Hydrogen peroxide (H2O2), tert-butylhydroperoxide (TBH) and
staurosporine (STS) are very common chemical oxidants, and have been shown previously to induce apoptosis in several cell types, including PC12 cells and fibroblasts (Makino et al., 1994; Lee et al., 2000).
Neurons are very vulnerable to these oxidative drugs for their relatively low levels of antioxidant enzymes and dependence on mitochondrial respiration (Whittemore et al., 1994). In order to study the apoptotic effect of these oxidative drugs in pathological conditions, we investigated three kinds of oxidative stresses on these clonal cells, respectively. Cell
viability under oxidative stress was measure by MTT method. Our study showed that viability of cells exposed to 20 µM H2O2, 10 µM TBH and 125 nM STS were significantly decreased in B1 overexpresses group compared to control group (Fig. 42). These results indicate that oxidative stress caused apoptosis by cytotoxity in this diseased-cell model.
Discussion
I. Mouse model
Our Bβ1 transgenic mice represent a model of cerebellar degeneration, due to loss of PCs caused by the overexpression of Bβ1.
The rotarod test, in which animals walk on a rotating rod, is widely used to assess motor status in laboratory rodents. In our research, motor learning was assessed in male mice started at 2 months of age by using an accelerating rotarod. This accelerating rotarod can test animals’ tolerance of the accelerated speed. The heterozygous mice had difficulty to stay on the rotating rod upon the more restricted condition (4 to 30 rpm in the first 5 min), even the wild type mice could not last for 600 sec. Thus, we modified the rotating speed to an easier condition (2 to 20 rpm in the first 5 min) which wild type mice can stay on for 600 sec. There was no
significant difference between transgenic and wild type mice when they were before 60 weeks old. Upon the easier condition, we found that heterozygous transgenic mice show significant shorter latency time compared to wild type mice at older stage, suggest these mice had a late onset disease.
To gain more severe phenotype and early onset disease mice, we generated homozygous transgenic mice in our study. Behaviorally,
compared to heterozygous mice, homozygous line TG-13 showed deficits
in rotarod testing and bad motor coordination since they were 2-month-old. Interestingly, homozygous line TG-20 showed better
performance on the rotating rod when they were 2-month-old. During the task of rotarod task, we observed that wild type mice knew how to get out of the rotating rod but not felled down when they were tired or loss
balance, suggesting an anxiety-like behavior. The limbic system is
involved in anxiety and risk evaluation tasks (Millan, 2003), with several classes of neurotransmitters participating in these processes (McEwen and Sapolsky, 1995). Previous research also revealed the correlation between anxiety and behavior. Spinocerebellar ataxia type 3 is a neurodegenerative disease caused by expansion of a polyglutamine domain in the protein ataxin-3 (ATXN3). Atxn3 knockout (ko) mice displayed no overt abnormalities. On the accelerating rotarod Atxn3 ko mice performed as well as wild type animals, but reduced exploratory behavior in the open field suggested a sense of heightened anxiety (Schmitt et al., 2007). G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels mediate the postsynaptic inhibitory effects of many neurotransmitters and drugs of abuse. Both GIRK1(-/-) mice and GIRK2(-/-) mice, however, showed elevated motor activity and delayed habituation to an open field (Pravetoni and Wickman, 2008). This
evidence suggests that, homozygous line TG-20 showed better
performance might due to anxiety. Thus, we cannot use motor activity to define how overexpression of Bβ1 affected the behavior of mice.
Furthermore, we may test mice using plus-maze test and light-dart
exploration test, which are useful to definite the anxiety level of the mice.
Also accompany with brain pathology analysis in areas correlated to
emotion control, such as frontal cortex and prefrontal cortex would be necessary to confirm our hypothesis.
On the other hand, PC has a main role participating in the motor function of cerebellum. PC of the cerebellum receives excitatory input from the inferior olivary nucleus via climbing fibers, and sends inhibitory projections to the deep cerebellar nuclei, which in turn project to postural and motor structures such as the vestibular nucleus and motor cortex (Patel and Hillard, 2001). This inhibitory projection was sometimes placed at decisive location within the motor system and was therefore important for programming and controlling movement (Linnemann et al., 2006). According to previous studies, due to loss of PC will suffer from functional cerebellar decortication resulting in ataxia and deterioration of cognitive functions (Cendelin et al., 2008). Since PC axons are the only efferent pathway of the cerebellar cortex, the loss of these cells doesn’t lead to complete functional cerebellar decortication. Brain still involves in this multiple motor and learning activity, such as motor cortex,
hippocampus and hypothalamus. Thus, we should also conduct brain pathology analysis in areas involved in multiple motor and learning activity in the future.
To demonstrate that the motor and cognitive deficits observed in Bβ1 transgenic mice were not caused by muscle weakness or some other restriction on general locomotor ability, we compared the open field exploratory activity between homozygous and wild type mice at 2 months of age. The measurements of total distance traveled showed no
differences between homozygous line TG-13 and wild type mice, but the total distance traveled of homozygous line TG-20 mice display more active in this test. These results suggest homozygous line TG-20 mice might have more anxiety than homozygous line TG-13 mice. However, rearing frequency of homozygous line TG-13 was significantly lower than that in wild type mice, but homozygous line TG-20 was significantly increased than two other groups, this results indicate that homozygous mice line TG20 have more exploring activity in a novel environment than others. Combine the results from rotarod and locomotor tests, neither the rotarod nor the activity in the open field indicate a sign of motor
dysfunction in homozygous line TG-20 mice. How does this distinct impaired locomotion results from two homozygous lines with the
alternation outcome of behavioral test happened, we may confirm by the anxiety behavior analysis and more brain pathology in the future.
In our IHC data, we can detect the transgene expression in cerebellar nuclei of transgenic mice by using flag antibody. The flag signals were significantly increased through quantitative analysis. Our mouse models also show some features with some extent of cerebellar pathology.
Only the PC mutant transgenic mice show an early loss of dendritic PC arborization and abnormal cell morphology with reduction of the
perikaryon and clarification of the nucleus (Weber et al., 2001). However, PC degeneration was not significant in both young heterozygous and homozygous mice. Although PC loss is not fully acknowledged, many experimental results point to apoptosis as the underlying process.
Experiments that measure apoptosis-related byproducts in PCs and
cerebellum (cytochrome c release, mRNA expression of pro- and
antiapoptotic genes, TUNEL assay, cleaved caspase-3) (Light et al., 2002;
Heaton et al., 2003) have shown that apoptotic events occur and in a similar pattern as the cellular degradation labeling. A more severe degeneration of PC was identified when these heterozygous transgenic mice were 7-month-old and homozygous transgenic mice were
6-month-old, or even older. The intensity of calbindin immunostaining in PC diminished as the mice aged, but only with slight absence of
arborization and pale cytoplasmic staining caused by the reduction of PC perikarya, which indicates that PC degeneration is caused by Bβ1
transgene overexpression.
We also examined the immunostaining intensity of GFAP in the Bβ1 transgenic mouse cerebella. GFAP is widely used as a marker of astrocyte activation or reactive gliosis. Astrocytes are the only cells in the brain that have the important ability to convert glutamate into glutamine via Glutamine synthetase (GS). Glutamine is released to neurons and used for the synthesis of glutamate (and then GABA, in GABAergic neurons). A cortical GS decrease has also been suggested as a characteristic in
Alzheimer’s disease (Robinson, 2001). A major finding of this study is that overexpression of Bβ1 is critically involved in degeneration-induced astrocyte changes similar to gliosis. Compared to control group, GFAP levels in cerebellar nucleui region of astrocytes were slightly higher in young transgenic mice, and were significantly higher when transgenic mice were aged. These results indicate that astrocyte pathology turned into more severity during the disease progressing and increased GFAP
immunostaining intensity, which are typical signs of astrogliosis and cerebellum degeneration.
Based on our microarray data, Growth hormone (GH) is upregulated and cocaine and amphetamine regulated transcript (CART) is
downregulated in transgenic mouse cerebella. This was also confirmed by our IHC results at different stages of mice. Both GH and insulin-like growth factor-1 (IGF-1) elicit pleiotropic actions in the CNS and may play a role in age-related changes in cognitive function (Kinney et al., 2001; Bartke, 2003). Unregulated GH-signaling in neural progenitor cells derived from SOCS-2 null mice results in impaired neurogenesis
(Turnley et al., 2002) GH-signaling in supporting hippocampal
neurogenesis can be evaluated irrespective of localized hippocampal GH synthesis (Ransome and Turnley, 2008). These evidences indicate that GH correlated with aging and neurogenesis, which suggest that GH upregulated in Bβ1 transgenic mouse cerebella may display a protective role to delay the degeneration.
The (CART) is an endogenous peptide which is widely expressed in the CNS and PNS (Douglass et al., 1995; Koylu et al., 1997). The
cerebellar coordinates motor behaviors and plays a key role in fine motor control, balance, posture and eye movements (Ito, 1984, 2006). CART peptides can localized in fiber like structures within the vestibular cerebellum (Koylu et al., 1998), part of the cerebellum involved in the maintenance of equilibrium and balance (Barmack, 2003). The presence of CART peptides in the inferior olivary complex has lead to the
suggestion that CART peptides are expressed by climbing fibers.
Climbing fibers play a central role in cerebellar processing and signal to the output neurons of the cerebellar cortex (PCs) by the production of a large membrane depolarization, termed a complex spike, (Koylu et al., 1998). CART is also protective against focal cerebral ischemia in vivo and against neuronal cell death (Mao et al., 2007). Thus, the
downregulation of CART in transgenic mouse cerebella is consistent to our rotarod data with transgenic mice had worse performance. These data further emphasis that maintaining normal PC activity is critical for
maintaining the whole coordination system.
In our HomeCageScan analysis, we have established a rigorous system for characterizing behavioral abnormalities in the mouse and have used it to demonstrate a contrasting array of behavioral changes in our transgenic mice model. Many of these behavioral alterations were previously
unreported in other disease models, such as awakening events were dramatically increased in Bβ1 transgenic mice. In addition, we detected differences in some exceedingly rare behaviors, such as stretching. Most of the behavioral testing of these mice is focus on their robust motor phenotypes, such as Hanging was reduced in our homozygous transgenic mice. Our analysis also revealed substantial psychological behaviors in homozygous transgenic mice, such as eating, drinking, turning, grooming, sniffing and rearing. One disadvantage of this system is that we cannot analysis two mice at the same time, mice must be singly housed during each testing. However, with this system we can detect multiple behavioral alterations of these mice and may greatly aid in testing and evaluation of
therapeutics of these disease mouse models.
According to our study, we found neurodegeneration of PCs, dendritic atrophy, and behavioral deficits in the transgenic mice. Therefore, these phenotypes will provide valuable parameters for future therapeutic evaluation for SCA12 disease.
II. Cell model
In our ICC results, we can detect cytoplasmic Bβ1 staining after NGF treatment 48 hrs followed by PonA induction for 48 hrs under both normal n and serum starvation condition. But there was only slight induction signal detected in normal condition. The western blot analysis show overexpression of Bβ1 was induced after PonA treatment. Although we observed significant Bβ1 expression, the cell survival and
morphological change were not identified during the PonA induction period, thus, we investigate whether oxidative stress would cause pathology of these disease-cell models.
The vast majority of eukaryotic organisms need atmospheric oxygen to survive. Oxygen is the terminal electron acceptor in the respiratory chain of mitochondria during the generation of ATP. An unavoidable by-product during respiration by the mitochondria is the generation of reactive
oxygen species (ROS), which are implicated in the pathophysiology of a diversity of chronic diseases (Boveris, 1984). Therefore oxidative stress is a ubiquitous phenomenon in all cell types. It has been pointed out that
the crucial balance between ROS generation and antioxidant defense is regarded as a force in a wide variety of chronic diseases (Machlin and Bendich, 1987). ROS may have several effects including alteration in the activity of transcription factors, phopholipases, protein tyrosine kinases, gene expression, apoptosis and cell injury (Finkel, 1998).Our study showed that viability of cells exposed to 20 µM H2O2, 10 µM TBH and 125 nM STS 48 hrs were significantly decreased in Bβ1 overexpressing group compared to control group. These results suggest that Bβ1
overexpressing cells are more vulnerable to these stress.
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