NOTE: End date changed to 11/30/2024 per NSSC information (Ed., 11/15/23)

NOTE: End date is 11/30/2023 (incorrectly listed in NSSC as 11/14/2021) per F. Hernandez/ARC (Ed., 7/27/21)

2. Genetic sensors of genotoxic stress were introduced into mice noninvasively and systemically. The mice were subsequently exposed to ground-based galactic cosmic radiation (GCR) analogs at sustained lunar operation (15 cGy), deep space habitat (30 cGy), and Mars-mission (75 cGy) relevant doses (50) in low (0.75 cGy/min) and high (3.2 cGy/min) dose rates, administered as either 5-ion simplified Galactic Cosmic Ray simulation (SimGCRsim) or 1000 MeV/n Fe passed through a moderator block to model intravehicular spallation. Another cohort of mice (6 months of age) were irradiated with 30 or 75 cGy moderated iron or 5-ion GCRsim. Sensorimotor, cognitive, social, and emotional behavior were monitored at two time points: 2-4 months and 10-14 months after irradiation. The oldest mice in the study lived up to 24 months, which is comparable to approximately 70 human years, making this the first long-term study to assess late-life central nervous system (CNS) risks across the entire lifespan of mice. Exposure to two methods of GCRsims induced robust genetic sensor labeling.

3. The brain regions with the highest labeling of cells with genomic instability include the cortex, striatum, thalamus, brain stem, and olfactory bulb, indicating that space radiation exposure can induce chronic brain-wide genotoxic stress. While marked neurodegeneration was not evident in the aged mice, sustained neural dysfunction in cognitive, emotional, and sensorimotor domains was noted 14 months after exposure. Alterations in emotional state and challenged movement were present at early time points and appeared to resolve over time. Other deficits – such as a prevalent alteration of spontaneous activity and sensorimotor function – persisted for more than a year, with a progressive decline in working memory.

4. The membrane tethering farnesylated/myristoylated derivatives of fluorescence proteins of the genetic sensor allows sensitive visualization and detection of the subtle neuropathology that cannot be detected by the traditional stereology-based method to count the loss of the cell number. A loss of dendritic stubby and thin spines of striatal medium spiny neurons was observed in the mice exposed to two methods of GCRsims. Single-cell pathology analysis further revealed sensor-labeled cells enriched with DNA damage, oxidative stress, senescence, and disruption of protein homeostasis, suggesting accelerated brain aging.

5. We found that GCRsim exposures disrupt brain transcriptomics in the mice 14 months after irradiation. Mice irradiated with 5-ion-GCRsim showed significantly more differentially expressed genes (DEG) in comparison to the group exposed to a modulated iron beam.

6. We are finalizing the first-ever brain-wide mapping of genetic sensor-labeled neurons with genotoxic stress after GCRsim exposures. The images of Artificially Recognized sensor-labeled neurons were registered to the Allen Brain Atlas.

7. We finished systemically characterizing and quantifying neuroinflammation in multiple brain regions at three time points after GCRsim exposures. Reactive microgliosis (indicated by increased diameter and surface area of Iba1-expressing microglia) was observed brain-wide in 24-month-old and 13-month-old mice, as well as many brain regions of 5-month-old mice – indicating a persistent and age-dependent neuroinflammation.

NOTE: End date is 11/30/2023 (incorrectly listed in NSSC as 11/14/2021) per F. Hernandez/ARC (Ed., 7/27/21)

• We have determined the dose-response curve and the baseline dose of AAV-G22-Cre PRISM to report the spontaneous DNA damage response in the mouse brain. We tested the gene dosage dependency of the genetic sensor with G22 repeats. We examined 6 different doses (5x1011, 1x1011, 5x1010, 1x1010, 0.5x1010 VG/mouse) in vivo. We have determined the best titer/dosage of 1x1010 VG/mouse for in vivo application.

• We acquired Th-Cre, CamKII-Cre, D1-Cre, D2-Cre, Aai9 mouse breeding pairs from the Jackson Laboratory (JAX). We have been breeding these mice in C57BL/6J background. We already accumulated a sufficient number of mice for the simulated space radiation exposure at the NASA Space Radiation Laboratory (NSRL) this October 2021. • We successfully constructed the genetic sensor (biodosimetry) of neuronal genomic instability and validated the Cre dependency in vitro. These sensors will be used to determine space radiation-induced neuronal genomic instability in the most vulnerable neuronal cell types in multiple neurodegenerative disorders.

• We generated a quantitative ratiometric sensor: AAV-G34/G22/G13- mScarlet/fWasabi/fBFP PRISM vector and packaged it in AAV-PhB.eB serotype in high titer and purity for non-invasive in vivo application (intravenous injection) in mice.

• Intravenous systemic administration of AAV-G34/ G13- mScarlet/ fBFP PRISM sensor in different Cre driver lines (Drd1a. Drd2, TH Cre, CamkII Cre lines) was performed and we successfully labeled major neuronal cell types vulnerable to neurodegeneration, striatal medium spiny neurons, nigral dopamine neurons, hippocampal and cortical pyramidal neurons.

• Developed assays to quantify and confirm the mechanism of action of Dn to induce neuronal genomic instability using T7E1, Next Generation Sequencing (NGS), and capillary electrophoresis.

• We have made significant progress in optimizing the volume imaging of the iDISCO cleared mouse brains labeled with the genetic sensor of neuronal genomic instability.

• We have demonstrated that radiation mimic, Bleomycin, can increase AAV-G22-Cre dependent expression of AAV-G34/ G13- mScarlet/ fBFP PRISM sensor (systemic administration) genetic labelling in the mouse brains.

• We have scheduled and are ready for our first beamline exposures on 10/29-10/20/2021.