Telencephalic histopathology and changes in behavioural and neural plasticity in a murine model for metachromatic leukodystrophy

Behavioural Brain Research 222 (2011) 309–314

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Telencephalic histopathology and changes in behavioural and neural plasticity in a murine model for metachromatic leukodystrophy Enrico Faldini a,1 , Stijn Stroobants a,1 , Renate Lüllmann-Rauch b , Matthias Eckhardt c , Volkmar Gieselmann c , Detlef Balschun a , Rudi D’Hooge a,∗ a

Laboratory of Biological Psychology, Department of Psychology, University of Leuven, Tiensestraat 102, B-3000 Leuven, Belgium Department of Anatomy, University of Kiel, Olshausenstraße 40, 24098 Kiel, Germany c Department of Biochemistry and Molecular Biology, University of Bonn, Nussallee 11, D-53115 Bonn, Germany b

a r t i c l e

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Article history: Received 5 November 2010 Received in revised form 24 March 2011 Accepted 27 March 2011 Available online 1 April 2011 Keywords: Synaptic plasticity Hippocampus-dependent learning and memory Mouse models Metachromatic leukodystrophy Lysosomal storage disorders

a b s t r a c t Arylsulfatase A-deficient (ASA−/− ) mice constitute an animal model for metachromatic leukodystrophy, a lysosomal storage disorder. We had previously examined the behavioural phenotype of these mice, but were unable to distinguish between proper cognitive symptoms and potentially interfering, solely neuromotor impairments. In the present study, T-maze delayed alternation (TMDA) showed that ASA−/− mice perform worse than controls already at the age of 6 months in a hippocampus-dependent task that does not require motor proficiency. In addition, long term potentiation (LTP) in the CA1 region of the hippocampus, a cellular correlate of learning and memory, was also impaired in ASA−/− mice. Finally, histological analysis of previously unexamined telencephalic and diencephalic structures illustrated sulfatide accumulation in brain areas that are important for cognitive functioning. These include the hippocampus, striatum, internal capsule and diencephalon as well as prefrontal, insular, and motor and somatosensory cortices. Together these data corroborate the usefulness of the model in preclinical evaluations of therapeutic strategies that aim to reverse cognitive defects in the human disease. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Metachromatic leukodystrophy (MLD) is a sphingolipidosis caused by lysosomal arylsulfatase A (ASA; EC 3.1.6.8) deficiency [1]. Lack of ASA triggers lysosomal accumulation of its substrate sulfatide, and subsequent progressive demyelination in the peripheral (PNS) and central nervous system (CNS). Four different forms of MLD have been defined, including late-infantile and juvenile forms as well as an adult form that presents with neurological and psychiatric symptoms [2]. Children affected with the typical late-infantile form of MLD display progressive symptoms that include ataxic gait, cognitive impairments, epileptic seizures, loss of speech, spastic quadriplegia, and final death in a decerebrated state. In contrast, patients suffering from juvenile and adult MLD show manifest heterogeneity of clinical symptoms, which was shown to be related to specific mutations in the ASA gene [3]. ASA-deficient (ASA−/− ) mice were generated by targeted disruption of the ASA gene, and develop a phenotype comparable to, but less severe than human MLD [4]. The most striking discrepancy is the lack of demyelination in these mice [5], likely due to relatively

∗ Corresponding author. Tel.: +32 16 326142; fax: +32 16 326099. E-mail address: [email protected] (R. D’Hooge). 1 Equal contribution. 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.03.059

low levels of sulfatide accumulation [6]. The mouse model proved to be useful for evaluation of experimental therapeutic manipulations including enzyme replacement [7,8], haematopoietic stem cell gene therapy [9–12], and direct CNS gene transfer [13–15]. Beyond 1 year of age, ASA−/− mice develop severely impaired motor coordination and altered gait that is proposedly related to cerebellar cell loss [16–18]. This progressive neuromotor disability is reminiscent of ataxic gait in human MLD. More recently, we were able to quantify motor incoordination (and emotional alterations) in ASA−/− mice as early as 6 months of age [19], which allowed functional evaluation of long-term enzyme replacement therapy in mice that were presymptomatic for the more severe late-developing neuromotor phenotype [8]. Cognitive decline is part of all clinical MLD phenotypes as well, but very little is known about its etiology or therapeutic reversibility. Unfortunately, previous studies in ASA−/− mice failed to demonstrate neurocognitive defects that could be unrelated to their neuromotor phenotype and/or cerebellar degeneration. The present study was therefore designed to detect early signs of cognitive dysfunction in ASA−/− mice that could be attributed to telencephalic sulfatide storage. We examined ASA−/− mice in a Tmaze delayed alternation (TMDA) task that is known to depend on hippocampal function and does not require motor proficiency [20]. We also included electrophysiological measurements of hippocampal synaptic plasticity that form an established cellular correlate

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Fig. 1. ASA−/− mice display changes in delayed alternation in a T-maze task. Although both ASA+/− controls (black symbols) and ASA−/− mice (white symbols) learned to alternate their visits to the maze arms, ASA−/− mice acquire the task much slower. Data are mean number of correct forced alternations (and SEM) during each training day (main effect of genotype, p < 0.05, two-way repeated measures ANOVA).

of learning and memory [21,22]. Finally, we inspected previously unexamined areas of the brain of ASA−/− mice for histopathological indications of sulfatide storage in telencephalic and diencephalic regions with proven involvement in such higher brain functions. 2. Materials and methods 2.1. Animals and behavioural testing ASA−/− mice were generated as described previously [4], and bred on a pure 129/OlaHsd background. For efficient littermate breeding, control ASA+/− and ASA−/− littermates were derived from ASA+/− × ASA−/− breeding pairs. ASA+/− mice have proved to be valid controls to study the pathological effects of sulfolipid accumulation because they do not display any sulfatide storage or functional defects whatsoever [19]. All animals were housed at standard laboratory conditions (12 h light–dark cycle, constant room temperature and humidity) with ad libitum access to water. All experiments were performed during the light phase of this cycle, and only female mice were used. During the behavioural experiments, mice were placed on a food restriction schedule to keep their weight at approximately 80–90% of its initial value. All procedures were approved by the ethical research committee of the K.U. Leuven. Behavioural testing was performed in 6-month-old mice (6 ASA−/− and 5 ASA+/− littermates). Mice were taught to alternate arm visiting in a T-shaped maze (50 cm × 50 cm × 5 cm). Arms were closed with sliding doors placed 10 cm from the crossing. During the first shaping trial, mice could freely explore the maze, which was baited with food rewards (20 mg MLab Rodent Tablet) at the crossing and at both arm ends. Six trials were then conducted with sliding doors closed and only the arm ends baited. When a mouse entered the arm with all four paws, the sliding door was opened. If the mouse did not proceed, the sliding door was closed again. A shaping trial ended when the mouse collected both food rewards. Shaping was considered successful when mice visited both arms without making turns when sliding doors were opened. Subsequently, 7 daily blocks of 10 trials were conducted. One trial consisted of two runs in which mice had the choice to enter one of the arms. During the first run one arm was randomly baited. Mice were confined to the chosen arm for a fixed period of 10 s and then placed back in the starting arm. This time, only the arm they did not visit during the initial run was baited. The maze was cleaned between trials to avoid confounding odour or food traces. 2.2. Electrophysiology For electrophysiological experiments, 11-month-old mice (8 ASA−/− and 6 ASA+/− littermates) were killed by cervical dislocation. Hippocampal slices were prepared as previously described [23], and rapidly transferred to a submerged-type chamber, where they were constantly perfused with 32 ◦ C artificial cerebrospinal fluid (ACSF) saturated with carbogen (95% O2 /5% CO2 ). After approximately 90 min of incubation, a lacquer-coated stainless-steel stimulation electrode and a glass recording electrode (filled with ACSF; 1–5 M) were placed in the stratum radiatum of the CA1 region to record field EPSPs (fEPSPs). Input/output curves were obtained and stimulation strength was set to elicit a fEPSP slope of 35% of maximal responses. Paired-pulse facilitation (PPF) was studied by applying 2 pulses in rapid succession every 120 s, with different interpulse intervals (10, 20, 50, 100, 200, and 500 ms

Fig. 2. Reduced LTP in hippocampal CA1 region in brain slices from ASA−/− mice. Neither input/output curves (A), nor PPF (B), measured as the ratio of second and first fEPSP slope, differed between groups (p > 0.05, t-tests for all stimulation strengths and interpulse intervals). However, LTP is diminished in ASA−/− mice compared to controls (C). Data are mean values and SEMs (main effect of genotype, p < 0.01, two-way repeated measures ANOVA, Holm–Sidak method for multiple comparison).

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Fig. 3. Alcianophilic storage material in 6-month-old ASA−/− mice. The photomicrographs show storage material already at this age in the lateral olfactory tract and to some extent in the anterior commissure (A and B). Sulfoglycolipid storage is most prominent in corpus callosum (C), but also detectable in striatum, internal capsule (D), and neocortex (E). Relatively weak alcian blue staining illustrates storage in hippocampus as well (F). Abbreviations: AC, anterior commissure; CC, corpus callosum; HC, hippocampus; IC, internal capsule; lo, lateral olfactory tract; PFA, prefrontal association cortex; PLCx, prelimbic cortex; SCx; somatosensory cortex; Str, striatum.

respectively). Baseline recordings lasted for 30 min and consisted of 3 single stimuli (0.1 ms pulse width; 10 s interval) that were averaged, every 5 min. Long term potentiation (LTP) was induced by theta burst stimulation (TBS, 10 trains of each 4 × 100 Hz stimuli; 200 ms inter-train interval; 0.2 ms pulse width) and thereafter fEPSPs recordings were resumed for 4 h.

buffer overnight. Followed, postfixation with 0.66% OsO4 for 30 s (in 24-month-old mice only), dehydration with xylol, and mounting with DePeX. Vibratome slices (100 ␮m thickness) from rostral to caudal were prepared from these fixed brains, and collected individually a 24-well tray (except for the small slices, most rostrally). 2.4. Statistical analysis

2.3. Histology Telencephalic and diencephalic storage of sulfatide substrates was examined as described previously [5]. Briefly, 6-month-old and 24-month-old ASA-deficient and control mice were transcardially perfused and their brains were dissected. After preincubation overnight in Scott buffer pH 5.7 with 0.3 M MgCl2 , brains were fully incubated overnight in Scott buffer plus 0.05% alcian blue, and rinsed with Scott

Data are presented as mean and standard error of the mean (SEM). Differences between groups were determined using unpaired t-tests and two-way ANOVAs with repeated measures and the Holm–Sidak method for multiple pair-wise comparisons when appropriate. In LTP experiments paired t-tests were used to measure differences within groups (mean responses 5 min before TBS were compared to mean responses obtained 10 or 240 min after conditioning). For the TMDA, outliers were

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Fig. 4. Alcianophilic storage material occurs throughout the telencephalon and diencephalon of 24-month-old ASA−/− mice. Photomicrographs of 100 ␮m coronal slices demonstrated the occurrence of alcian blue-stained storage material in several brain regions. A slice at IA 6.5 mm [47] displays slight storage in deeper layers of prefrontal cortex (A). At IA 4.5 mm (B), alcian staining is prominent in corpus callosum, the medial part of the septum and the anterior commissure, but also in striatum and cortex. At IA 2.1 mm (C), prominent staining occurs in corpus callosum, internal capsule, habenula and thalamus. Hippocampus is remarkably pale, but the fimbria is prominently stained, and an enlargement of the CA3 region illustrates the presence of storage throughout the hippocampal layers (D). Abbreviations: AC, anterior commissure; CC, corpus callosum; CCx, cingulate cortex; Hab, habenula; HC, hippocampus; IC, internal capsule; ICx, insular cortex; MCx, motor cortex; OCx, orbital cortex; PFA, prefrontal association cortex; PLCx, prelimbic cortex; SCx; somatosensory cortex; Sept, septum; Str, striatum; Thal, thalamus.

calculated for each testing day and were determined as a deflection of more than 2 standard deviations of the mean. If performance was deviant for more than 2 trial blocks, the mouse was excluded from the data, which was the case for one poorly performing ASA−/− mouse. For electrophysiological experiments, slices from ASA−/− mice were interleaved with control slices. Statistical significance levels were at p < 0.05.

3. Results 3.1. T-maze delayed alternation (TMDA) In order to study neurocognitive defects that do not require motor proficiency, ASA−/− mice were examined in a TMDA task. During shaping trials, mice from both genotypes did not differ in the amount of time needed to retrieve all food pellets (p > 0.05). A significant effect of day indicated successful learning in both groups during acquisition (p < 0.001, two-way ANOVA with repeated measures). However, ASA−/− mice performed worse than ASA+/− mice (p < 0.05, two-way ANOVA with repeated measures), indicating that they were slower in acquiring the task (Fig. 1). 3.2. Hippocampal LTP Consistent with the deficit in TMDA, an hippocampusdependent learning task, ASA−/− mice also showed impaired LTP of fEPSPs in the CA1 region of the hippocampus (Fig. 2C). Although both groups showed a significant potentiation following a single TBS (10 min ASA+/− : 183.2 ± 8.7%, p < 0.005; ASA−/− : 150.7 ± 3.8%, p < 0.001; paired t-test vs. baseline), the magnitude of potentiation of ASA−/− mice was significantly reduced, and this difference was maintained throughout the whole duration of the experiments

(p < 0.01, two-way ANOVA with repeated measures, Holm–Sidak method for multiple comparisons). In fact, by the time recordings were finished, ASA+/− mice still kept a significant residual potentiation (p < 0.05, paired t-test, 240 min vs. baseline), whereas this was clearly not the case for ASA−/− mice (p > 0.05, paired t-test, 240 min vs. baseline). The impairment of LTP in ASA−/− mice was unrelated to changes in neuronal excitability. Indeed, excitability was not different between groups as assessed in input/output curves (Fig. 2A), and paired pulse facilitation (PPF, Fig. 2B), a form of pre synaptic short term plasticity. 3.3. Alcian blue staining We inspected telencephalic and diencephalic brain regions in 6-month-old (Fig. 3) and 24-month-old (Fig. 4) ASA−/− mice with special emphasis on structures that were relevant within the scope of the present study. As some of us had noted previously [5], neocortex and hippocampus did not display the prominent alcian blue staining that occurred in white matter tracts like corpus callosum or anterior commissure (Figs. 3A–C and 4A and B). However, alcianophilic material was definitely observed in many neocortical regions including prefrontal, insular, and motor and somatosensory cortices. A rather prominent stripe of alcianophilic material was observed around cortical lamina 5 in 24-month-old mice (Fig. 4B). Hippocampus was relatively pale (Figs. 3C and F and 4C and D), but close inspection did reveal the unquestionable occurrence of alcian blue-stained cells in several hippocampal layers including the pyramidal cell layers of the cornu ammonis (CA). Also, prominent staining was clearly evident in the fimbria hippocampi of 24-month-old mice, a region known to affect hippocampal func-

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tion. Other notable observations in the scope of the present study is the occurrence of storage material in striatum, internal capsule, and diencephalon in 24-month-old ASA−/− mice (esp. thalamus and habenula). Storage material was more prominent in the older mice, but it is clear that storage was already evident at 6 months of age. We inspected a few ASA+/− and wildtype mice using the same procedures as well, but these preparations failed to show any alcian blue staining whatsoever (not shown in figure). 4. Discussion Cognitive decline occurs in all forms of human MLD, regardless of variations in onset and progression or symptom heterogeneity. Previous studies in ASA−/− mice of approximately 1 year of age demonstrated impaired visuo-spatial and passive avoidance learning [13,17], but acquisition and extinction of conditioned emotional responding was normal in these mice [19]. Slight impairment of water maze acquisition suggested that changes in cognitive function might be present in these mice as early as 6 months of age [17]. However, as Suzuki et al. [24] had already noted with regard to the first behavioural observations in ASA-deficient mice, defects in learning tasks that require motor proficiency might be, at least partly, attributed to their prominent and progressive neuromotor or other non-cognitive impairments. Our recent demonstration of subtle neuromotor changes at 6 months further challenged the previous assumption that neurocognitive defects might occur in ASA-deficient mice independent of neuromotor defects [19]. It is therefore significant that we were now able to demonstrate cognitive dysfunction in 6-month-old ASA−/− mice using measures of delayed alternation performance that solely require rather basic perambulation in the tested mice. Importantly, ASA-deficient mice show a general decline of their normal speed of aquatic [16], but not terrestrial locomotion [19]. This was also confirmed by similar reward retrieval latencies in both genotypes during the shaping phase of TMDA. Nevertheless, the subtle gait defects observed in these mice might have triggered an adaptive cognitive resource allocation to keep their balance and postural control that did affect their TMDA performance. However, it should be noted that mice displaying various degrees of ataxia and motor incoordination can show intact T-maze cognitive performance [25,26]. Since neuronal sulfatide storage is widespread in ASA−/− brain, the presently observed learning abnormality cannot be attributed to storage and/or neuronal loss in any specific neural structure. However, brain regions considered to be most prominently involved in this type of rodent task include the hippocampus [20,27] and the prefrontal cortex [28,29]. In our present histological inspections, we indeed observed sulfatide accumulation in (prefrontal) cortex and hippocampus. Previous studies reported contradictory evidence for hippocampal neurodegeneration in ASA−/− mice. Consiglio et al. [13] showed pathological alterations in the CA3 region from 9 months onwards, but this was not confirmed in two other studies that, in agreement with the present observations, also failed to find evidence for extensive lipid accumulation in this part of the brain [5,30]. Yet, the mild lipid storage that we now observed in parts of the hippocampal formation, starting already at 6 months of age, might have caused functional learning deficits, regardless of neuron loss. Also, sulfatide storage that became evident in 24-month-old ASA−/− mice in fimbria and fornix may have contributed to the deficits in water maze learning and delayed alternation [31–33], and it should be noted that the marked sulfatide accumulation in habenula could have played a role in the observed learning deficit as well. Habenula is linking forebrain to midbrain regions, and has been increasingly implicated in the regulation of reward-based decision making [34]. Deficient LTP in the CA1 region of 11-month-old ASA−/− mice is indicative of impaired hippocampal synaptic plasticity that might

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have formed the basis of the observed learning defect. Clearly, none of this can be attributed to any gross structural defect in hippocampus, but the observed storage or its consequences appeared to be sufficient to induce changes in hippocampal synaptic plasticity. LTP was diminished, but not entirely absent, possibly due to the slight degree of sulfatide storage in ASA−/− hippocampus. It is also possible that this reduction in hippocampal LTP is due to the influence of secondary effects of sulfatide storage. For example, reduced brain cholesterol content was reported in ASA−/− mice [35], and recent studies indicate the importance of cholesterol homeostasis in regulating hippocampal synaptic plasticity [36–38]. The question remains why 6-month-old ASA−/− mice are now so clearly distinguishable from their heterozygous littermates in spatial delayed alternation, whereas this was not the case in different tests of fear conditioning, and only marginally in water maze acquisition [17,19]. We have previously suggested that different forms of learning might be differentially influenced by brain sulfatide accumulation [19]. Water maze acquisition is a form of allocentric learning, whereas TMDA learning is mainly based on egocentric strategies [39]. It has been observed that hippocampal damage/dysfunction in rodents more clearly affects allocentric water maze learning than egocentric water maze learning, whereas the reverse applies for deficits in medial prefrontal cortex [40]. In a series of studies [31–33], Mogensen and co-workers examined rats following single and double lesions of hippocampus and prefrontal cortex. The objective was to investigate whether structures with some functional similarity would reciprocally contribute to the functional recovery following damage to the other structure. They concluded that allocentric place learning in the water maze recovered fully, whereas egocentric spatial orientation and spatial delayed alternation returned only partially. It should also be noted that the test environment is much more restricted in T-maze tasks and contains less extra-maze cues, thus diminishing the opportunities to develop alternative solution strategies [33]. Additionally, TMDA is a task claimed to be typically requiring short-term working memory function [41], as well as reference memory and flexibility of responding [42]. Prefrontal cortex and hippocampus might subserve different components of working memory separately and in conjunction [43,44]. Interestingly, it has been shown in rats that the initial water maze performance increase, expressed as a decrease in escape latencies, is mainly related to efficient spatial working memory, whereas sufficiently delayed probe trials are indicative of long-term reference memory function [45]. In general, the results presented here are compatible with a working memory deficit in ASA−/− mice at the age of 6 months, which might further deteriorate and involve a wide variety of CNS functions at a later age. Different ages of onset of deficits in spatial learning and memory, visual recognition memory and nonassociative memory were recently also reported in a mouse model of Hurler syndrome [46]. In conclusion, we were able to distinguish ASA−/− mice from controls already at the age of 6 months using a TMDA task that might relate to defects in hippocampal LTP, and sulfatide storage in telencephalic brain structures. This study provides the first unequivocal proof of cognitive deficit and impaired synaptic plasticity in an MLD mouse model. These observations will be of use in future preclinical evaluation of therapeutic strategies that aim to reverse cognitive defects in this devastative disease.

Acknowledgements This work was supported by the University of Leuven research council (Impulse Fund IMP/04/006; OT/06/23 to DB), and Fonds voor Wetenschappelijk Onderzoek (FWO) grants G.0271.06 and G.0496.07 to DB and RD.

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