detected by S100 antibody, however we could only observe the S100 expression signal was reduced in transgenic mice no matter in treatment or in vehicle group (Figure 7K).
Evaluation the molecular effect of 4% trehalose treatment in SCA17 transgenic mice
We also checked the trehalose molecular effect in SCA17 transgenic mice. HSP70 is considered the major chaperon protein and played the important neuron protection role in neuron degenerative diseases
(Friedman et al., 2007, Huang et al., 2011). However, we could not detect significant differences between in wildtype and transgenic groups (Figure 8A). Although the p-ERK was upregulated in transgenic mice, the
trehalose could not affect the expression level (Figure 8A). The levels of GAD67, β-catenin and pp38 were also not effected after trehalose
treatment (Figures 8B-8C). However, the MnSOD was resumed to the normal expression level after trehalose treatment, which indicated that the trehalose might ameliorate the oxidative stress in SCA17 transgenic mice.
Furthermore, it is interesting that the p-JNK was downregulated in
wildtype mice after trehalose treatment; nevertheless, trehalose treatment upregulated the p-JNK protein expression in SCA17 transgenic mice (Figure 8D).
27
Discussion
SCA17 is an autosomal dominant hereditary disease caused by abnormal amplification of CAG repeats in TBP gene and resulted in neuron degeneration. Although the molecular pathogenesis of SCA17 had not been clarified yet, cerebellar atrophy, Purkinje cell loss and TBP nuclear aggregation were obvious markers in SCA17 patient (Nakamura et al., 2001). In our previous study, we could also detect the TBP puncta, ataxia and neuronal degeneration in TBP-109Q transgenic mice (Chang et al., 2011). In this study, we further found the TBP puncta was
co-localized with 1C2 and ubiquitin (data not show) which were reported to be detected in SCA17 patients. These results confirmed that the
abnormal TBP puncta detected in the SCA17 transgenic mice was the aggregation. Furthermore, the presence of vacuoles was reported as a sign of degeneration of cells of SCA1 and tottering mice (Florez-McClure et al., 2004, Vig et al., 2006, Hoebeek et al., 2008, Vig et al., 2009). We could also found vacuoles within the Purkinje cells with intensive aggregation in our transgenic mice (data not show). These observations reveal that Our SCA17 transgenic mice represent anideal animal model in studying pathogenesis or screening potential treatments for polyQ
mediated SCA diseases.
To identify potential treatments for SCA17, an in vitro model may be established for a screening platform. However, the Purkinje cell was difficult to be maintained in its normal function and morphology without co-culture with its surrounding glia cell. For example, the Bergmann glia
28
cell plays the role of scaffold in development stage of Purkinje cell (Lippman et al., 2008).Therefore, we tried to set up the organotypic cerebellar slice culture for the drug screen platform. The slice culture maintained the normal cerebellar neuronal morphology at DIV7, and we could observe the TBP aggregation was formed in SCA17 transgenic slice between DIV1 and DIV3, indicating that the slice culture could be a suitable system to screen the potential drugs for SCA17.
Trehalose is analpha-linked disaccharide synthesized by fungi, plants and invertebrates. There were reports suggested that the trehalose had low toxicity and could help the cell to protect against the stress threatening the cell survival (Chen and Haddad, 2004). In addition, the trehalose had been reported to have potential in rescuing neuron
pathology, molecular dysfunction and abnormal behavior in lots of neurodegenerative diseases, such as AD (Beranger et al., 2008), prion disease (Aguib et al., 2009), HD (Tanaka et al., 2004) and SCA14 (Seki et al., 2010). After applying the trehalose in slice culture at DIV1, we could observed the TBP aggregation was significantly reduced at DIV7, indicating that the trehalose might prevent the aggregation formation.
Previous study reported that trehalose might play a role as a
chaperon to prevent the abnormal protein aggregation formation (Tanaka et al., 2004, Seki et al., 2010). To further understand whether trehalose coulddecrease TBP aggregation and rescue the SCA17 pathology in vivo, we applied the 2% and 4% trehalose, respectively, into mouse drinking
29
water. The trehalose was stable during our treatment condition; however, we could not observe significant rescuing effect of trehalose determined by rota-rod test. It could be that the first rota-rod condition was too strict to distinguish treatment group from vehicle group. After modifying the rota-rod condition from 4-30 rpm accelerated speed to 26 rpm fixed speed, we could observe the differences between these two groups. In addition, the footprint of trehalose treated mice had better performance than vehicle treated mice. These data reveal that although trehalose had some neuron protective effect on SCA17 mice, which could only be detected by a mild analysis protocol.
In our previous study, hyperactivity was reported to be one of behavior markers in our SCA17 transgenic mice (Chang et al., 2011). In the present study, we could also observe that after 4% trehalose treatment, the total distance analyzed by locomotor was slightly reduced in
transgenic group, indicating that trehalose could ameliorate the hyperactivity of SCA17 mice.
From the in vivo study, we found the dendritic tree of transgenic mouse Purkinje cell had better performance after trehalose treatment, however, the TBP aggregation was not significantly reduced as the results found in slice culture. Trehalase is suspected to be the reason to cause the effect difference between mice and slice culture. Trehalase is the enzyme to digest trehalose into two glucoses. It was reported that the trehalase is present in the intestine of mammals including rabbit (Ruf et al., 1990), rat
30
(Oesterreicher et al., 1998), mouse (Oesterreicher et al., 2001) and human (Ishihara et al., 1997), which could digest the trehalose drunk by SCA17 transgenic mice. It might reduce the trehalose concentration and explain why there was no significant improvement in transgenic mice identified from both the rota-rod and locomotor test after trehalose treatment.
Although the glucoses also showed some positive effect on HD mice (Tanaka et al., 2004), the trehalose was much better than it (Tanaka et al., 2004, Kruger et al., 2011). Therefore, finding a compound with similar potent as trehalose and also working as a trehalase inhibitor might be a potential strategy to solve the polyQ aggregation. For example,
validamycin A is the trehalase inhibitor and was used to improve the trehalose biosynthesis (Xue et al., 2005) and accumulation (Lopez et al., 2009). It would be interesting to see whether the validamycin A could inhibit the abnormal TBP aggregation.
In this study, we also monitor the trehalose drinking level (data not show). We found that the mice drunk more water than vehicle treatment.
However, the blood glucose did not change after treatment, indicated that the trehalose treatment did not affect the blood glucose and harm the mice.
The interesting thing is that we found the blood glucose was significantly reduced in transgenic group at 5-week-old. Although there was no report pointed out the blood glucose was affected in SCA patients, the
hypometabolism phenomena was observed by Positron emission tomography (PET) with 2-[fluorine18]-fluoro-2-deoxy-D-glucose in cerebellum of SCA patients (Wang et al., 2007), indicating that the
31
dysfunction of energy metabolism might be another pathology of SCAs.
However, at this moment, we do not know whetherour SCA17 mice have any defect in energy metabolism and the trehalose treatment would have any effect on the hypometabolism phenomena of cerebellum or not.
Gliosis is observed as a neuron degenerative marker in SCA17 mice (Friedman et al., 2007, Chang et al., 2011). In this study, we could also detect the astrocytes was highly activated in the transgenic mice. After trehalose treatment, we found the activation of astrocytes was reduced, indicating that the trehalose could delay the neuron degeneration.
However, we could also detect the microglia cells were activated after trehalose treatment, especially in SCA17 transgenic mice. The microglia contributed about 12% cells in whole brain and was known as biosensors in the central nervous system (Penfield, 1932). However, it had not been clarified yet that the activation of microglia would exert positive or negative effect on neurons (Li et al., 2007). For example, many studies reported that activating microglia cells would increase neuronal cell death through releasing glutamate, nitric oxide and toxic cytokines (Chao et al., 1992, Piani et al., 1992, Viviani et al., 1998). In contrast, some evidences indicated activating microglia cells could secrettrophic factor which is good for neurons, such as neurotrophins (Elkabes et al., 1996) and transforming growth factor-β (TGF-β) (Lehrmann et al., 1998). In a previous study, the astrocyte gliosis upregulated and neurodegeneration phenomena were observed when the hippocampal slice was cultured in microglia cell-depleted condition (Montero et al., 2009), indicating a
32
protective role of microglia cell in this system. In this study, we could also observe the astrocytes activation was reduced and microglia cells activation was upregulated.
Taking together, our data suggest that the trehalose treatment has positive effect in our neuronal pathology of SCA17 transgenic mice. This natural disaccharide might have potential to delay the polyQ diseases.
However, adding the trehalase inhibitor with trehalose might be a more efficiency way than trehalose only for the treatment.
33
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43 Table 1. Primary antibody list in this study
Protein Manufacturer Titer Source MW (kDa)
Calbindin Sigma 1:1000 mouse 28 Calbindin Sigma 1:1000 rabbit 28 IP3R1 SantaCruz 1:1000 goat 313 1TBP18 QED 1:30000 mouse
GFAP Millipore 1:1000 mouse 51 S100 Millipore 1:1000 mouse 10
Iba1 Wako Pure Chemical
1:1000 rabbit 17
β-actin Millipore 1:1000 mouse 42 MoSOD Cell signaling 1:1000 mouse 24 pp38 Cell signaling 1:1000 rabbit 38 GAD67 Millipore 1:1000 rabbit 65 p-ERK (Thr202/Tyr204) Cell signaling 1:1000 rabbit 42/44
ERK 1/2 Cell signaling 1:1000 rabbit 42/44 HSP70 Cell signaling 1:1000 rabbit 72 p-JNK (Thr183/Tyr185) Cell signaling 1:1000 rabbit 46/54
JNK Cell signaling 1:1000 rabbit 46/54
44 Table 2. Secondary antibody list in this study
Antibody Manufacturer Titer Source
anti-mouse IgG, Alexa Fluor 488 Invitrogen 1:500 donkey anti-mouse IgG, Alexa Fluor 555 Invitrogen 1:500 donkey anti-rabbit IgG, Alexa Fluor 555 Invitrogen 1:500 donkey anti-goat IgG, Alexa Fluor 488 Invitrogen 1:500 donkey anti-goat IgG, Alexa Fluor 555 Invitrogen 1:500 donkey Biotinylated Goat Anti-Mouse IgG Vector 1:200 goat
anti-mouse IgG, Alexa Fluor 488 Invitrogen 1:500 donkey anti-mouse IgG, Alexa Fluor 555 Invitrogen 1:500 donkey anti-rabbit IgG, Alexa Fluor 555 Invitrogen 1:500 donkey anti-goat IgG, Alexa Fluor 488 Invitrogen 1:500 donkey anti-goat IgG, Alexa Fluor 555 Invitrogen 1:500 donkey Biotinylated Goat Anti-Mouse IgG Vector 1:200 goat