression levels of the sphingomyelinases A-SMase and N-SMase, the transcriptional lipid regulator PPAR{delta}, and of the fatty acid binding protein BLBP were identified. PPAR{delta} is a receptor of lipid-signals (51), and BLBP expression is modulated by Notch signaling (52). Sphingolipids, ganglioside GM1 and cholesterol are characteristic constituents of neuronal membranes which are involved in signaling and neurodegenerative diseases (53–57). Specifically, ganglioside GM1 and related a-series gangliosides inhibit the Insr (31), although this occurs with unchanged Insr levels through reduced phosphorylation. Thus, it might be argued that altered insulin signaling occurs as a consequence of the lipid anomalies induced by ataxin-2 deficiency. Insulin signaling in the brain has been implicated in the activity modulation of excitatory and inhibitory receptors, synaptic plasticity, memory and even neurodegeneration (58,59). The notion that signaling is altered in specific brain regions is supported by our observation of locomotor hyperactivity in Sca2–/– mice.

The obesity appears to be the consequence of increased feed efficiency or a lower basal metabolic rate, since even with increased locomotor activity and unchanged food intake, Sca2–/– developed a higher body weight than their wild-type littermates. How is this metabolic phenotype compatible with the available data on ataxin-2 function? It has been shown that ataxin-2 assembles with polyribosomes and interacts with poly(A)-binding protein PABP-1 (8); it might therefore be speculated that ataxin-2 affects the efficiency of protein synthesis or processing. This function would be highly selective, as the levels of various proteins analysed were not altered by the ataxin-2 deficiency. In concordance with this hypothesis, we found increased levels of Insr mRNA but decreased levels of protein in liver and in cerebellum, suggesting a role of ataxin-2 in post-transcriptional pathways. However, ataxin-2 might not exert an effect on the synthesis of proteins but rather on their degradation. It has been found that ataxin-2 directly interacts with endophilin A1 (11) and parkin (60), proteins known to regulate the endocytosis of receptor tyrosine kinases similar to Insr (61,62). Therefore, the most attractive explanation of the downregulated Insr levels in Sca2–/– liver and cerebellum would be an effect of ataxin-2 on the endocytic degradation of Insr. Indeed, altered internalization of Insr resulting from a K44A-dynamin mutation is known to increase lipogenesis and glycogen synthesis (63,64), and defects in Insr internalization have been demonstrated in patients with obesity (65). Thus, it is conceivable that altered insulin signaling through reduced Insr levels might be a consequence of ataxin-2 deficiency.

Similar to our findings in Sca2–/– mice, progressive weight changes and lipid pathology affecting cerebellum rather than neocortex are also features of the disease SCA2. A progressive loss of subcutaneous fat tissue can be observed in SCA2 mutation carriers from presymptomatic stages (66), with consequent weight loss due to reduced body fat as documented also for other polyglutamine neurodegenerations (67,68). The selective cerebellar demyelination early in the course of SCA2 includes changes in sphingomyelin tissue concentrations. It is widely accepted that the biological function of the disease proteins is relevant for polyglutamine pathogenesis, and therefore it will be interesting to study lipid metabolism in SCA2 patients and mouse models further and assess insulin receptor dysfunction. It would also be important to extend the study of mice with genetically altered insulin signaling to determine whether the enrichment of insulin receptors in the cerebellum (69) results in differential pathology of lipid metabolism as observed in Sca2–/– mice. Such data would help to decide whether the cerebellar lipid alterations observed are secondary to insulin resistance or constitute a primary consequence of ataxin-2 loss-of-function.

In conclusion, the lack of ataxin-2 leads to obesity, diminished Insr protein in parallel to elevated Insr mRNA levels, hepatosteatosis and dyslipidemia. These findings are compatible with a scenario where ataxin-2 directly regulates Insr activity and degradation at the endocytosis machinery. The cerebellar changes in sphingomyelin, gangliosides, sulfatides, cholesterol, sphingomyelinases, PPAR{delta} and BLBP suggest altered signaling at neuronal membranes and may correlate to the locomotor hyperactivity of Sca2–/– mice. It remains unclear whether these ataxin-2 physiological function findings are relevant for the pathogenesis of the neurodegenerative disease SCA2, where an unstable expansion of the polyglutamine domain within ataxin-2 leads to progressive cerebellar atrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Generation of constitutive and conditional Sca2 knock-out mice
Generation of the pKO-Sca2-vector: an 8.4 Kb BglII fragment containing exon 1 was used to generate the targeting construct pKO-Sca2 (Fig. 1A). The 8.4 kb BglII fragment was cloned into the pV4n vector, which contains a diphtheria toxin A (dta) gene cassette as negative selection. A conditional targeting strategy was used in which the targeting vector contained exon 1 flanked by loxP-sites, as well as an FRT-flanked neomycin (neo) selection cassette. For homologous recombination, the targeting vector pKO-Sca2 was linearized and transfected into 129/Ola ES cells by electroporation. ES cells were grown under neo selection. Neo-resistant clones were screened by an optimized PCR analysis for positive recombination events (details available upon request). Three ES cell clones with complete integration of the targeting vector were obtained. Correct recombination and the lack of additional, randomly integrated copies were verified by Southern blotting. All three clones had a complete set of chromosomes as determined by karyotype analysis. For the constitutive KO, the ES cell lines with homologous recombination were transiently transfected with Cre-recombinase expression vector. Removal of the loxP flanked sequences was assayed by a duplex PCR analysis. Two recombinant clones were obtained for further blastocyst injection. Ten-to-twelve ES cells were injected pro blastocyst from C57BL6 mothers. With each floxed ES cell line, chimeric mice were generated, and from each line germ line transmission was obtained (2+3). The mice were maintained in a mixed 129/Ola/C57BL6 (50/50) genetic background and littermates were compared for each experiment. This transgenic work was out-sourced to the company Murinus GmbH, Hamburg-Eppendorf, now Genoway Inc.

Animals and genotyping
Animals were housed in individually ventilated cages, 4–6 animals per cage, under a 12 h light cycle with food (Ssniff® M-Z, calories from protein 36%, fat 11% and carbohydrates 53%) and water provided ad libitum, in compliance with all relevant federal guidelines and institutional policies. All phenotypic analysis was performed on male mice with a mixed C57BL/6 x 129/Ola background. Genotyping was performed with tail biopsies by PCR with three sets of primers: Sca-In0-F3 (5'-TGA CCT CTA TCT CCC GAA TGC T-3'), Sca-In0-R2 (5'-GCT CGC TCG GAC TTC TTA GTT-3') and Sca-In1-R3 (5'-TGC TAT CTC AAA CAT GAC GCC T-3') (Supplementary Material, Fig. S3).

Reverse transcriptase (RT) PCR of the Sca2 transcript in mice
Total RNA from cerebellum of WT, homozygous Sca2–/– (KO) and heterozygous Sca2+/– (HET) mice was isolated using TRIZOL Reagent. cDNA was generated from 5 µg RNA template using the First-Strand cDNA Synthesis Kit (Amersham Bioscience). One microliter of the first-strand mixture was added to 50 µl of the PCR mix containing 200 µM of dNTPs and 10 µM of sense and antisense primers (see Fig. 1B), corresponding to PCR product 1 of 490 bp (exon 1) (forward: 5'-CAG CCC GGG TCC CAT GCG TTC GT-3', reverse: 5'-CGG CTG CGG CTG CGG CTT CA-3'), PCR product 2 of 741 bp (exon 7–12 according to NCBI Entrez Evidence Viewer) (forward: 5'-TCG GGG CCA AAA CGT GAA GAA ATA-3', reverse: 5'-AGG TGG GCG AGA GGA AGG AGA TGG-3') or PCR product 3 of 710 bp (exon 18–22) (forward: 5'-TCA GCC AAA GCC TTC TAC TAC CC-3', reverse: 5'-CAT GCT GGC TTT GCT GCT GTC-3'). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (forward: 5'-TTC ACC ACC ATG GAG AAG GC-3', reverse: 5'-GGC ATC GAC TGT GGT CAT GA-3'). Amplified products were resolved on 1% agarose-gels.

RNA preparation and first strand cDNA synthesis
RNA was extracted using the Trizol Reagent (Invitrogen, Karlsruhe, Germany) method. Two microgram total RNA were digested with DNaseI Amplication Grade (Invitrogen, Karlsruhe, Germany) with 1x DNaseI buffer [20 mM Tris–HCl (pH 8.4), 2 mM MgCl2, 50 mM KCl] and 2 Units DNaseI (1 U/µl) Amplication Grade in reaction volumes of 20 µl. cDNA synthesis was performed by using First Strand cDNA Synthesis Kit (GE Healthcare, UK).

Quantitative real-time RT–PCR (qRT–PCR)
Twenty-five to thirty nanogram cDNA were used to perform a Taqman® assay in a final reaction volume of 20 µl. The expression analysis was carried out using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany) and 96-well Micro Plates (Applied Biosystems, Singapore). All reactions were carried out using the Taqman® Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems). Primers and probes for TaqMan® PCR were obtained by using Applied Biosystems pre-designed TaqMan® Gene Expression Assays (Insr, Mm00439693_m1; glucokinase, Mm00439129_m1; malic enzyme, Mm00782380_s1; pyruvate kinase, Mm00443090_m1; SCHAD, Mm00492535_m1; Insig1, Mm00463389_m1; A-SMase, Mm00488321_g1; N-SMase, Mm00448042_g1; PPAR{delta}, Mm00803184_m1; LXRβ, Mm00437262_m1; BLBP/FABP7, Mm00445225_m1; PGC-1, Mm00447183_m1). The PCR conditions were 50°C for 2 min and 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 40 s. All assays were run in triplicates. Analysis of relative gene expression data was performed using the {Delta}{Delta}CT method with TATA box binding protein (TBP) (Mm00446973_m1) as endogenous control/reference assay (70).

For the expression analysis of other genes, the PCR mix consisted of SYBRGreen® Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), cDNA (corresponding to the 25 ng RNA used for synthesis, each sample in triplicate), and the corresponding primer pairs for FAS (forward, 5'-TTG CTG GCA CTA CAG AAT GC-3'; reverse, 5'-AAC AGC CTC AGA GCG ACA AT-3') and SCD1 (forward, 5'-GCC TCT TCG GGA TTT TCT AC-3'; reverse, 5'-GTC ATT CTG GAA CGC CAT G-3'). Data were normalized according to Livak and Schmittgen (70), using a β-actin expression assay (Mm00607939_s1; Applied Biosystems) and primers for 18sRNA (forward, 5'-TGA GGC CAT GAT TAA GAG GG-3'; reverse, 5'-TTC TTG GCA AAT GCT TTC G-3') as endogenous controls.

Western blots
Tissue samples from wild-type (WT) control, heterozygous (HET) and homozygous KO mice (n = 4) were homogenized with a tissue mincer in modified RIPA-buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, a tablet of protease inhibitor cocktail (Roche), 1 mM PMSF and 1 mM Na3VO4] and, after 15 min incubation on ice, centrifuged at 16 000 g for 20 min at 4°C. The protein concentration in the crude preparation was measured according to the standard Bradford assay. Lysates were resolved by SDS–PAGE under reducing conditions and transferred to PVDF membranes. Blots were incubated with mouse monoclonal anti-ataxin-2 antibody (1:500, BD Transduction Laboratories), rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1:10000, Sigma-Aldrich), rabbit anti-insulin-receptor-β (1:500, Santa Cruz Biotechnology), monoclonal anti-β-actin (1:10000, Sigma-Aldrich), rabbit anti-{alpha}-tubulin (1:500, Abcam), rabbit anti-brain lipid-binding protein (BLBP) antibody (1:1000, Chemicon International) or monoclonal anti-GAPDH (1:15000, Calbiochem) and visualized using the ECL method (Pierce). The data are illustrated giving the means±SD from at least n = 4 independent tissues.

Body-weight measurement and behavioral observation
The body weight was measured just before animals were sacrificed. At the age of 3 months, 10 WT and 10 KO mice were weighed before tissue dissection. At the age of 6 months, 19 WT and 14 Sca2–/– mice were used. Observational assessment of the phenotype was performed according to the primary screen from the SHIRPA test battery, where muscle and lower motor neuron, spinocerebellar, sensory, neuropsychiatric and autonomic functions are comprehensively scored, first with undisturbed behavior in a viewing jar and then during a sequence of manipulations within an arena (43).

Open-field test
Digiscan open-field arenas (20 x 20 cm, Omnitech, Columbus, OH, USA) were used to assess the spontaneous motor performance of naïve animals without previous training for 5 min, starting immediately after placing the mice in the arena.

Measurement of blood glucose, insulin and leptin levels
Blood was sampled from the retroorbital vein. Glucose levels were determined with a glucometer FreeStyle Mini (Abbott, AG). Insulin (insulin mouse ultrasensitive ELISA, DRG Diagnostics Instruments GmbH, Germany) and leptin (mouse leptin ELISA, DRG Diagnostics Instruments GmbH, Germany) were analyzed by colorimetric assays (SpectraMax Plus384, Molecular Devices). For each of the analyses, 4 to 11 animals were used.

Analysis of total insulin in pancreas
Whole pancreas was homogenized in acid ethanol (about 150 mg pancreas in 0.5 ml 70% ethanol in 0.1 N HCl) at 4°C for 24 h. After centrifugation and dilution (1:1000; 1:5000; 1:10 000 in diabetes sample buffer, DRG Instruments GmbH, Germany), insulin was measured in the supernatant with ELISA (Insulin mouse ultrasensitive ELISA, DRG Instruments GmbH, Germany).

Immunohistochemistry of pancreas
Pancreatic tissue was harvested from 6-month-old mice, immersed in tissue-tek O.C.T. (Bayer, Elkhart, IN, USA) and quick-frozen on dry ice. Six millimeter tissue sections were fixed with 4% paraformaldehyde. After washing in PBS, an avidine-biotin blocking step was included (Vector laboratories, Burlingame, CA, USA). The polyclonal guinea pig anti-swine insulin antibody (DakoCytomation, Glostrup, Denmark) and the biotinylated secondary antibody (Vector laboratories, Burlingame, CA, USA) were incubated with the sections for 1 h each and the color reaction was obtained by sequential incubation with avidine-peroxidase conjugate (Vector laboratories) and diaminobenzidine-hydrogen peroxide (Sigma-Aldrich, Germany).

Staining of mouse liver
Livers were dissected, frozen and kept at –80°C until use. Cryostat sections of liver (15 µm) were used and stained with Oil Red O to visualize the fat or lipid content of liver cells (71). Hematoxylin counterstain was used to detect cell nuclei. Periodic Acid Schiff (PAS)-staining was used for the detection of glycogen (72).

Extraction and analysis of sterols in serum and brain
Cholesterol and cholestanol, its precursors lanosterol, lathosterol and desmosterol, its metabolites 24SOH and 27OH (27-hydroxycholesterol), as well as the plant sterols campesterol and sitosterol, were extracted from brain, liver and serum (n = 6 for each genotype and for each case) by chloroform/methanol and determined after derivatization to the corresponding trimethylsilyl-ethers by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry as reported previously (34). Dry weight of brain and liver specimen was determined after drying them to constant weight overnight in a SpeedvacTM (Servant Instruments, Inc., Farmingdale, NY, USA) ultracentrifuge dryer.

Brain lipid analysis
Analysis of brain lipids followed a previous protocol (73), summarized as follows: samples of mouse cortex and cerebellum (pool of 3–4 mice, n = 2) (150–200 mg) were homogenized with a homogenizer (type 853202, B. Braun, Melsungen, Germany) at 1,000 rpm in a mixture of 600 µl water, 2 ml methanol and 1 ml chloroform. After extraction for 24 h at 37°C, the liquid phase was separated from insoluble tissue components by filtration. The solvent was evaporated in a stream of nitrogen and the residues were desalted by reversed phase chromatography on LiChroprep RP18 columns (Merck, Darmstadt, Germany) (74). Lipids were then applied to high performance thin layer chromatography (HPTLC) Silica Gel 60 plates (Merck, Darmstadt, Germany), which were pre-washed twice in chloroform/methanol (1:1, v/v). Each lane of the TLC plate was loaded with the equivalent of 5 mg wet weight of cortex or cerebellum tissue. For separation of cholesterol and ceramide, the TLC solvent system used was chloroform/methanol/glacial acetic acid (190:9:1, v/v/v). Since glycerophospholipids cannot be completely separated from sphingolipids in thin layer chromatograms, glycerolipids were degraded by alkaline hydrolysis with 2.5 ml of a 100 mM solution of sodium hydroxide in methanol for 2 h at 37°C. After neutralization with acetic acid, the mixtures were desalted again on RP18 columns. Lipids were then separated into acidic and neutral fractions by anion exchange chromatography on DEAE-cellulose columns (GE Healthcare, Uppsala, Sweden) (75), with some modifications. The lipid mixture was dissolved in 1 ml of chloroform/methanol/water (3:7:1, v/v/v) and applied to the columns. Neutral lipids were eluted with 7 ml of the same solvent, and acidic lipids were eluted with 8 ml of chloroform/methanol/0.8 M ammonium acetate in water (3:7:1, v/v/v). After further desalination on RP18 columns, lipids were applied to pre-washed HPTLC plates, loading each lane with the equivalent of 8 mg wet weight of mouse brain tissue. The TLC solvent system used for the separation of neutral sphingolipids (sphingomyelin, galactosylceramide) was chloroform/methanol/0.22% calcium chloride in water (60:35:8, v/v/v). For the separation of the acidic sphingolipids (gangliosides, sulfatide), TLC plates were developed in chloroform/methanol/0.22% calcium chloride in water (55:45:10, v/v/v). For quantitative analytical TLC determination, increasing amounts of standard lipids (ceramide 3, Cosmoferm, Delft, The Netherlands, now Degussa-Goldschmidt, Essen, Germany; cholesterol, sphingomyelin, glucosylceramide, all three Sigma, Taufkirchen, Germany) were applied to the TLC plates in addition to the lipid samples. After development, plates were dried under reduced pressure. For detection of lipid bands, the TLC plates were sprayed with a phosphoric acid/copper sulfate reagent (15.6 g of CuSO4(H2O)5 and 9.4 ml of H3PO4 (85%, w/v) in 100 ml of water) and charred at 180°C for 10 min (76). Bands were quantified in a photo-densitometer (Shimadzu, Kyoto, Japan) at 595 nm wavelength.

Brain morphology
Adult mouse brains (3 months old) were obtained from wild-type and homozygous Sca2–/– animals and frozen directly by immersion in 2-methylbutane at –80°C, and kept at –80°C until use. Coronal sections at different regions from frozen brains were cut at 15 µm, mounted onto SuperFrost Plus microscopic slides (Menzel GmbH & Co. KG, Germany) and stained with Klüver-Barrera staining.

Statistical analyses
The Graph-Pad software package (version 4.03, GraphPad Software Inc., San Diego, CA, USA) was used to perform unpaired Student's t-tests when normal distribution and equal variances were fulfilled, or the non-parametric Mann–Whitney test, and to represent data with bar graphs, illustrating mean values and standard deviations. Significant differences were highlighted with asterisks (*P < 0.05; **P < 0.01; ***P < 0.005).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by the EU project EuroSCA LSHM-CT-2004-005033, the DFG project AU 96/9-1, the German Ministry of Education, Research and Technology (0313128B) and the DFG project SFB 645 (B3). I.L.-B. was a fellow of the Alexander von Humboldt Foundation.


   ACKNOWLEDGEMENTS
 
We are grateful to Monica Fittschen, Anja Kerksiek and Birgitt Meseck-Selchow for experimental assistance and to David Nonis, Florian Eich and Suzana Gispert for helpful discussions.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
This article has been versioned to include alterations made during the peer review process.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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