We received an approval for one year no-cost extension of our project ending in January 31, 2015.
Dr. Badaut, PhD, has been a co-investigator and lead for the immunohistochemical (IHC) aspect of our project. In July, Dr. Badaut had announced his intention to relocate to France and on his request his participation in our project was terminated as of July, 2013. Dr. Shalini Mehrotra, PhD, a first year postdoctoral fellow, was trained by Dr. J. Badaut. She was working with us since June 2012 and she was mostly responsible for performing the IHC analyses. Dr. Mehrotra had announced terminating her employment with LLU in August, 2013. Thus, the IHC part of our study was paused since Dr. Badaut's laboratory equipment (e.g., fluorescent microscope with Mercator software package) became unavailable. Nonetheless, we intend to complete the IHC part in full in collaboration with Dr. Nelson (co-investigator) and students trained by Dr. Badaut.
Re-allocation of funds allowed us to hire part time Mr. Gordon Harding as of September 2013, a senior research associate with significant experience in Western blotting and other protein quantification techniques. Mr. Harding performed initial experiments with presynaptic marker synaptophysin in mice irradiated with protons.
Technical progress: Summary Results by Aims.
Aim 1 & Aim 3 Activities. In accord with our statement of work (SOW), we completed all irradiations, behavioral testing, and in vitro electrophysiological experiments with protons and HZE (iron 600 MeV/n & silicon 250 MeV/n) particles. Proton irradiated APP/PSEN1 transgenic (TG) and wild-type (WT) mice were behaviorally tested pre- and 3 and 6 months post-irradiation followed by electrophysiological testing either at 6 or 9 months post-irradiation, as planned. HZE-irradiated mice (TG only) were behaviorally tested 3 and 6 months post-irradiation, followed by electrophysiological testing at 6-7 months post-irradiation. Pooling two electrophysiological time points (6 and 9 months) to one in HZE-irradiated animals was inevitable to boost the power of consequent statistical analyses. This change to the original SOW was opted for after observing considerable variability in electrophysiological results along with increased mortality specifically in iron-irradiated TG mice (likely unrelated to irradiation) that reduced the total numbers of experimental subjects per group. The change to the electrophysiological time point was applied for both HZE species to allow for direct comparisons of the radiation effects on relevant endpoints.
In total, from 2010-2013 we irradiated 78 TG and 16 WT mice with protons at Loma Linda University, Proton Treatment Facility. Twelve TG mice died; thus, electrophysiological testing was successfully performed in 82 proton-irradiated mice. We irradiated 120 TG with HZE particles at Brookhaven National Laboratories. Seven TG mice died spontaneously and thus electrophysiological testing was successfully performed in 113 animals. Ninety of these TG animals were electrophysiologically tested in 2013. We used conventional extracellular recordings to monitor both evoked synaptic responses and spontaneous activity. The behavioral and electrophysiological data from all proton- irradiated animals, including statistical evaluation, have been 95% completed. The analyses of electrophysiological and behavioral data from HZE-irradiated mice have been ~65% completed.
Behavioral analyses of proton and HZE-irradiated animals have been completed. Data from the water maze (WM) and the Barnes maze confirmed previously described deficits in spatial memory in control (0Gy) APP/PSEN1 TG mice (increased swim distance to the target area) when compared to the WT mice. We also observed that proton radiation (0.5 Gy) affected the performance of WT mice, but did not affect the performance of APP/PSEN1 TG mice. This may indicate that low radiation may not necessarily worsen the AD-like pathology, or that such pathology trumps any radiation-induced effects. In APP/PSEN1 TG mice irradiated with 600 MeV/n iron particles we surprisingly observed improved performance in WM (reduced cumulative distance to the target platform), the effect became significant at 6 months post-irradiation at the dose of 1 Gy. Interestingly, the TG mice irradiated with 250 MeV/n silicon particles exhibited reduced performance in WM at 3 months; the decrement was statistically significant at 0.1 Gy only and appeared to be transient as it could not be detected at 6 months post-irradiation. No significant differences were observed for either HZE species in the Barnes maze or zero maze.
Electrophysiological data show that proton radiation at doses from 0.1 to 1 Gy may impact synaptic excitability and short term synaptic plasticity mediated by presynaptic glutamate release, but it likely does not affect long-term potentiation (LTP; reported previously), the widely used cellular correlate of memory formation in the hippocampus. We observed that proton radiation-induced changes in synaptic excitability are qualitatively different in APP/PSEN1 TG and WT mice. In accord with our behavioral findings, the WT mice exhibit different sensitivity to radiation and, for example at 0.5 Gy we observed increased postsynaptic excitability in CA1 neurons, whereas the TG mice exhibited opposite responses at the same radiation dose. The stimulation paradigms using two (paired) stimulation pulses were used to evaluate the effect of proton radiation on presynaptic glutamate release (paired-pulse facilitation; PPF). In TG mice at 6 months post-irradiation with protons we observed reduced PPF indicating increased glutamate release and this change became more pronounced at 9 months post-irradiation. Changes in PPF were not detected in WT mice. On the other hand, WT mice exhibited sensitivity to proton radiation because at the dose of 0.5 Gy we observed radiation-induced decrements in frequency of sharp wave-ripple complexes, which are implicated in memory consolidation process in the hippocampus. Interestingly, in TG mice, a radiation exposure to protons or HZE particles had no effect on these spontaneous oscillations.
Aim 2 Activities. We partly completed immunohistological evaluations of ß-amyloid deposits in the brain samples (the cortex and the hippocampus) of APP/PSEN1 TG mice irradiated with protons using thioflavin-S staining (fibrillar form of amyloid) and by IHC using 6E10 monoclonal antibody (total amyloid). Both methods confirmed amyloid depositions in the brains of APP/PSEN1 TG mice at 6 and 9 months post irradiation. In the dorsal cortex (but not the hippocampus) at 1 Gy of protons we observed significant increase of total amyloid by 9 months post-irradiation detected by 6E10 antibodies. The IHC on brain samples irradiated with HZE particles was temporarily paused due to departure of Drs. Badaut and Methorta. Nonetheless, the IHC analyses of HZE irradiated samples is planned for the fourth year of the project (the no-cost extension has been approved) by hardware provided by Dr. Nelson (co-investigator) and performed by other team members trained in Dr. Badaut’s lab and by student volunteers.
Neuroinflammation and neurodegenerative changes in TG (and WT) brains (cortex only) exposed to radiation have been assessed by determination of five cytokines/chemokines (IL-1 beta, IL-6, TNF alpha, MCP-1, and IL-10). These molecules have been previously reported to be elevated in irradiated brains and/or have been shown to affect synaptic plasticity in the hippocampus, thus their elevation may be associated with functional decrements observed in these animals. The Luminex assays have been completed in samples irradiated with protons, the assays with HZE-irradiated brains will be completed by December, 2013. In a cohort of proton-irradiated mice we observed differences in the expression of chemokine IL-10 between TG and WT mice at 9 months, but the effect was not dependent on the radiation exposure. The other chemokines were not affected by either genotype or radiation, indicating that at 9 months radiation effects on the CNS are not associated with elevated levels of pro-inflammatory cytokines. This also indicated that the electrophysiological and behavioral decrements reported above are not due to elevated levels of cytokines within the CNS, as previously suggested by us and other investigators.
We are currently performing the analyses of synaptic markers in WT and TG mice irradiated with protons by Western blotting. The initial analyses in APP/PSEN1 TG mice irradiated with protons indicates that such exposure may increase the expression of synaptic vesicle glycoprotein and presynaptic marker synaptophysin, which may explain the radiation-induced changes in PPF described above. This marker has been previously shown to be affected by exposure to iron radiation, which awaits confirmation in APP/PSEN1 TG mice planned for the next year. Analyses in cortices irradiated with 0.1 and 1 Gy of protons and with HZE particles will be ensuing.
Abstracts for Journals and Proceedings
Rudobeck E, Szücs A, Vlkolinsky R. "Effects of Proton Radiation on Evoked and Spontaneous Neuronal Activity in the Hippocampus of APP/PSEN1 Transgenic Mice." HITSRS2013--Heavy Ion in Therapy and Space Radiation Symposium 2013, Chiba, Japan, May 15-18, 2013.
HITSRS2013--Heavy Ion in Therapy and Space Radiation Symposium 2013, Chiba, Japan, May 15-18, 2013. , May-2013