NOTE: End date changed to 8/31/2016 per S. Monk/HRP and NSSC information (Ed., 6/21/16)

NOTE: End date changed to 3/31/2016 per NSSC information (Ed., 7/9/15)

NOTE: Extended to 1/21/2016 per PI and NSSC information (Ed., 3/12/2015)

This proposal will utilize a well-characterized and widely used Alzheimer's disease transgenic mouse model (Tg2576) to address the following research objectives: (1) examine the long-term impact of space radiation (SR) on hippocampal-dependent spatial learning and memory, (2) evaluate the potential of SR to accelerate Alzheimer's disease pathogenesis and neuropathology, (3) evaluate a novel non-invasive laser-based eye scanner to detect and monitor molecular changes in the lens of the eye induced by radiation exposure and Alzheimer's disease pathology (Goldstein, et al., Lancet, 2003).

A complementary companion study will utilize the same cohort of animal subjects to: (1) evaluate electrical communication between neurons, and changes in function and fine structure of neurons, including dendritic spines where synaptic contacts enable neuronal communication, (2) determine whether SR, in reducing neurogenesis, also alters the functionality of newly-born neurons, and (3) assess whether SR differentially affects electrical or physical function of neurons, and/or accelerates the Alzheimer's disease process.

Our proposed studies directly address key objectives of the NASA Human Space Flight Program, including determination of potential space-related SR dependencies related to late central nervous system (CNS) risks such as early-onset dementia or Alzheimer's disease, assessment of SR effects on molecular, cellular, and tissue environment changes in hippocampus indicative of increased risk of dementia or Alzheimer's disease, and evaluation of biological models of Alzheimer's disease or other forms of dementia that occur in humans.

The existing knowledge gap is immense and presents a major obstacle to rational assessment of short- and long-term risk to the central nervous system posed by SR exposure expected during extended human space travel. Our experiments will examine, for the first time, the mechanisms by which SR impairs synaptic function in normal brain, assess whether SR does, in fact, enhance long-term risk of Alzheimer's disease, and provide an experimental system to identify and evaluate new radiation countermeasures. The proposed interdisciplinary research program will provide an integrated scientific foundation to assess and reduce SR-induced risk to the brain, thus enabling a safe path forward for extended human space exploration.

Reference

Goldstein LE, Muffat JA, Cherny RA, Moir RD, Ericsson MH, Huang X, Mavros C, Coccia JA, Faget KY, Fitch KA, Masters CL, Tanzi RE, Chylack LT Jr, Bush AI. Cytosolic beta-amyloid deposition and supranuclear cataracts in lenses from people with Alzheimer's disease. Lancet. 2003 Apr 12;361(9365):1258-65.

Reference

Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 1996 Oct 4;274(5284):99-102.

This project investigated brain vulnerability in the setting of exposure to low-dose GCR (galactic cosmic radiation). Extended human space flight is predicated on establishing a solid scientific foundation for rational assessment of CNS risk and development of prophylactic countermeasures relevant to GCR exposure expected during long-duration human space travel. This information represents a critical prerequisite for mission success of long-duration human space travel beyond the Earth’s magnetosphere.

While acute CNS damage is a hallmark of exposure to high-dose ionizing radiation, the effects of low-dose GCR on brain structure and function remain largely unknown. An emerging body of evidence suggests that low-dose GCR induces deleterious effects on neuronal function and accelerates brain pathologies associated with AD and other neurodegenerative diseases. Our research involving neuropathological analysis of post-mortem human brains and experimental animals indicates that exposure to low-intensity neurotrauma triggers specific neurodegenerative disease mechanisms and pathologies that degrade brain structure and function (Goldstein et al., Science Transl Med, 2012; McKee et al., Brain, 2013 ; Kondo et al., Nature, 2015). These effects manifest acutely, progress chronically, and trigger brain pathologies, cognitive dysfunction, and frank neurodegeneration. Experimental evidence developed during the course of this NASA-funded project indicate that the same pathogenic pathways and mechanisms are activated in laboratory mice exposed to low-dose GCR. We used non-transgenic C57BL/6 mice and Tg2576 AD transgenic mice to determine whether and to what extent exposure to low-dose GCR influences brain structure, function, pathology, risk, latency, progression, and penetrance relevant to these questions. The project generated new information that is directly relevant to maintaining astronaut health and optimal performance during and after long-duration space travel. Project insights are critically important for successful long-duration human space travel and the NASA Human Space Program. Further studies are warranted to confirm and extend project results and translational strategies.

Goldstein LE, Fisher AM, Tagge CA, Zhang XL, Velisek L, Sullivan JA, Upreti C, Kracht JM, Ericsson M, Wojnarowicz MW, Goletiani CJ, Maglakelidze GM, Casey N, Moncaster JA, Minaeva O, Moir RD, Nowinski CJ, Stern RA, Cantu RC, Geiling J, Blusztajn JK, Wolozin BL, Ikezu T, Stein TD, Budson AE, Kowall NW, Chargin D, Sharon A, Saman S, Hall GF, Moss WC, Cleveland RO, Tanzi RE, Stanton PK, McKee AC. Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model. Sci Transl Med. 2012 May 16;4(134):134ra60. [non-NASA supported]

McKee AC, Carreras I, Hossain L, Ryu H, Klein WL et al. (2008) Ibuprofen reduces Abeta, hyperphosphorylated tau and memory deficits in Alzheimer mice. Brain Res 1207: 225-236. [non-NASA supported]

Kondo A, Shahpasand K, Mannix R, Qiu J, Moncaster J, Chen CH, Yao Y, Lin YM, Driver JA, Sun Y, Wei S, Luo ML, Albayram O, Huang P, Rotenberg A, Ryo A, Goldstein LE, Pascual-Leone A, McKee AC, Meehan W, Zhou XZ, Lu KP. Antibody against early driver of neurodegeneration cis P-tau blocks brain injury and tauopathy. Nature. 2015 Jul 23;523(7561):431-436. [non-NASA supported]

NOTE: End date changed to 8/31/2016 per S. Monk/HRP and NSSC information (Ed., 6/21/16)

NOTE: End date changed to 3/31/2016 per NSSC information (Ed., 7/9/15)

NOTE: Extended to 1/21/2016 per PI and NSSC information (Ed., 3/12/2015)

This proposal will utilize a well-characterized and widely used Alzheimer's disease transgenic mouse model (Tg2576) to address the following research objectives: (1) examine the long-term impact of space radiation (SR) on hippocampal-dependent spatial learning and memory, (2) evaluate the potential of SR to accelerate Alzheimer's disease pathogenesis and neuropathology, (3) evaluate a novel non-invasive laser-based eye scanner to detect and monitor molecular changes in the lens of the eye induced by radiation exposure and Alzheimer's disease pathology (Goldstein, et al., Lancet, 2003).

A complementary companion study will utilize the same cohort of animal subjects to: (1) evaluate electrical communication between neurons, and changes in function and fine structure of neurons, including dendritic spines where synaptic contacts enable neuronal communication, (2) determine whether SR, in reducing neurogenesis, also alters the functionality of newly-born neurons, and (3) assess whether SR differentially affects electrical or physical function of neurons, and/or accelerates the Alzheimer's disease process.

Our proposed studies directly address key objectives of the NASA Human Space Flight Program, including determination of potential space-related SR dependencies related to late central nervous system (CNS) risks such as early-onset dementia or Alzheimer's disease, assessment of SR effects on molecular, cellular, and tissue environment changes in hippocampus indicative of increased risk of dementia or Alzheimer's disease, and evaluation of biological models of Alzheimer's disease or other forms of dementia that occur in humans.

The existing knowledge gap is immense and presents a major obstacle to rational assessment of short- and long-term risk to the central nervous system posed by SR exposure expected during extended human space travel. Our experiments will examine, for the first time, the mechanisms by which SR impairs synaptic function in normal brain, assess whether SR does, in fact, enhance long-term risk of Alzheimer's disease, and provide an experimental system to identify and evaluate new radiation countermeasures. The proposed interdisciplinary research program will provide an integrated scientific foundation to assess and reduce SR-induced risk to the brain, thus enabling a safe path forward for extended human space exploration.

Our next Brookhaven National Laboratory (BNL) campaign allowed us to investigate possible sources of acute injury that might trigger chronic changes in neurophysiological and cognitive function. We exposed C57BL/J6 mice, 4 months-of-age, to 28Si (300 MeV/u, 70 keV/µm, 100 cGy, or 0 cGy sham control). Mice were sacrificed and brain inflammatory responses analyzed by flow cytometry (+3 days post-irradiation). We found that while space radiation does not alter microglia morphology or increase monocyte infiltration into the brain, we did detect a significant increase in CNS-infiltrating F4/80+ macrophage.

Results of these studies will be included in a paper that we are preparing now with collaborator Patric Stanton, Ph.D., New York Medical College (NASA NNX13AB66G).

This proposal will utilize a well-characterized and widely used Alzheimer's disease transgenic mouse model (Tg2576) to address the following research objectives: (1) examine the long-term impact of space radiation (SR) on hippocampal-dependent spatial learning and memory, (2) evaluate the potential of SR to accelerate Alzheimer's disease pathogenesis and neuropathology, (3) evaluate a novel non-invasive laser-based eye scanner to detect and monitor molecular changes in the lens of the eye induced by radiation exposure and Alzheimer's disease pathology (Goldstein, et al., Lancet, 2003).

A complementary companion study will utilize the same cohort of animal subjects to: (1) evaluate electrical communication between neurons, and changes in function and fine structure of neurons, including dendritic spines where synaptic contacts enable neuronal communication, (2) determine whether SR, in reducing neurogenesis, also alters the functionality of newly-born neurons, and (3) assess whether SR differentially affects electrical or physical function of neurons, and/or accelerates the Alzheimer's disease process.

Our proposed studies directly address key objectives of the NASA Human Space Flight Program, including determination of potential space-related SR dependencies related to late CNS risks such as early-onset dementia or Alzheimer's disease, assessment of SR effects on molecular, cellular and tissue environment changes in hippocampus indicative of increased risk of dementia or Alzheimer's disease, and evaluation of biological models of Alzheimer's disease or other forms of dementia that occur in humans.

The existing knowledge gap is immense and presents a major obstacle to rational assessment of short- and long-term risk to the central nervous system posed by SR exposure expected during extended human space travel. Our experiments will examine, for the first time, the mechanisms by which SR impairs synaptic function in normal brain, assess whether SR does, in fact, enhance long-term risk of Alzheimer's disease, and provide an experimental system to identify and evaluate new radiation countermeasures. The proposed interdisciplinary research program will provide an integrated scientific foundation to assess and reduce SR-induced risk to the brain, thus enabling a safe path forward for extended human space exploration.

Project Hypotheses We hypothesize that exposure to low-dose particle space radiation will negatively and synergistically impact: (i) hippocampal-dependent learning and memory, and (ii) Alzheimer’s disease (AD)-linked pathology in the brain and lens. We anticipate that these effects will be dose- and time-dependent. Furthermore, we hypothesize that cerebral microvasculature disruption and reactive neuroinflammation are critical radiation-induced pathogenic mechanisms by which hippocampal neurocognitive dysfunction and age-dependent AD pathology are synergistically accelerated. Understanding of these relationships is essential for rational assessment of CNS risk and efficient development of prophylactic countermeasures for extended human space travel.

Specific Aim 1. Identify and characterize the effects of low-dose particle space radiation exposure on hippocampal-dependent spatial learning and memory using the Morris water navigation task (or alternative hippocampal-dependent spatial reference paradigm) in Tg2576 AD transgenic mice compared to age-matched wild-type controls.

Specific Aim 2. Identify and characterize the effects of low-dose particle space radiation exposure on AD biochemistry and histopathology in Tg2576 AD transgenic mice compared to age-matched wild-type littermate controls.

Specific Aim 3. Identify, characterize, and track the effect of low-dose particle space radiation on AD progression using a novel noninvasive laser-based eye scanner to quantitatively assess AD-linked Aß pathology in the lens.

Our team discovered AD-linked Aß pathology in the lenses of human subjects with AD (Goldstein, 2003). We subsequently confirmed identical AD-linked lens pathology and age-dependent Abeta accumulation in Down syndrome (100% risk of developing early-onset AD; (Moncaster, 2010) and Tg2576 mice (Moncaster, submitted). These discoveries led us to develop an innovative scanning laser ophthalmoscope with quasi-elastic light scattering analytical capabilities. This noninvasive instrument allows safe, simple, and extremely sensitive analytical assessment of AD-linked Aß pathology in the lenses of non-anesthetized mice. In a recently completed series of experiments utilizing this noninvasive technology in Tg2576 mice (Moncaster, submitted), we detected and tracked pre-cataractous AD-linked Aß lens pathology in Tg2576 mice before onset of detectable amyloid pathology in the brain or lens. Our proposal affords a unique opportunity to evaluate this innovative technology in the context of two possibly interactive variables (i.e., low-dose space particle radiation and AD) both of which are primary targets of the proposed research program. Potential for informative covariate and correlative analyses involving other study endpoints (i.e., behavior, histopathology, biochemistry, neurophysiology) is high. This aim is easily justified given the noninvasive nature of the technology and the low-risk/high-yield potential of experimental deployment.

B. YR2 PROGRESS TO DATE (Sep 1, 2012 through Aug 31, 2013)

Fall 2012 Campaign: We whole-body irradiated 3-month-old male and female mice Tg2576 and wild-type controls using Beamline 56Fe (600 MeV/u, 181 keV/µm) at Brookhaven National Laboratory (BNL). These studies were conducted in consultation with NASA and Eleanor Blakely, Ph.D., Senior Staff Biophysicist, Lawrence Berkeley National Laboratory, who serves as Radiobiology Collaborator on this project. We did 0 and 100 cGy. Each group comprised of n=8 mice and will be sacrificed at 18 months-of-age (+14 months s/p irradiation). Behavior in these mice will be assessed at the midpoint between irradiation, and again just prior to sacrifice and tissue harvest. Animal numbers are calculated as follows: 2 groups (Wt, Tg) x 2 genders (F, M) x 1 irradiation types (Fe) x 2 doses (0, 100 cGY) x 1 timepoints (18mos) = 8 groups x 10 per group = 80 mice total.

Summer 2013 Campaign: We whole-body irradiated 3-month-old male and female C57Bl/6 mice with Beamline 28Si (300 MeV/u, 70 keV/µm) at BNL using doses 0,10, 50, 100 cGY. Animal numbers were calculated as follows: 2 genders (F, M) x 1 irradiation types (Si) x 4 doses (0,10, 50, 100 cGY) x 2 timepoints (10mos, 18mos) = 16 groups x 10 per group = 160 mice total. Expt objectives: (i) evaluate background effects of 28Si exposure on cerebral microvasculature and neuroinflammation assessed by ultrastructural (EM) neuropathological analysis.

Dr. Patric Stanton and a lab member of his assisted and observed this Beamline run. This was the first time at NASA BNL, NSRL and the Goldstein group mentored them through the process. Dr. Stanton has a collaborative NASA grant from which some of the Goldstein mice irradiated at BNL will be shared with Dr. Stanton who will be studying electrophysiological changes in the brain.

All mice will be behaviorally assessed on a hippocampal-dependent spatial learning and memory task using the Barnes Maze and evaluated using a battery of neurobehavioral, neuropathological, and biomarker endpoints at selected post-irradiation intervals endpoints as a function of: (i) particle radiation exposure (Z, energy, dose), (ii) genotype (Tg, Wt), (iii) post-exposure interval, (iv) age and gender. Details of each of these assessments are included in the original proposal. See our publication for additional details regarding immunohistochemical, ultrastructural, and neurobehavioral index metrics (Goldstein, 2012, Science Transl Med).

YR3 EXPERIMENTAL PLAN

Summer 2014 Campaign: We whole-body irradiate 3-month-old male and female mice Tg2576 and wild-type controls using Beamline 28Si (300 MeV/u, 70 keV/µm) at BNL using doses 0, 10, 50, 100 cGY. Each group comprises of n=10 mice and will be sacrificed at 18 months-of-age (+14 months post- irradiation). Behavior in these mice will be assessed at the midpoint between irradiation, and again just prior to sacrifice and tissue harvest. Animal numbers are calculated as follows: 2 groups (Wt, Tg) x 2 genders (F, M) x 1 irradiation types (Si) x 4 doses (0, 10, 50, 100 cGY) x 1 timepoints (18mos) = 16 groups x 10 per group = 160 mice total.

Data analysis: All tissue analyses from mice tissues collected in Yr 1-3 will be analyzed using a battery of neuropathological and biomarker endpoints as a function of: (i) particle radiation exposure (Z, energy, dose), (ii) genotype (Tg, Wt), (iii) post-exposure interval, (iv) age and gender. Details of each of these assessments are included in the original proposal. See our publication for additional details regarding immunohistochemical, ultrastructural, and neurobehavioral index metrics (Goldstein, 2012, Science Transl Med). All Barnes Maze neurobehavioral data collected in Yr 1-3 will be analyzed and prepared for publication.

This proposal will utilize a well-characterized and widely used Alzheimer's disease transgenic mouse model (Tg2576) to address the following research objectives: (1) examine the long-term impact of space radiation (SR) on hippocampal-dependent spatial learning and memory, (2) evaluate the potential of SR to accelerate Alzheimer's disease pathogenesis and neuropathology, (3) evaluate a novel non-invasive laser-based eye scanner to detect and monitor molecular changes in the lens of the eye induced by radiation exposure and Alzheimer's disease pathology (Goldstein, et al., Lancet, 2003).

A complementary companion study will utilize the same cohort of animal subjects to: (1) evaluate electrical communication between neurons, and changes in function and fine structure of neurons, including dendritic spines where synaptic contacts enable neuronal communication, (2) determine whether SR, in reducing neurogenesis, also alters the functionality of newly-born neurons, and (3) assess whether SR differentially affects electrical or physical function of neurons, and/or accelerates the Alzheimer's disease process.

Our proposed studies directly address key objectives of the NASA Human Space Flight Program, including determination of potential space-related SR dependencies related to late CNS risks such as early-onset dementia or Alzheimer's disease, assessment of SR effects on molecular, cellular and tissue environment changes in hippocampus indicative of increased risk of dementia or Alzheimer's disease, and evaluation of biological models of Alzheimer's disease or other forms of dementia that occur in humans.

The existing knowledge gap is immense and presents a major obstacle to rational assessment of short- and long-term risk to the central nervous system posed by SR exposure expected during extended human space travel. Our experiments will examine, for the first time, the mechanisms by which SR impairs synaptic function in normal brain, assess whether SR does, in fact, enhance long-term risk of Alzheimer's disease, and provide an experimental system to identify and evaluate new radiation countermeasures. The proposed interdisciplinary research program will provide an integrated scientific foundation to assess and reduce SR-induced risk to the brain, thus enabling a safe path forward for extended human space exploration.

Project Hypotheses

We hypothesize that exposure to low-dose particle space radiation will negatively and synergistically impact: (i) hippocampal-dependent learning and memory, and (ii) Alzheimer’s disease (AD)-linked pathology in the brain and lens. We anticipate that these effects will be dose- and time-dependent. Furthermore, we hypothesize that cerebral microvasculature disruption and reactive neuroinflammation are critical radiation-induced pathogenic mechanisms by which hippocampal neurocognitive dysfunction and age-dependent AD pathology are synergistically accelerated. Understanding of these relationships is essential for rational assessment of CNS risk and efficient development of prophylactic countermeasures for extended human space travel.

Specific Aim 1

Identify and characterize the effects of low-dose particle space radiation exposure on hippocampal-dependent spatial learning and memory using the Morris water navigation task (or alternative hippocampal-dependent spatial reference paradigm) in Tg2576 AD transgenic mice compared to age-matched wild-type controls.

Specific Aim 2

Identify and characterize the effects of low-dose particle space radiation exposure on AD biochemistry and histopathology in Tg2576 AD transgenic mice compared to age-matched wild-type littermate controls.

Specific Aim 3

Identify, characterize, and track the effect of low-dose particle space radiation on AD progression using a novel noninvasive laser-based eye scanner to quantitatively assess AD-linked Aß pathology in the lens.

B. YR1 PROGRESS TO DATE (Sep 1, 2011 through Aug 31, 2012)

Gain of Experience at the Brookhaven National Laboratory (BNL) NSRL facility

September 2011 – June 2012: All Boston University and BNL IACUC protocols were submitted and approved for this project. Dr Goldstein’s team conducted all necessary training online and on-site to be eligible to conduct experiments safely at BNL NSRL. Submission of beamline time request was submitted by Dr Goldstein and approved for 2 sessions. First session was conducted in June 2012 (Summer 2012) and the second in November 2012 (Fall 2012).

April 2012: Prior to the start of this project, Dr Lee Goldstein and his team had never worked at the BNL NSRL facility. Dr Goldstein has a longstanding collaboration with Dr Eleanor Blakely, Lawrence Berkeley National Laboratory, who is Co-I on this project. Her role is to advise Dr Goldstein regarding radiobiology experimental design and execution based on her prior experience in the field and at the BNL NSRL facility. In early April 2012, Dr Goldstein and his research technician Mark Wojnarowicz observed Dr Eleanor Blakely and her team conduct experiments at the NSRL facility.

First Experiments Conducted at BNL NSRL

June 2012: Dr Goldstein, Dr Juliet Moncaster, Mark Wojnarowicz (senior research technician), and Andrew Fisher (BU bioengineering doctoral candidate in the Goldstein laboratory) conducted their first experiments on the BNL NSRL beamline. The experiment involved whole-body irradiation vs sham irradiation of 2-month-old male C57BL/6 mice using beamline 56Fe (600 MeV/u, 181 keV/µm). Experiment objectives: (i) establish detailed workflow strategy and standard operating protocols (SOPs) for experiments conducted at NSRL, and (ii) evaluate background effects of 56Fe exposure on cerebral microvasculature and neuroinflammation assessed by ultrastructural (EM) neuropathological analysis. In consultation with Eleanor Blakely, Ph.D., Lawrence Berkeley National Laboratory (Dr. Blakely serves as Radiobiology Collaborator on this project), 2 particle radiation doses were chosen: 0 and 100 cGy. Each group was comprised of n=12 mice and will be sacrificed at 4, 10, and 20 months-of-age. Animal numbers were calculated as follows: 1 irradiation types (Fe) x 2 doses (0, 100 cGY) x 3 timepoints (4, 10, 20 mos) = 6 groups x 12 per group = 72 mice total. This pilot study will not only establish team familiarity with NSRL procedures and protocols, but also provide important information for our subsequent studies.

Experimental Changes: In our original proposal, we identified the Morris water maze (MWM) (Morris, 1984; Goldstein, 2003) as our behavioral assay of hippocampal-dependent spatial learning and memory. While the MWM is widely used for this purpose, this behavioral assay was designed specifically for rats, a species well adapted for navigating water environments. The MWM works poorly in certain mouse strains that are not water tolerant and innately assume a motionless floating posture when placed in a water environment. This behavioral response defeats the assay and undermines experimental utility. To overcome this obstacle in our studies, we now plan to deploy the Barnes maze (Barnes CA, J Comp Physiol Psychol, 1979), a well-characterized hippocampal-dependent, spatial learning and memory assay that does not utilize a water environment for behavioral testing. The Barnes maze provides a robust and reliable test of hippocampal-dependent spatial learning and memory in many different inbred mouse strains (Fox, 1999; Nguyen, 2000; Holmes, 2002; Koopmans, 2003; O'Leary, 2011; O'Leary, 2012) and Alzheimer’s disease (AD) transgenic mouse models (Pompl, 1999; Brown, 2007; Reiserer, 2007; O'Leary, 2009). Importantly, the Barnes maze also provides secondary measures of gross locomotor function, exploratory behavior, higher-order working memory, and thigmotaxis (a sensitive measure of murine anxiety), that are relevant to our research objectives and NASA program priorities. We recently deployed the Barnes maze to assess hippocampal-dependent spatial learning and memory deficits in C57BL/6 mice exposed to a single experimental blast ((Goldstein, 2012), Science Translational Medicine). We will use the same behavioral testing paradigm in our NASA studies.

Barnes Maze Test of Hippocampal-Dependent Spatial Learning and Memory Retrieval: Neurobehavioral assessment will be performed using a combination open-field test and Barnes maze (Med-Associates, Inc., St. Albans, VT, USA). Open-field testing enables assessment of baseline locomotor functioning (average velocity), exploratory activity (total distance), and thigmotaxis (number of central zone entries). The test is performed by placing each test mouse in the middle of a 42.5 cm x 42.5 cm open arena and monitoring movement for 10 min using a 3D infrared diode motion detector system (Any-Maze, Stoelting Co., Inc., Wood Dale, IL). The Barnes maze utilizes the same experimental system to quantitatively assess hippocampal-dependent spatial learning and memory retrieval. Barnes maze evaluation is conducted using a 20-box apparatus illuminated with 900 lux surface light intensity. Mice are familiarized with the test apparatus by placement on the platform and gentle guidance to the escape box. Training sessions are conducted across four training trials per day for four days. The order of testing of individual subjects is the same throughout daily sessions, but randomized across the four test days for a total of 16 trials. To initiate testing, a single mouse is placed in the start box in the middle of the maze and released. Test subjects are evaluated while locating a single escape box placed at a constant position. Spatial learning is assisted by visual cues that remain constant during and across test sessions. Movement is tracked and recorded electronically as in the open-field test. Latency to find the escape box, trajectory velocity to the escape box, and total trajectory distance is assessed and recorded daily. Memory retrieval is evaluated by replacing the escape box with a blank box 24 hours after the last training session. Memory retrieval is electronically assessed by recording the number of nose pokes into the blank box as a percentage of total nose pokes.

We will test the influence of exposure to low-dose 56Fe particle radiation on cognitive impairment using the Barnes Maze in the male C57/bl mice we irradiated in June 2012. Behavioral results will be correlated with ex vivo analysis of brains via immunohistopathology, electron microscopic ultrastructural pathology focusing on microvasculopathy, and assessment of neuroinflammation using a variety of specific markers and techniques, including TSPO imaging of activated microglia and astrocytes (Chen, 2008).

C. YR2 EXPERIMENTAL PLAN

Fall 2012 Campaign: We will irradiate 4-month-old mice Tg2576 and wild-type controls using Beamline 56Fe (600 MeV/u, 181 keV/µm) at Brookhaven National Laboratory (BNL). These studies will be conducted in consultation with NASA and Eleanor Blakely, Ph.D., Senior Staff Biophysicist, Lawrence Berkeley National Laboratory, who serves as Radiobiology Collaborator on this project. We have chosen 4 particle radiation doses: 0, 10, 50, and 100 cGy. Each group will be comprise of n=12 mice and will be sacrificed at 10 months-of-age (+6 months s/p irradiation), and 18 months-of-age (+14 months s/p irradiation). Behavior in these mice will be assessed before irradiation, at the midpoint between irradiation, and again just prior to sacrifice and tissue harvest. Animal numbers are calculated as follows: 2 groups (Wt, Tg) x 2 genders (F, M) x 1 irradiation types (Fe) x 4 doses (0,10, 50, 100 cGY) x 2 timepoints (10mos, 18mos) = 32 groups x 12 per group = 384 mice total.

Spring 2013 Campaign: We plan to irradiate Tg2576 and wild-type controls mice with Beamline 28Si (300 MeV/u, 70 keV/µm) at BNL using the same doses (0,10, 50, 100 cGY) and age timepoints. Animal numbers are calculated as follows: 2 groups (Wt, Tg) x 2 genders (F, M) x 1 irradiation types (Si) x 4 doses (0,10, 50, 100 cGY) x 2 timepoints (10mos, 18mos) = 32 groups x 12 per group = 384 mice total.

All mice will be behaviorally assessed on a hippocampal-dependent spatial learning and memory task using the Barnes Maze before irradiation and evaluated using a battery of neurobehavioral, neuropathological, and biomarker endpoints at selected post-irradiation intervals endpoints as a function of: (i) particle radiation exposure (Z, energy, dose), (ii) genotype (Tg, Wt), (iii) post-exposure interval, (iv) age and gender. See our recent publication (non-NASA support) for additional details regarding immunohistochemical, ultrastructural, and neurobehavioral index metrics (Goldstein, 2012, Science Transl Med; http://dx.doi.org/10.1126/scitranslmed.3003716 ).

This proposal will utilize a well-characterized and widely used Alzheimer's disease transgenic mouse model (Tg2576) to address the following research objectives: (1) examine the long-term impact of space radiation (SR) on hippocampal-dependent spatial learning and memory, (2) evaluate the potential of SR to accelerate Alzheimer's disease pathogenesis and neuropathology, (3) evaluate a novel non-invasive laser-based eye scanner to detect and monitor molecular changes in the lens of the eye induced by radiation exposure and Alzheimer's disease pathology (Goldstein, et al., Lancet, 2003).

A complementary companion study will utilize the same cohort of animal subjects to: (1) evaluate electrical communication between neurons, and changes in function and fine structure of neurons, including dendritic spines where synaptic contacts enable neuronal communication, (2) determine whether SR, in reducing neurogenesis, also alters the functionality of newly-born neurons, and (3) assess whether SR differentially affects electrical or physical function of neurons, and/or accelerates the Alzheimer's disease process.

Our proposed studies directly address key objectives of the NASA Human Space Flight Program, including determination of potential space-related SR dependencies related to late CNS risks such as early-onset dementia or Alzheimer's disease, assessment of SR effects on molecular, cellular and tissue environment changes in hippocampus indicative of increased risk of dementia or Alzheimer's disease, and evaluation of biological models of Alzheimer's disease or other forms of dementia that occur in humans.

The existing knowledge gap is immense and presents a major obstacle to rational assessment of short- and long-term risk to the central nervous system posed by SR exposure expected during extended human space travel. Our experiments will examine, for the first time, the mechanisms by which SR impairs synaptic function in normal brain, assess whether SR does, in fact, enhance long-term risk of Alzheimer's disease, and provide an experimental system to identify and evaluate new radiation countermeasures. The proposed interdisciplinary research program will provide an integrated scientific foundation to assess and reduce SR-induced risk to the brain, thus enabling a safe path forward for extended human space exploration.