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Human Molecular Genetics, 2011, Vol. 20, No. 7 doi:10.1093/hmg/ddr019 Advance Access published on January 20, 2011 1375–1386 Spatial and temporal correlation between neuron loss and neuroinflammation in a mouse model of neuronopathic Gaucher disease Tamar Farfel-Becker 1, Einat B. Vitner 1, Sarah N.R. Pressey 3, Raya Eilam 2, Jonathan D. Cooper 3 and Anthony H. Futerman 1,∗ 1 Received December 29, 2010; Revised and Accepted January 12, 2011 Gaucher disease (GD), the most common lysosomal storage disorder, is caused by a deficiency in the lysosomal enzyme glucocerebrosidase (GlcCerase), which results in intracellular accumulation of glucosylceramide (GlcCer). The rare neuronopathic forms of GD are characterized by profound neurological impairment and neuronal cell death, but little is known about the neuropathological changes that underlie these events. We now systematically examine the onset and progression of various neuropathological changes (including microglial activation, astrogliosis and neuron loss) in a mouse model of neuronopathic GD, and document the brain areas that are first affected, which may reflect vulnerability of these areas to GlcCerase deficiency. We also identify neuropathological changes in several brain areas and pathways, such as the substantia nigra reticulata, reticulotegmental nucleus of the pons, cochlear nucleus and the somatosensory system, which could be responsible for some of the neurological manifestations of the human disease. In addition, we establish that microglial activation and astrogliosis are spatially and temporally correlated with selective neuron loss. INTRODUCTION Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by mutations in the gene encoding the lysosomal hydrolase, glucocerebrosidase (GlcCerase), which results in accumulation of glucosylceramide (GlcCer). Patients with GD are usually classified into three types, based on the presence or absence of neurological manifestations and their rate of progression. Types 2 and 3, which comprise only a small percent of GD patients, are collectively referred to as neuronopathic GD (nGD), with both displaying central nervous system (CNS) involvement in addition to systemic disease. Patients with type 2 GD typically present either prenatally or during infancy and usually die before 3 years of age (1). Type 2 patients fail to thrive, and display severe and rapidly progressive brainstem degeneration (2). The most frequent initial clinical signs are hyperextension of the neck, swallowing impairment and strabismus (2). The most common cause of death is prolonged spontaneous apnea which occurs with increased frequency in the later stages of the disease (2,3). Type 3 patients present with similar signs but with a later onset and decreased severity, and these patients usually survive until adolescence or adulthood (3). Eye movement abnormalities are common in nGD (4) and their detection is diagnostic of this disorder. In type 3 nGD, oculomotor signs may precede the appearance of overt neurological signs by many years. The main sign is severe difficulty (in type 3) (5), or a total inability (in type 2) (6) to generate saccades (ocular motor apraxia). Auditory brainstem response (ABR) abnormalities are also an early neurological sign in nGD. These findings may be isolated, or appear together with developmental delay and seizures (3,5). ∗ To whom correspondence should be addressed. Tel: +972 89342704; Fax: +972 89344112; Email: [email protected] # The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Department of Biological Chemistry and 2Department of Veterinary Resources, Weizmann Institute of Science, Rehovot 76100, Israel and 3Pediatric Storage Disorders Laboratory, Department of Neuroscience, Centre for the Cellular Basis of Behavior, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King’s College London, London, UK 1376 Human Molecular Genetics, 2011, Vol. 20, No. 7 RESULTS Microglial activation and astrogliosis are spatially and temporally correlated To determine the time course of neuropathological changes in nGD, we used a mouse model in which GlcCerase deficiency is restricted to neurons and macroglia, with normal GlcCerase activity in microglia (the Gbaflox/flox; Nestin-Cre mouse, hereafter referred to as the 2/2 mouse) (8). These mice exhibit rapid motor dysfunction including rigidity of limbs and abnormal gait, leading to seizures and paralysis by 21 days of age, at which time mice exhibit massive microglial activation, astrocytosis and neuron loss (8). To accurately define disease onset and progression, we monitored mouse body weight over a 21-day period. There was no difference between 2/2 and +/2 mice between 10 and 16 days (Fig. 1); however, at 18 days, 2/2 mice started to loose weight and by 21 days of age, 2/2 mice weighed 73% of +/2 mice. A similar pattern was obtained for brain weight, with no difference at 12 days, and a significant reduction of brain weight at 21 days (378 + 12 mg for +/2 mouse brains and 324 + 9 mg for 2/2 brains). We thus classified the nGD mice as presymptomatic at 12 days, early symptomatic at 16 days and late symptomatic at 21 days, the age at which they were euthanized. Neuroinflammation, including microglial activation and astrocytosis, is a common feature of neurodegenerative diseases (9), which may serve as a marker of ongoing neuronal damage or dysfunction (10). To determine the age and the brain areas where neuropathological changes first occur, we stained sections throughout the rostrocaudal extent of brains with the microglial marker, Mac2 (11). While +/2 brains showed no immunoreactivity for Mac2, faint but detectable Mac2 labeling was observed as early as 12 days in 2/2 mice (i.e. at the pre-symptomatic stage) (Fig. 2A). By 16 days, intense and localized microglial activation was evident Figure 1. Progressive reduction in body weight in a mouse model of nGD. ∗ P , 0.05, ∗∗ P , 0.01, ∗∗∗ P , 0.001, n ¼ 13–15 + SEM. in many brain areas, spreading to additional brain areas by 21 days (Fig. 2A). A similar pattern was observed for the astrocyte marker glial fibrillary acidic protein (GFAP) (Fig. 2B), with astrocytosis apparent at the same age and in the same brain areas as microglial activation (Fig. 2A). Indeed, astrocytosis was invariably observed with a similar pattern to microgliosis, but since the latter gave a more localized and distinct labeling pattern, most of the data below document microgliosis. Although the identity and sequence in which areas displayed microglial activation and astrocytosis was maintained between 2/2 mice, there was considerable variation in the time of onset and rate of progression of these reactive phenotypes. Nevertheless, the most striking microglial activation and astrocytosis were seen in the thalamocortical area, which prompted us to investigate the relationship between glial activation and neuron loss in these brain regions. Progressive glial activation and neuron loss in the nGD cortex Within the cortical mantle, astrocytosis and glial activation displayed both regional and laminar specificity. As previously reported (7,8), layer V of the cortex showed the highest degree of microglial activation. Occasional Mac2 immunoreactive microglia were present in this layer of the somatosensory and motor cortex as early as 12 days (data not shown). By 16 days, many more intensely stained Mac2-positive microglia were evident, but were confined specifically to layer V (Figs 3A and 4A), and were more frequent in motor and somatosensory areas than more caudal cortical regions, such as the visual cortex (Figs 2A and 3A). This distribution of Mac2 staining was closely paralleled by a band of GFAP staining in layer V of the same cortical regions (Figs 2B and 3A). By 21 days, Mac2 immunoreactivity had spread ventrally into layer VI, as well as in more superficial cortical layers (Fig. 4A). A similar subfield specificity was also revealed by cortical thickness measurements, with motor and somatosensory subfields displaying greater atrophy than the visual cortex (Fig. 3B). No cortical atrophy was detected in 16-day-old 2/2 mice (Fig. 3B). Unbiased optical fractionator estimates of the number of Nissl-stained layer V neurons in S1BF revealed that these reactive changes were accompanied by progressive neuron loss. There was no difference in the Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Little is known about the neuropathological events that cause these neurological abnormalities. A systematic neuropathological analysis of human GD brain (7) highlighted specific patterns of astrogliosis and neuron loss, in addition to non-specific gray and white matter gliosis. However, since this study (7) was performed on autopsy material, it was not possible to determine anything about the temporal sequence of pathogenesis. To address this issue, we have systematically examined the onset and progression of neuropathological events (microglial activation, astrogliosis and neuron loss) in a mouse nGD model (8). We identify several brain areas and pathways that may be responsible for some of the neurological manifestations in nGD, and show that microglial activation and astrogliosis are spatially and temporally correlated with selective neuron loss. This study may pave the way for the identification of disease markers for use in animal studies, since investigation of the areas affected at different stages of disease progression will allow determination of the efficacy of experimental therapies. In addition, it will direct further investigations into the molecular mechanisms by which GlcCer accumulation leads to the death of specific neuronal populations. Human Molecular Genetics, 2011, Vol. 20, No. 7 1377 nucleus [ventral posteromedial/posterolateral (VPM/VPL)], which relays somatosensory information to the regions of the sensory cortex that were most severely affected. Microglial activation in the thalamus progressed rapidly, particularly in the VPM/VPL, such that it was densely stained by 16 days and was the most affected brain area at this age (Fig. 5A). Optical fractionator estimates of neuron number revealed that the robust microglial activation in the VPM/VPL was accompanied by massive and rapid neuron loss, with a 57% reduction at 16 days and 78% at 21 days (Fig. 5B). Other thalamic nuclei also exhibited localized microglial activation from 16 days onwards (Table 1), including the auditory relay nucleus [medial geniculate (MGN)] (Fig. 5A), whereas no immunoreactive microglia were found in the visual relay nucleus (dorsal lateral geniculate) up to 21 days. Figure 2. Progressive glial activation in nGD mice. Low magnification of coronal sections of 12-, 16- and 21-day-old nGD mice reveals onset and progression of immunohistochemical staining for markers of microglial activation (Mac2, A) or astrocytosis (GFAP, B). Pronounced and localized microglial activation and astrocytosis were evident in multiple brain regions, occurring at the same stage of disease progression and becoming more intense with increased age. In contrast, no staining was observed in control brains (B). Schematic diagrams indicate the rostrocaudal level of each section. Scale bar ¼ 1 mm. number of these neurons at 12 days, but there was a loss (30% reduction) of layer V S1BF neurons in 2/2 mice at 16 days, which became more pronounced (58% reduction) at 21 days (Fig. 4B). Progressive glial activation and neuron loss in the thalamus Late symptomatic 2/2 mice displayed localized and progressive microglial activation in many individual thalamic nuclei (Figs 2A and 5A). Microglial activation began in the reticular thalamic nucleus (Rt), which provides inhibitory input to the rest of the thalamus, and in the ventral posterior We next used Mac2 immunostaining to survey other areas of the CNS. The basal ganglia, the nuclei globus pallidus and the substantia nigra started showing faint Mac2 immunoreactivity from 12 days (Figs 5A and 6A), which became more intense and widespread with increased age. Microglial activation was evident in both pars compacta [substantia nigra compacta (SNC)] and reticularis [substantia nigra reticulata (SNR)] of the substantia nigra of 2/2 mice, spreading from the dorsolateral parts to the whole region between days 16 and 21 (Fig. 6A), the time at which neuronal loss was first detected in SNR (Fig. 6B). In contrast, microglial activation was far less prominent in the striatum, another major component of the basal ganglia, with only a few scattered Mac2stained microglia at 21 days in the dorsolateral part of the caudate putamen (data not shown). The SNR is involved in the initiation of saccadic eye movements (12,13), suggesting that the pathology seen in this area maybe of clinical significance in nGD. Another key area of the oculomotor circuits which regulate horizontal saccades is the reticulotegmental nucleus of the pons (RtTg, nucleus Bechterew) (14), which contained many intensely stained Mac2positive microglia at 16 days (Fig. 7A). Other areas that may be related to neurological manifestations in nGD are the cochlear nucleus and the inferior colliculus, which may contribute to brainstem auditory abnormalities (15). Microglial activation was first detected in the cochlear nucleus of 2/2 mice at 12 days (data not shown), with very little increase in activation versus time (Fig. 7B). Microglial activation also occurred in the inferior colliculus at 16 days (Fig. 7C), but was not pronounced and did not increase with age. Several cerebellar-related nuclei also displayed specific microglial activation, among them the red nucleus, pontine nucleus and external cuneate nucleus (Fig. 7D– F). While the red nucleus displayed microglial activation only at 16 days (Fig. 7D), the pontine nucleus and external cuneate nucleus displayed Mac2-immunoreactive microglia at 12 days (data not shown), which became more pronounced with disease progression (Fig. 7E and F). Additional cerebellarrelated structures that displayed Mac2-positive microglia were the lateral reticular nucleus (LRt) (Fig. 8), vestibular nucleus and spinal cord (data not shown). Microglial activation in the LRt began at 12 days, became more pronounced Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Midbrain and brainstem pathology 1378 Human Molecular Genetics, 2011, Vol. 20, No. 7 with disease progression (Fig. 8) and was accompanied by severe neuronal loss, such that this structure, readily detectable in 12-day-old 2/2 mice, was barely detectable by Nissl staining at 16 days (data not shown) and was completely absent at 21 days (Fig. 8). The facial nucleus, an adjacent nucleus to the LRt, was clearly detected by Nissl staining in late symptomatic 21-day-old 2/2 mice (Fig. 8) and no microglial activation was detected (data not shown), further emphasizing the correlation between microglial activation and neuron loss and the specificity of neuronal loss within the brainstem. Another severely affected brainstem structure was the sensory spinal trigeminal nucleus (SP5). Microglial activation was detected as early as 12 days and rapidly progressed, with numerous intensely stained Mac2-positive microglia evident at 21 days (Fig. 9A). The increase in microglial activation was accompanied by abnormal development of the SP5 in 2/2 mice, which did not show the progressive increase in the number of Nissl-stained SP5 neurons that was evident in control mice (Fig. 9B). The changes within individual brainstem nuclei were not accompanied by an overall reduction in brainstem volume (Fig. 9C, P ¼ 0.13). Figure 4. Loss of cortical layer V S1BF neurons in nGD mice. (A) Double immunofluorescence labeling for NeuN (red) and Mac2 (green) reveals the loss of neurons in layer V, together with pronounced staining for Mac2 in this layer, which was absent in age-matched controls. Cortical layers are indicated. Scale bar ¼ 100 mm. (B) Unbiased optical fractionator estimates of the number of Nissl-stained neurons in layer V of the S1BF. ∗∗ P , 0.01, n ¼ 3– 4 + SEM. Additional brain areas displaying microglial activation A unique and distinct pattern of Mac2 immunoreactivity was observed in the olfactory bulb at 12 days, which was highly specific to the mitral cell layer and progressed to some extent with age (Fig. 10A). Another area that showed early Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Figure 3. Cortical subfield specificity of glial activation and cortical atrophy in nGD mice. (A) Immunohistochemical staining for Mac2 and GFAP reveals activated microglia and astrocytosis in the primary motor cortex (M1), somatosensory barrel field cortex (S1BF) and primary visual cortex (V1) of nGD mice, with an earlier and more pronounced staining in M1 and S1BF versus V1. (B) Cortical thickness measurements reveal late onset thinning of the cortical mantle to a larger extent within M1 and S1BF in late symptomatic animals. ∗∗ P , 0.01, ∗∗∗ P , 0.001, n ¼ 5 + SEM. Scale bar ¼ 100 mm. Human Molecular Genetics, 2011, Vol. 20, No. 7 1379 and robust microglial activation was the caudomedial entothinal cortex (CEnt) (Fig. 10B). The hippocampus displayed a similar pattern of pathology as reported in human patients (7) [and in a previous study on the nGD mouse (8)], with microglial activation in subfields CA2 – CA3 and in the hippocampal hilus (CA4) (Fig. 10C). Microglial activation in the hippocampus started at 16 days and was most pronounced in the caudal hippocampus, progressing to only a mild extent by 21 days at which time it had also spread to CA1 (Fig. 10C). A summary of the progression of microglial activation versus time in various brain areas is given in Table 1. colliculus (Fig. 11A) and the periaquaductal gray (PAG) (Fig. 11A), displaying little or no Mac2 immunoreactivity. Likewise there was specificity in which brainstem nuclei displayed microglial activation, with no activation even at the end-stage of the disease in nuclei, such the facial nucleus and the motor trigeminal nucleus. This lack of microglial activation suggested that there was no neuronal loss, which was confirmed by optical fractionator analysis of the number of PAG neurons (Fig. 11B). Together, our data strongly suggest a direct correlation between the extent of microglial activation and neuronal loss. DISCUSSION Several brain areas are resistant to GlcCerase deficiency Even in late symptomatic 2/2 mice, microglial activation remained localized, with many brain regions, such as the hypothalamus [with the exception of the mammillary nucleus, which showed some microglial activation at 16 days (data not shown)], superficial layers of the superior In this study, we provide the first detailed systematic description of the temporal and spatial progression of neuropathological changes in a mouse model of nGD. We have identified a number of brain areas that display pronounced microglial activation which were not previously known to be involved in nGD pathology, among them regions that may be of Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Figure 5. Thalamic pathology in nGD. (A) Immunohistochemical labeling for Mac2 shows faint, but detectable staining (arrows) in the reticular thalamic nucleus (Rt), globus pallidus (GP) and ventral posterior nucleus (VPM/VPL) in 12-day-old 2/2 mice which increased with age, while staining in the MGN was only apparent from 16 days onwards. No staining was observed in control brains (data not shown). Scale bar ¼ 200 mm. (B) Unbiased optical fractionator estimates of the number of Nissl stained neurons in VPM/VPL. ∗ P , 0.05, ∗∗ P , 0.01, n ¼ 3 –5 + SEM. 1380 Human Molecular Genetics, 2011, Vol. 20, No. 7 Table 1. Distribution of Mac2-immunoreactive microglia in brain regions during disease progression Brain area ++ ++ ++ 2/+ 2 + + 2 2 2 2 + +++ ++ +++ +++ + ++ ++ ++ ++++ ++++ ++++ ++++ ++++ ++ +++ +++ +++ ++++ 2 2 2/+ +/++ + 2 ++/+++ ++ +/++ 2 +++ ++/+++ 2 2 2 2 2 2 ++ ++ 2 +++ ++ +++ ++/+++ 2 ++++ 2 2 2 2 2 2 2 2 2 ++/+++ 2 2 2 2 +++ 2 2 2 2/+ + ++/+++ +++ 2 ++/+++ ++++ ++++ ++++ 2 +++ ++++ 2 2 2 2 2 ++/+++ 2 ++ ++ 2 +++ 2 +++ +++ ++ 2 ++/+++ +++ 2 2 ++ 2 ++ 2 2 2 2 2 2 ++ ++ 2 +++ 2 ++++ ++++ + + 2 2 2 + + 2 ++++ +/++ 2 2 ++ ++/+++ +++ 2 ++++ ++ 2 2 ++/+++ +++ ++++ 2 2 2 2 ++++ 2 ++++ Continued Brain area Principal sensory trigeminal nucleus (Pr5) Sp5 Dorsal motor nucleus of vagus (10N) Hypoglossal nucleus (12N) Spinal cord Intensity of Mac2 labelinga 12 days 16 days 21 days 2 ++ +++ + 2 2 + +++ 2 2 ++ ++++ 2 2 +++ a The number of plus signs indicates the extent of Mac2 immunoreactivity, with + indicating a few scattered Mac2-positive cells and ++++ indicating a high density of Mac2-positive cells. A minus sign indicates no Mac2 immunoreactivity. significance for understanding the clinical neurological manifestations observed in type 2 and 3 GD patients. In the nestin-Cre Gbaflox/flox mice, only neurons and macroglia were engineered to have defective GlcCerase activity, with microglia unaltered (8). Nestin-Cre Gbaflox/flox mice develop similar neurological signs and brain pathology to another nGD mouse, which does contain defective GlcCerase activity in microglia (the K14-Cre Gbalnl/lnl mouse) (8). Similar regions appear to be affected in both models, although the Nestin-Cre Gbaflox/flox mice show a higher extent of microglial activation than K14-Cre Gbalnl/lnl mice (8). The main advantage of the nestin-Cre Gbaflox/flox mouse is that it lives considerably longer than the K14-Cre Gbalnl/lnl mouse (8), allowing more in-depth analysis of the neuropathological changes over a longer period of time. Our data suggest that the affected brain areas that we have identified in nGD mice may also be affected in human GD patients. Human nGD brain shows a selective and specific loss of neurons in cortical layers III and V (7), consistent with our data that cortical layer V is severely affected in nGD mice. However, we were also able to detect a regional specificity, with motor and somatosensory cortex more affected than caudal cortical regions, such as the visual cortex. Human nGD patients also show selective loss of pyramidal neurons in CA2 – CA4, but not in the CA1 regions of the hippocampus (7), and a similar pattern was observed in nGD mice with respect to microglial activation, although this occurred late in disease progression and was not as pronounced as in other brain areas. Finally, both substantia nigra and red nucleus pathology were observed in our study and also previously in the human brain (7). Considering the similarities in the pathological changes between the mouse model used in this study and in the human disease, important lessons about the progress of the human disease can be derived, along with lessons concerning the time of possible therapeutic intervention. Thus, neuroinflammation was detected well before noticeable symptoms in the mouse [although it was not present in the brains of younger [10-day-old) nGD mice (data not shown)], which was followed by neuronal loss in defined areas prior to symptom onset. This suggests that early therapeutic intervention, even before the appearance of clinical symptoms, will be necessary once such interventions become available. An additional implication of our study Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Olfactory bulb Mitral cell layer Cortex Frontal association (FrA) Orbital Motor Sensory Piriform Parietal Auditory Visual CEnt Basal ganglia Caudate putamen Nucleus accumbens Globus pallidus (GP) Substantia nigra (SN) Hippocampus CA1 CA2/CA3 Hilus (CA4) Dentate gyrus (DG) Dorsal subiculum (DS) Hypothalamus Anterior hypothalamic area (AHA) Lateral nucleus (LA) Medial preoptic nucleus (MPO) Ventromedial nucleus (VMH) Mammillary nucleus Thalamic nuclei Anteroventral (AV) Anterodorsal (AD) Paraventricular, anterior part (PVA) Reticular (Rt) Ventral posteromedial/posterolateral (VPM/VPL) Mediodorsal (MD) Habenular Latrodorsal (LD) Posterior (Po) Lateral geniculate, dorsal part (LGND) MGN Midbrain Red nucleus Superficial area of superior colliculus (SC) Intermediate and deep layers of SC Periaqueductal gray (PAG) Inferior colliculus (IC) Pons and medulla Reticulotegmental nucleus of the pons (RtTg) Pontine nucleus Ventral cochlear nucleus (VCA) 8 nerve Facial nucleus (7N) Vestibular nucleus (Ve) External cuneate (ECu) LRt Parvicellular reticular nucleus (PCRt) Motor trigeminal nucleus Motor root of the trigeminal nerve (m5) Intensity of Mac2 labelinga 12 days 16 days 21 days Table 1. Continued Human Molecular Genetics, 2011, Vol. 20, No. 7 1381 Figure 7. Midbrain and brainstem pathology in nGD mice. (A– F) Immunohistochemical staining for Mac2 in 16-day-old 2/2 mice. No staining was observed in control brains (data not shown). Sections in (A) and (D) were counterstained with Nissl (purple). Scale bar ¼ 100 mm for A, B, D and F and 200 mm for C and E. Figure 8. LRt pathology in nGD mice. Immunohistochemical staining for Mac2 revealed pronounced microglial activation in 21-day-old 2/2 mice, which was accompanied by the loss of neurons, evident by Nissl staining. In contrast, the adjacent facial nucleus was unaltered. Scale bar ¼ 200 mm. Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Figure 6. Substantia nigra pathology in nGD mice. (A) Nissl (purple) stained sections labeled for Mac2 (brown) revealed microglial activation in the substantia nigra of 12-day-old 2/2 mice (arrows), which increased with age. Borders between the SNC (C) and the reticulata (R) are indicated. No staining was observed in the control brain (data not shown). Scale bar ¼ 200 mm. (B) Unbiased optical fractionator estimates of the number of Nissl-stained neurons in SNR. ∗ P , 0.05, n ¼ 3 + SEM. 1382 Human Molecular Genetics, 2011, Vol. 20, No. 7 Figure 10. Additional brain regions affected in nGD mice. Immunohistochemical staining for Mac2 revealed localized microglial activation in (A) the mitral cell layer of the olfactory bulb and in (B) the CEnt of 12-day-old 2/2 mice. (C) Microglial activation in CA2–CA3 and in the hippocampal hilus (CA4) was apparent in 2/2 mice at 16 days, and had spread to CA1 by 21 days. No staining was observed in control brain (data not shown). Scale bar ¼ 200 mm. concerns the efficacy of a stem cell therapy approach in treating neuronal forms of GD, as well as LSDs in general. Such a therapeutic approach is based on GlcCerase secretion by healthy (stem) cells, and its subsequent uptake by unhealthy Gaucher cells (cross-correction). However, since the mouse model used in the current study contains normal GlcCerase activity in microglia, our data would suggest that secretion of GlcCerase by microglia is not sufficient to rescue Gaucher neurons, as massive neuronal death occurs after microglial proliferation, raising questions concerning the suitability of stem cell therapy in treating acute forms of nGD. Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Figure 9. Sensory trigeminal nucleus pathology in nGD mice. (A) Immunohistochemical staining for Mac2 revealed microglial activation (arrows) in the SP5 of 12-day-old 2/2 mice, which became more pronounced with age. No staining was observed in control brains (data not shown). Scale bar ¼ 200 mm. (B) Unbiased optical fractionator estimates of the number of Nissl-stained neurons in the interpolar part of SP5 (SP5I). ∗ P , 0.05, ∗∗ P , 0.01, n ¼ 3 + SEM. (C) Unbiased Cavalieri estimates of brainstem volume. n ¼ 4– 5 + SEM. Human Molecular Genetics, 2011, Vol. 20, No. 7 1383 Figure 11. Not all brain areas exhibit microglial activation and neuron loss at the end-stage of the disease. (A) Immunohistochemical staining for Mac2 demonstrated that in contrast to adjacent areas of the midbrain reticular formation, the superficial layers of the superior colliculus (SC) and the PAG displayed very little microglial activation in 21-day-old 2/2 mice. Scale bar ¼ 500 mm. (B) Unbiased optical fractionator estimates of the number of Nissl stained neurons in PAG. n ¼ 2 + SEM. One of the most important findings from our study is identification of pathological changes within several brain structures that may explain early clinical signs in nGD patients. The most consistent and well characterized of these are abnormalities in eye movements, specifically a failure to initiate saccadic eye movements, slow saccades and an abnormality in horizontal saccades, which can also be followed by abnormality in vertical saccades (3). We have demonstrated early microglial activation, astrocytosis and neuron loss in the SNR, an area that plays a vital role in the initiation of saccades (12,13). We also show pronounced and localized microglial activation in the RtTg, another area involved in horizontal saccades (14). Abnormality in ABRs is also an early neurological sign in nGD (16– 18), and these abnormalities could result from damage to nuclei in the brainstem auditory pathway, i.e. the eighth cranial nerve (vestibulocochlear nerve), cochlear nucleus, superior olivary complex, lateral lemniscus or inferior colliculus (15). Although we found that the vestibulocochlear nerve is apparently unaffected, the cochlear nucleus displayed microglial activation and astrocytosis at the pre-symptomatic stage, in agreement with a study in one nGD patient (19). The inferior colliculus, another component of the brainstem auditory pathway, also displayed microglial activation at the mid-stage of disease progression. A key feature of our study was the early and pronounced targeting of the somatosensory system, which relays information about touch, pain, temperature and body position from somatosensory receptors (20). Many components of this pathway showed significant early pathology in nGD mice (Fig. 12), including the sensory trigeminal nucleus SP5, which receives somatosensory information from the face and sends it to the thalamic nucleus VPM (20). Both SP5 and VPM/VPL displayed intense microglial activation and a significant reduction in neuron number in nGD mice by the mid-stage of disease progression. While the VPM receives somatosensory information via the sensory trigeminal nuclei, the VPL receives somatosensory information from the limbs and trunk via the dorsal column nuclei and spinal cord (20). One of these dorsal column nuclei is the external cuneate nucleus, which also showed microglial activation at early stages. The VPM/VPL sends its major output to the primary somatosensory cortex, a region in which pronounced microglial activation, astrogliosis and neuron loss were all detected, although these phenomena were not confined to the somatosensory cortex. Pathological targeting of the VPM/ VPL and the cortical layers that receive projections from it, or project to it, is a consistent feature in mouse models of LSDs (21 – 23). In contrast, in nGD mice, inflammation in the cortex was largely confined to layer V, but not layer IV, which receives direct input from VPM/VPL (20). Interestingly, nGD patients show a greatly increased amplitude of somatosensory evoked potentials (24). Another anatomical characteristic of the structures that display microglial activation in nGD mice is that many of them send mossy fibers to the cerebellum. Cerebellar mossy fibers form large glutamatergic synapses that contact numerous granule cells in the cerebellum (25). These structures include the pontine nuclei [which receives its major input from cortical layer V (26)], the RtTg, the vestibular nuclei, the LRt, the dorsal column nuclei, the trigeminal nuclei and the spinal cord (26), which all displayed early microglial activation. The fact that only specific sub-populations of neurons were lost upon GlcCerase deficiency further strengthens the need to carefully choose the correct cellular model for nGD research. We previously demonstrated that conclusions based on study of fibroblasts were not relevant for neuronal models (27), and we now suggest that further research on nGD should Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 Figure 12. A summary of somatosensory system components that displayed early and pronounced microglial activation in nGD mice. 1384 Human Molecular Genetics, 2011, Vol. 20, No. 7 MATERIALS AND METHODS Nestin-flox/flox mice Nestin-Cre Gbaflox/flox mice were used as a model of nGD (8). Flox/flox Cre 2/2 mice were crossed with wild-type/flox Cre +/2 mice to generate flox/flox Cre +/2 mice (referred to as 2/2 mice) and wild-type/flox Cre +/2 mice (referred to as +/2 mice), which served as healthy controls. Genotyping was performed by PCR using genomic DNA extracted from mouse tails (27). The colony was maintained in the Experimental Animal Center of the Weizmann Institute of Science. Histological analysis Brains were removed and immersion fixed in 2.5% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 2 days, then cryoprotected in 30% sucrose in 50 mM Tris-buffered saline (TBS), pH 7.6, for several days and kept frozen at 2208C prior to sectioning. Brains were bisected along the midline, and 40 mm frozen coronal sections (through the rostrocaudal extent of the hemi-brain) were collected in 96-well plates (one section per well) containing a cryoprotectant (30% ethylene glycol, 15% sucrose, 0.05% sodium azide in TBS). Nissl staining A series of every sixth section through each brain was mounted and stained with cresyl violet (23). Slides were incubated with 0.05% cresyl fast violet (Merck, Germany), 0.05% acetic acid in water for 45 min at 608C, rinsed in deionized water, differentiated through an ascending series of alcohols, before clearing in xylene, and cover slipped with DPX (VWR, West Chester, PA, USA). Immunohistochemistry A series of every sixth section was collected in six-well plates, and endogenous peroxidase activity was quenched with 1% hydrogen peroxidase in TBS for 30 min. Sections were rinsed in TBS and blocked with 15% normal horse serum (Vector, Burlingame, CA, USA) in TBS with 0.3% Triton X (TBS-T) for 30 min, and then incubated with primary antibodies: rabbit anti-GFAP (1:4000, AbD Serotec, Oxford, UK) or rat anti-Mac2 (1:1000, Cedarlane, Ontario, Canada) in 10% normal horse serum in TBS-T, overnight at 48C. Sections were rinsed in TBS and incubated with the appropriate biotinylated secondary antibodies: biotinylated donkey antirabbit (1:200, Jackson Immunoresearch, West Grove, PA, USA) or biotinylated donkey anti-rat (1:200, Jackson) in 10% normal horse serum in TBS-T for 2 h at room temperature. Sections were rinsed in TBS followed by incubation with avidin – biotin – peroxidase (Vectastain Elite ABC kit, Vector, 1:1000) in TBS for 2 h and rinsed in TBS. To visualize immunoreactivity, sections were incubated with 0.05% 3,3′ -diaminobenzidine tetrahydrochloride containing 0.001% hydrogen peroxide in TBS for 10 min, and rinsed in ice-cold TBS. Sections were mounted on gelatin – chrome-coated Superfrost microscope slides, air-dried overnight, cleared in xylene and cover slipped with DPX. In some cases, Nissl Downloaded from hmg.oxfordjournals.org at King's College London on March 7, 2011 focus on the same specific neuronal sub-populations that show the most acute response to GlcCerase deficiency, such as cortical layer V, VPM/VPL and neurons which project mossy fibers. A major remaining challenge is to determine the mechanistic basis for the sensitivity of specific neuronal subpopulations to a loss of GlcCerase. One plausible explanation may be the relative level of GlcCerase activity/expression in these neuron populations. It might be predicted that neurons which normally contain high levels of GlcCerase would be most vulnerable when this enzyme is deficient. Unfortunately, there is no information available about GlcCerase expression (or of GlcCer levels) at the resolution of the specific brain areas identified in our study. Most mouse neurons were reported to show high GlcCerase mRNA expression at 2 days and at 4 weeks (28), but this contrasts with another study in 56-day-old mice that revealed significant heterogeneity between different brain regions (29). Indeed, some of the pathological findings that we describe match data from this latter study. For instance, the mitral cell layer of the olfactory bulb, CEnt, red nucleus of the midbrain, RtTg and pontine nucleus, which each display distinct microglial activation, all have relatively high GlcCerase expression (29). Distinct patterns of GlcCerase expression are also apparent in cortical layer V and to a lesser extent in layers II/III, in addition to high levels throughout the hippocampus (29). In the human brain, strong GlcCerase immunoreactivity was observed in hippocampal regions CA2 – CA4, in cortical layer V and to a lesser extent in layer 3. However, not all brain regions that displayed pathology in the nGD mouse had high GlcCerase expression levels in the adult mouse (http://mouse.bra in-map.org), including the thalamus and the trigeminal sensory nuclei. Levels of expression during development may be of more relevance for disease progression, or pathology in these areas may be a secondary process, or expression levels of GlcCerase mRNA may not correspond with GlcCerase activity. Another area that displays distinct and relatively high levels of GlcCerase expression is the SNC (http://mouse. brain-map.org). The loss of dopaminergic neurons in this area causes Parkinson disease (PD), which may be of particular importance since mutations in the GlcCerase gene were found to be a risk factor for PD (30). Another area that may be involved in both disorders is the olfactory bulb, since a marked decrease in olfaction is highly characteristic of PD (31). The olfactory bulb mitral cell layer has relatively high GlcCerase expression (22), and in nGD mice it presented with early onset and specific microglial activation and astrocytosis. Finally, neuroinflammation has emerged as a common feature in LSDs with CNS pathology (32), and antiinflammatory drugs were shown to benefit mice with various LSDs, including Sandhoff disease (33). Our study demonstrates that neuroinflammation, i.e. microglial activation and astrocytosis, occurs in the same brain regions where the neuron loss takes place. Presently, we do not know whether this inflammation precedes neuron death or is concomitant with it. Irrespective of the precise time course, we suggest that the use of anti-inflammatory drugs may be beneficial to alleviate neurological manifestations in nGD. Human Molecular Genetics, 2011, Vol. 20, No. 7 staining was also performed on the same sections to allow better identification of brain structures. A standard immunofluorescence protocol was used for double labeling of neurons and activated microglia. Twenty micrometer frozen coronal sections were blocked using 20% normal horse serum and 0.2% Triton X-100 in PBS for 2 h, and then incubated with a mixture of mouse anti-NeuN (1:50, Chemicon, Temecula, CA, USA) and rat anti-Mac2 (1:200, Cedarlane) in 2% normal horse serum containing 0.5% Triton X-100 in PBS overnight at room temperature. For detection of NeuN, sections were incubated with biotinylated donkey anti-mouse secondary antibody (1:200, Jackson) for 90 min followed by Cy3-conjugated streptavidin (1:200, Jackson) for 60 min. Mac2 was detected using a Cy2-conjugated donkey anti-rat antibody (1:200, Jackson) (2 h). Unbiased stereological estimates of regional volume or neuron number were obtained using Stereo Investigator software (Microbrightfield Inc., Williston, VT, USA) with regions of interest defined by referring to the neuroanatomical landmarks described by Paxinos and Franklin (34). All measurements were made blind (with respect to genotype) using Nisslstained sections. Cavalieri estimates of volume were obtained using a sampling grid superimposed over the brainstem region and the number of points covering the relevant area was assessed. Brainstem volume was estimated from one in three series of sections through the brainstem using a 200 mm sampling grid. To measure cortical thickness, the length of perpendicular lines extending from the white matter to the pial surface of the cortex was measured in M1, S1BF and V1 cortical subfields with Stereo Investigator software, using 10 lines placed on three consecutive sections spanning each cortical region. Results are expressed as the mean cortical thickness (mm per region). The design-based optical fractionator probe was used to estimate cell number (35) in Nissl-stained sections. Nisslstained cells were only counted if they had a neuronal morphology and a clearly identifiable nucleolus. A line was traced around the boundary of the region of interest, a grid was superimposed and cells were counted using a 100× objective within a series of dissector frames placed according to intersections of the sampling grid. Different grid and dissector sizes were determined according to each brain region using a coefficient of error (CE) value of ,0.1 to indicate sampling efficiency. Layer V of the S1BF grid was 200 × 200 mm, frame 60 × 40 mm; the VPM/VPL grid was 175 × 175 mm, frame 70 × 42 mm; the SNR grid was 150 × 150 mm, frame 73 × 44 mm; the SP5I grid (extending from immediately caudal to the facial nucleus to the first appearance of area postrema) was 220 × 220 mm, frame 65 × 35 mm for 21- and 16-day-old animals, and 190 × 190 mm, frame 65 × 35 mm for 12-day-old animals; the PAG grid (extending from immediately caudal to mammillary bodies to the first appearance of the inferior colliculus) was 175 × 173 mm, frame 65 × 35 mm. A 1:3 series was used for SP5I analysis, and a 1:6 series was used for all other brain regions. Statistical analysis The mean CE for all individual optical fractionator and Cavalieri estimates was calculated according to the method of Gundersen and Jensen (36) and was ,0.1 in the majority of cases, except for few 21-day-old 2/2 brains, in which neurons were largely absent. P-values were calculated using a one-tailed, two-independent sample Student’s t-test. A P-value ≤ 0.05 was considered statistically significant. ACKNOWLEDGEMENTS We would like to thank Drs Andrew Wong and Zvi Farfel for helpful discussions, and Dr Stefan Karlsson for supplying the Gbaflox/flox;Nestin-Cre mice. A.H. Futerman is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science. Conflict of Interest statement. None declared. FUNDING This work was supported by the Children’s Gaucher Research Fund (A.H.F.), the Batten Disease Support and Research Association, Batten Disease Family Association and the Natalie Fund (J.D.C.). REFERENCES 1. Gupta, N., Oppenheim, I.M., Kauvar, E.F., Tayebi, N. and Sidransky, E. (2011) Type 2 Gaucher disease: phenotypic variation and genotypic heterogeneity. Blood Cells Mol. Dis., 46, 75– 84. 2. Mignot, C., Doummar, D., Maire, I. and De Villemeur, T.B. (2006) Type 2 Gaucher disease: 15 new cases and review of the literature. 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