Altered level of GM2 ganglioside
A previous study showed significantly higher levels of GM2 ganglioside accumulation in the brain of 4.5-month-old Hexa-/-Neu3-/- mice compared to Hexa-/- using HPTLC and mass spectrometric analysis [5]. In this study, 10 μm coronal brain sections from 4.5-month-old WT, Hexa-/-, Neu3-/- and double-deficient Hexa-/-Neu3-/- mice were immunostained with anti-GM2 antibody. As shown in Supp. Fig. 1, the number of neuron cells with GM2 accumulation was significantly increased in CA1, CA2, and CA3 hippocampal regions (7-fold), primary motor and somatosensory area of the cerebral cortex (7-fold), thalamus (7.5-fold), Purkinje and granular layers of the cerebellum (18-fold) and pons (7.5-fold) of Hexa-/-Neu3-/- mice compared with that in Hexa-/- mice. GM2 ganglioside accumulation was also observed in the retrosplenial cortex and secondary motor cortex (data not shown). No GM2 was detected in WT and Neu3-/- mice.
Altered levels of the inflammatory cytokines and chemokines
The presence of neuroinflammatory conditions such as astrogliosis in the hippocampus, cortex and cerebellum of 4.5-month-old Hexa-/-Neu3-/- mice has been previously shown [5]. In this study we found that activated astrocytes are present in the hippocampus, cortex and cerebellum of both 2.5- and 4.5-month-old Hexa-/-Neu3-/- mice (Supp. Fig. 2). Neuroinflammation involves the release of inflammatory cytokines and chemokines [17]. Therefore, we evaluated the levels of pro- and anti-inflammatory cytokines and chemokines in the cortex and cerebellum of 4.5- month-old Hexa-/- and Hexa-/-Neu3-/- mice. We used a preformatted gene pathway array to compare the expression of 84 inflammatory chemokine, cytokine and interleukin receptor genes relative to that in Hexa-/- mice. After disregarding unregulated, non-detectable gene products and evaluating the relative levels of gene expression across all samples, we identified 42 inflammatory genes, which we categorized into 3 distinct groups.
Pro-inflammatory cytokines and chemokines, secreted mostly by activated macrophages in microglial cells, are involved in the up-regulation of inflammatory reactions (Zhang and An 2007). Our data showed that Hexa-/-Neu3-/- mice compared to Hexa-/- mice displayed an altered expression profile for pro- and anti-inflammatory cytokines and chemokines in both cortex (Fig. 1A) and cerebellum (Fig. 1B) regions. Major increases were seen in Ccl2 (11.3-fold), Ccl3 (12-fold), Ccl4 (24.4-fold), and Cxcl10 (17.2-fold) in cortex of mice (Fig. 1A). Ccl3 (17.2 fold), Ccl4 (41 fold), and Cxcl13 (5 fold) were predominantly expressed in the cerebellum. It has been established that Ccl2 cytokine displays chemotactic activity for monocytes, lymphocytes and neutrophils while Ccl3 is involved in the migration of monocytes, lymphocytes, and neutrophils together with Ccl2 [19] and activates T cells and macrophages with Ccl4 [20]. Increased expression of Ccl3 and Ccl4 in brain and cerebellum were also shown in different lysosomal storage diseases such as MPSIIIA [21] and MPSIIIB [22] and Gaucher Disease [23, 24]. In addition, Cxcl10, an inflammatory chemokine produced by astrocytes which, recruits activated T lymphocytes by increasing their migration to the site of tissue damage, is increased in both cortex and cerebellum of Hexa-/-Neu3-/- [25, 26]
Anti-inflammatory cytokines and chemokines regulate the expression of pro-inflammatory cytokines to arrest or slow down the immune response [18]. We found not only up-regulation of pro-inflammatory cytokines and chemokines in Hexa-/-Neu3-/- mice (Fig.1A, B); but also down-regulation of anti-inflammatory cytokines and chemokines which provides a more complete pictures of the inflammatory conditions in the cortex (Fig.1C) and cerebellum (Fig. 1D). Analysis of anti-inflammatory cytokines and chemokines revealed a significant decrease in the levels of IL10 (2.6-fold), IL11 (3.6-fold), IL13 (3.2-fold), IL2 (3.1-fold), IL22 (2.9-fold) and IL4 (3.2-fold) in the cortex of double deficient Hexa-/-Neu3-/- mice compared to Hexa-/-. In addition, we found that, IL10 (1.2-fold), IL11 (1.3-fold), IL13 (7.8- fold), IL2 (5.3-fold) and IL24 (5-fold) were significantly decreased in the cerebellum of Hexa-/-Neu3-/- mice, compared to Hexa-/-. IL10 protect tissues by preventing excessive inflammation to [27], and IL13 has a similar immunosuppressant activity [28]. Decreased expression of IL10 and IL13 in cortex and cerebellum was thought to contribute to the neuroinflammation seen in Hexa-/-Neu3-/- mice.
Expression levels of growth factors in cortex (Fig. 1E) and cerebellum (Fig. 1F) of Hexa-/-Neu3-/- mice were also analyzed. We found that the expression ratio of Bmp2 (1.2- fold), Bmp4 (2.3-fold), Bmp6 (2.5-fold), Bmp7 (1.7-fold), Csf3 (7.1-fold) in the cortex was lower than Hexa-/- mice, whereas the expression ratio of Csf1 (1.6-fold) and Lif (3.4-fold) was higher. In the cerebellum, Bmp2 (2.2-fold), Bmp4 (1.1-fold), Bmp6 (1.3-fold), Csf3 (2.2-fold) were decreased while Csf1 (1.3 fold) was increased compared with that in Hexa-/- mice. Csf1, a cytokine that controls the production, differentiation, and function of macrophages [29], was increased in cortex (1.6-fold) and cerebellum (1.3-fold) of Hexa-/-Neu3-/- mice, which may account for the higher number of macrophages seen in these regions (Fig. 1H, P). The Bmp protein family is responsible for the regulation of bone formation, maintenance and repair [30]. Decreased expression of Bmp2, 4, 6, and 7 may be a contributing factor to the hunched posture observed in Hexa-/-Neu3-/- mice [5]. Increased level of Lif under inflammatory conditions has been shown to inhibit cell proliferation [31], a similar increase was observed in the cortex of Hexa-/-Neu3-/- mice. Csf3 is responsible for the production and differentiation of granulocytes and is involved in defense against pathogens. Decreased inexpression of Csf3 might result in predisposition of Hexa-/-Neu3-/- mice to bacterial infection
Microglial activation
Microglial cells are the immune cells of the CNS responsible for sensing stress signals released from damaged or dying neurons [20]. They migrate to sites of injury where they release cytokines to promote removal of dead and dying cells by phagocytosis [32]. Once activated, they can act to activate neighboring microglia, astrocytes, neurons, or oligodendrocytes. Lysosomal storage disorders, including gangliosidosis, result in activation of neuroinflammatory response in the CNS [20]. Under normal conditions microglial cells remain inactive, however the accumulation of undegraded macromolecules activates these cells, causing an increased inflammatory response [17]. Here, we found that the accumulation of GM2 in the cortex and cerebellum led to microglial activation and consequent production of pro-inflammatory cytokines (Fig. 1A, B). To visualize the location of active microglia in Hexa-/-Neu3-/- mice, brain sections from WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice were immunostained with the anti-Moma2 and anti-Lamp1 antibodies, which are specific to the activated microglial/macrophage system [33]. The number of Moma2-positive cells was significantly increased in the hippocampus, cortex, thalamus, cerebellum and pons of Hexa-/-Neu3-/- compared with that in WT, Hexa-/- and Neu3-/- mice at both mice at 2.5 and 4.5-months of age (Supp. Fig. 3 and Fig. 2, respectively).
Microglial activation was statistically analyzed using Image J software. We found significantly increased microgliosis in the brains of 4.5-month-old Hexa-/-Neu3-/- mice compared with that in age-matched Hexa-/- mice, with the following ratios: approximately 60-fold in hippocampus (Fig. 2V), 9-fold in cortex (Fig. 2W), 13-fold in thalamus (Fig. 2X), 19-fold in pons (Fig. 2Z). In the cerebellum, microgliosis was observed only in Hexa-/-Neu3-/- mice (Fig. 2P, Y). Significantly increased amount of microgliosis was also observed at 2.5 months of age Hexa-/-Neu3-/- brain compared to age-matched Hexa-/-Neu3-/- mice as approximately 37 fold in hippocampus (Supp. Fig. 3V), 4 fold in the cortex (Supp. Fig. 3W), and 5 fold in the thalamus (Supp. Fig. 3X). In cerebellum (Supp. Fig. 3P, Y) and pons regions microgliosis was observed only in Hexa-/-Neu3-/- mice (Fig. 2U, Z). There was no microglial activation in the hippocampus, cortex, thalamus and cerebellum of WT and Neu3-/- mice, with the exception of in the pons area.
Increased IL-6 expression in the neuron
Elevated IL6 (proinflammatory cytokine), production due to activated microglia was observed in lysosomal storage disorders, e.g, in the brains of Gaucher disease mouse model, in the serum of Gaucher, Fabry and mucopolysaccharidosis type IVA patients [20, 34–36]. To detect neuronal expression of IL6, brain sections 4.5-month-old WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice were immunostained with anti-IL6 antibody.
The number of IL6-positive cells was significantly increased in the hippocampus, cortex, thalamus, and pons of Hexa-/-Neu3-/- compared with that in WT, Hexa-/- and Neu3-/- (Supp. Fig. 4). There was no significant change in number of IL6-positive cells in the cerebellum of WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice (Supp. Fig. 4). We found, approximately 3-fold increase in IL6-positive cells in the hippocampus (Supp. Fig. 4V), cortex (Supp. Fig. 4W) and thalamus (Supp. Fig. 4X) of Hexa-/-Neu3-/- mice compared with that in age-matched Hexa-/- mice.
Increased infiltration of peripheral blood mononuclear cells (PBMCs) in the brain
Neuroinflammation contributes to disease progression in several brain disorders. Specifically, activated immune cells induce the infiltration of T and B cells into the CNS, promoting the release of chemokines and stimulating the migration of more immune cells to the CNS via a disrupted BBB [37]. Previous studies showed that neuroinflammation leads to significant infiltration of PBMC in the brain of the Sandhoff mouse model [38]. PBMCs consist of lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells [39].
The PCR Array analysis revealed that transcription levels of Ccl2, Ccl3, and Cxcl10 were significant infiltration in both cortex and cerebellum of Hexa-/-Neu3-/- mice compared with that in Hexa-/- mice (Fig. 2A, B, respectively). Ccl2, Ccl3 and Cxcl10 play various roles in the attraction, migration, and activation of monocytes, lymphocytes, and neutrophils in the CNS. To analyze the presence of activated PBMC in Hexa-/-Neu3-/- mice, 10µm coronal brain sections from 4.5-month-old WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- were immunostained with anti-CD45 antibody. The average number of CD45-positive cells in the hippocampus, cortex, and thalamus of Hexa-/-Neu3-/- mice was significantly increased compared with that in WT, Hexa-/- and Neu3-/- (Fig. 3). Similarly, activated PBMC were significantly increased in the cerebellum (2.5-fold) of Hexa-/-Neu3-/- mice (Fig. 3P) compared with that in Neu3-/- mice (Fig. 3Y) as well as in the pons (3-fold) of Hexa-/-Neu3-/- mice (Fig. 3U) compared with that in WT mice (Fig. 3Z). Readily detectable numbers of CD45-positive cells were also identified in the cerebellum and pons of WT (Fig. 3M, R, respectively) Hexa-/- (Fig. 3N, S, respectively) and Neu3-/- (Fig. 3O, T, respectively). Thus, progression of TSD could be associated with disruption of the BBB, and widespread infiltration of inflammatory immune cells into the CNS.
Decrease number of oligodendrocyte and neurons
To characterize the degree to which neuroinflamation affected neuron cells and oligodendrocytes, 10 µm coronal brain sections from 2.5 and 4.5-month-old WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice were immunostained with NeuN and CNPase antibodies, which recognize nonspecific neurons and oligodendrocytes, respectively. As shown in Fig. 4, 4.5-month-old mice showed loss in neuronal density compared to age-matched control groups, in different regions of the central nervous system. It was demonstrated that the number of NeuN-positive neurons was significantly reduced by approximately 50% in cortex, by approximately 40% in thalamus and by approximetly 50% in pons of Hexa-/-Neu3-/- mice (Fig. 4D, H, P, respectively) compared with that in WT mice (Fig. 4A, E, M, respectively). There was no significant change neuronal density, in the granular layer of the cerebellum (Fig. 4T). Furthermore, there was no significant change in the number of neuron in 2.5-month-old mice compared with that in age-matched control groups in different regions of the central nervous system (Supp. Fig. 5). Loss of NeuN immunoreactivity could be explained by neuronal death in damaged areas of the brain [40]. Thus, a decrease in NeuN immunostaining correlates with increased numbers of apoptotic cells. In previous study, TUNEL assay showed that there was apoptotic cell death in the brain of Hexa-/-Neu3-/- mice compared with that in WT, Hexa-/- and Neu3-/- mice [5]. Furthermore, NeuN-positive neurons in the cortex (Fig. 4D), thalamus (Fig. 4H), and hippocampus (Fig. 4P) appeared swollen, with accumulated storage material as previously shown in the Sandhoff mice brain [41].
Oligodendrocytes are, types of glial cells, that function in the formation of myelin which then provides support and insulation to axons [32]. Oligodendrocytes provide axons with myelin sheaths, and have the ability to renew their myelin sheaths three times within a day. Myelination is essential for optimal signal transduction in the CNS. Activated microglia produces various pro-inflammatory mediators, chemokines and cytokines. In such environments, oligodendrocytes are particularly susceptible to microglia-derived factors, resulting in the production of poor-quality myelin sheaths and oligodendrocyte death [42]. As shown in Fig.5 Hexa-/-Neu3-/- mice showed lower numbers of 2',3'-cyclic-nucleotide 3'-phosphodiesterase-positive cells compared with age-matched WT, Hexa-/- and Neu3-/- mice. The number of oligodendrocytes was reduced by approximately 45% in the cortex, by approximately 55% in thalamus and cerebellum and by approximately 35% in pons area of Hexa-/-Neu3-/- mice (Fig. 5D, H, L, P, respectively) compared with that in WT mice (Fig. 5A, E, I, M, respectively). The number of oligodendrocytes was significantly decreased in the cortex of Hexa-/- mice by approximately 30% compared with that in WT mice. We observed no significant changes in the number of oligodendrocytes in the thalamus, cerebellum and pons of Hexa-/- (Fig. 5F, J, N, respectively) and Neu3-/- (Fig. 5G, K, O, respectively) mice, compared with that in WT mice. These results are consistent with previous studies. Gene expression profile studies in cerebral cortex of normal and GM2 gangliosidosis (Tay-Sachs and Sandhoff) patients revealed that, the myelin basic protein gene, expressed by oligodendrocytes, was also significantly depressed [8]. Sialiated gangliosides, especially GD1a and GT1b, are present on the axonal membrane, and interact with the MAG on the periaxonal surface of to promote myelin sheath stability [43]. GM2 is also a sialic acid containing ganglioside; thus accumulation of GM2 could lead to instability of myelin sheath. We found no significant changes in the number of oligodendrocytes in 2.5-month-old Hexa-/-Neu3-/- mice compared with that in age-matched control groups (Supp. Fig. 6).
Increased number of microglial reaction in the retina
Neuroinflammation or neurodegeneration lead to reactive gliosis in the retina through hypertrophy, causing a thickening and enlargement of processes via Müller cells and astrocytes [44]. To characterize neuroinflammatory conditions affected the retinas, coronal eye sections from 4.5-month-old WT and Hexa-/-Neu3-/- mice at were immunostained with anti-lectin (vessels and glia) and anti-phalloidin antibody (vessels and actin filaments). We found significantly higher numbers of microglia cells in the retinas of Hexa-/-Neu3-/-, mice compared with that in WT mice (Fig. 7A, D). Microglial staining was prominent around the vessels. However, sagittal sections of Hexa-/-Neu3-/- mice showed no anatomical alterations in their retinal layers (Fig. 7G).
Impairments in spatial learning and memory
Up-regulation of cytokines and their receptors within the CNS during inflammation and concomitant effects on brain function have been reported [6]. Morris water maze task was used to detect spatial learning and memory deficits [45–48]. We used a two-way ANOVAS t0 analyze the date. Here we report that both 2.5 and 4.5-month-old Hexa-/-Neu3-/- mice displayed deficits in spatial learning and memory. Also, we found that WT (p<0.001), Hexa-/- (p<0.025) and Neu3-/- (p<0.05) mice learned to use the visual clues to quickly reach the visible platform in the first 3 days of training, whereas Hexa-/-Neu3-/- mice took a longer time to swim toward the platform (Fig. 7A, C). All groups initially had difficulty in finding the exact location of the hidden platform on day 4. While WT, Hexa-/- and Neu3-/- mice groups quickly improved their ability to find the platform, both (p<0.01) 2.5 and (p<0.025) 4.5-month-old Hexa-/-Neu3-/- mice were not able to learn the location of the hidden platform compared to WT. Also, 2.5-month-old WT (p<0.025), Hexa-/- (p<0.05) and Neu3-/- (p=ns) mice and 4.5-month-old WT (p<0.025) and Neu3-/- (p=ns) mice spent continuously less time from day one to five, to find the hidden platform (Fig. 7B, D). Our date showed that WT, Hexa-/- and Neu3-/- mice used distal clues more efficiently than Hexa-/-Neu3-/- mice.
Swimming speeds recorded in the Morris water maze were significantly lower at 2.5 and 4.5-month- old Hexa-/-Neu3-/- (7.8 ± 0.8; 8.7 ± 0.6, respectively) mice compared with that in age-matched WT (18.6 ± 1.3, p < 0.0001; 16.8 ± 1, p< 0.0001, respectively), Hexa-/- (12.7 ± 0.9, p < 0.0001; 12 ± 0.9, p< 0.001, respectively) and Neu3-/- (10.8 ± 1.2, p < 0.05; 20.2 ± 1.3, p = ns, respectively) (Fig. 7E, F). However, the average distance spent to find the target platform was similar in WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice in both 2.5 and 4.5-month-old mice groups (Fig. 7G, H).
Typical swim patterns revealed that both 2.5 and 4.5-month-old Hexa-/-Neu3-/- mice were unable to find the escape platform by the final day of the test, whereas age-matched WT, Hexa-/- and Neu3-/- mice were successful at using clues to find it. In addition, 4.5-month-old mice showed more anxiety-related behaviors compared with that in WT and single deficient Hexa-/- and Neu3-/- mice (Fig. 7I, J).
The 5 min open-field test was performed to measure levels of anxiety [49]. A significant difference was detected between WT and Hexa-/-Neu3-/- mice (p<0.001 for 2.5 months; p< 0.025 for 4.5 months) in age-independent manner. Moreover, Hexa-/-Neu3-/- (291.2 ± 1.8 s for 2.5 months; 288.4 ± 2.1 s for 4.5 months) mice spent most of the time in the periphery of the open field compared to WT (256.3 ± 7.5 s for 2.5 months; 268.3 ± 4.1 s for 4.5 months), Hexa-/- (275.4 ± 3.2 s for 2.5 months; 284.4 ± 3.1 s for 4.5 months) and Neu3-/- (273 ± 7.2 s for 2.5 months; 277.2 ± 3.3 s for 4.5 months). The walking patterns and the amount of time spent in the center and the periphery of the open field area indicated that both, 2.5 and 4.5-month-old Hexa-/-Neu3-/- mice displayed more anxiety-related behaviors, compared to the other genotypes according to the walking patterns and the amount of time spent in the center and the periphery of the open field area (Supp. Fig. 7).
Deficits in cognitive learning and memory
The passive avoidance test was used to detect cognitive learning and memory deficits [47]. We found that 2.5-month-old WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice re-entered the dark compartment at the average times: 219 ± 41.3 s, 203 ± 31.8 s, 132 ± 21.8 s, and 118 ± 60 s, respectively, after having previously received the electric shock through the feet. These results may indicate that the younger age group could not tolerate light and prefer the dark compartment despite of the 0.2 mA electric shock, for 2 s (Fig. 8A). Nearly all the 4.5-month-old WT mice avoided entrance into the dark compartment during the 300 s observation period, whereas the Hexa-/-Neu3-/- mice re-entered the chamber at an average time of 160 s (292.1 ± 7.9 s in WT and 159 ± 33.5 s in Hexa-/-Neu3-/-; p<0.001). In addition, Hexa-/- and Neu3-/- mice re-entered the chamber in slightly less time than WT mice (Fig. 8B). These results may show that Hexa-/-Neu3-/- mice display a significant deterioration in memory function compared with that in other groups.
Deficits in neuromotor behaviour
In a previous work, Hexa-/-Neu3-/- mice exhibited progressively impaired performance on the rotarod test [5]. This test is used to evaluate motor skill learning and involves endurance, motor coordination and muscle strength [50]. The Morris water maze test showed that both 2.5 and 4.5-month-old Hexa-/-Neu3-/- mice lost their swimming speed, which could be related with deterioration in motor coordination and muscle strength (Fig. 7E, F). For a more specific measure of muscle strength, grip strength tests were performed [47, 50]. The fore limb assessments demonstrated no significant change in strength of mice at the 2.5 months of age (Fig. 9A). The strength of Hexa-/- and Hexa-/-Neu3-/- mice was significantly impaired compared to WT mice at 4.5 months of age (87 ± 4.7 in WT, 56.2 ± 6.1 in Hexa-/- , 82 ± 11.2 in Neu3-/- and 26.5 ± 10.5 in Hexa-/-Neu3-/-). Overall, Hexa-/-Neu3-/- mice displayed the most dramatic muscle strength impairment and functional deterioration (p< 0.025). This result could be related to abnormal GM2 accumulation in the muscles of Hexa-/-Neu3-/- mice [5].