Experimental Design for Carboxyfullerene Study

By Sarah Constantin

The following is excerpted from Dr. Laura Dugan's grant application.


Age-related diseases are arguably the single greatest challenge for biomedicine in the 21st Century. The number of Americans over age 65 will be 70 million by 2030 - nearly 25% of the population1. While the expected lifespan of individuals in the US and most developed countries continues to increase, an increasing number of older adults are living with chronic conditions which can rob them of much of the benefits of longer life. Development of interventions whose goal is to enhance healthy aging – the health span – of individuals, however, has only recently emerged as a focus of biomedical research.

Here we propose to study the benefits of a novel class of compounds, the carboxyfullerenes, on both longevity and health span, in a natural aging model. The lead carboxyfullerene, C3, is a unique and highly potent anti-inflammatory agent, which has shown efficacy in not only mouse and rat models of age-related conditions, but also several other species, including primates. Low-grade inflammation occurs during aging in organisms ranging from flies to humans, and has been proposed be a fundamental feature of aging2-8. More importantly, many diseases which adversely impact older adults, such as cancer, diabetes, arthritis, involve low-grade, systemic inflammation. The anti-inflammatory effects of C3, therefore, are especially relevant to multiple aging- related conditions due to this nearly ubiquitous role of inflammation in aging. Here we propose studies to document the maximal efficacy of C3 treatment on both lifespan and health span. The project will quantify how C3modifies inflammation, and inflammation-associated diseases such as cancer transformation, muscle wasting, and end-organ fibrosis, in aging. We will also assess how well treatment preserves the functional status of the animals, physically and cognitively throughout the study. Ultimately, results from this research should allow the contribution of inflammation to aging to be assessed, but could provide a springboard for additional research into how globally effective strategies against, for example inflammation, could enhance health span.

Aim 1 will determine the maximum benefit that can be achieved in health span and longevity with sustained C3 treatment. In our previous trial, C3 treatment was initiated in middle-aged (12 month old) animals, and used only one (low) dose. However, it is well known that calorie restriction, for example, which almost universally produces anti-aging benefits, has its maximal effects when started in early adulthood. Plan:

1) Start treatment with C3 starting at 3 months of age, when mice have reached maturity.
2) Test 3 doses of C
3, including the original dose (1 mg/kg/day), and 3/mg/kg/day and 10 mg/kg/day.
3) Lifespan itself will be monitored in each cohort, and we will use two statistical approaches to this. Both

Censored and non-censored survival will be analyzed. The first allows all mice to survive their full natural lifespan, but generally precludes other analyses as there is no way to predict date of death. The other approach, censored survival, allows mice to be taken out of the cohort and studied before natural death, permitting “health span” phenotypes to be studied. This Aim will determine the most effective dose, and will test maximal effect of C3 on longevity. All previous studies using the C3 compound in mice, rats, and primates have always been carried out in a blinded manner to minimize unconscious bias; the studies proposed here will be carried out similarly.

Aim 2 will study multiple aspects of aging with clinical relevance to heathier aging – with the goal of documenting improved health span with treatment. Mice develop many features of “poor” aging that are quite similar to humans. These include decline in cardiac and renal function, glucose intolerance (pre-diabetes), cataracts, predisposition to cancer, and cognitive decline, among others. Aim 2 will:

1) Carry out complete autopsies (necropsies) on mice at 22 months of age to track effects on aging-related pathologies, including cancer, renal and cardiac fibrosis, muscle loss (sarcopenia) and cataracts. Autopsies will be carried out by an experienced veterinary pathologist. A total of 100 mice will undergo necropsies, to provide a robust read-out of global effects on aging pathology.

2) Comprehensive cognitive testing using conventional memory and learning tasks. These will performed by the VUMC Mouse Behavioral Phenotyping Core, using state-of-the-art tracking and analysis software.

3) Motor performance will also be tested using the conventional rotorod “mouse treadmill” task. 4) Overall health rating: pelt, grooming, cage mobility, weight will all be scored.

Aim 3 will characterize anti-inflammatory effects of C3 in various key organs, including heart, kidney, muscle and brain. If reducing inflammation in these organs is associated with less age-related damage, and presumably preserved function, this would provide strong support for targeting inflammation to enhance healthy aging, This Aim will provide mechanistic information on how C3 modifies aging, as well as providing a foundation for translational studies in humans.



Inflammation and activation of innate immunity may be key processes in aging. Increasing evidence indicates that adverse health outcomes in older adults are strongly associated with the development of a state of chronic, mild inflammation9-11. In humans, circulating markers of inflammatory pathway activation, including the inflammatory cytokine interleukin-6 (IL-6), are associated with, or predict, enhanced risk of frailty, loss of muscle mass and strength, disability and early mortality in dozens of studies12. Inflammatory mediators have also been linked to both acute and chronic cognitive decline, development of Alzheimer’s disease, and drug- and stress-induced delirium in older adults13-15. There are multiple etiological factors that may activate inflammatory pathways systemically and in the brain in older adults, including chronic disease states, genetic variation, increased numbers of senescent cells and fat cells, changes in hormone status and diet16,17.

Genome-wide association studies (GWAS) have also repeatedly shown that genes associated with inflammation and innate immunity confer increased risk for development of aging-related diseases. Risk genes identified include PU.1 (monocyte-lineage transcription factor)18, CD33, CR1, ABCA7, MS4A, and TREM2 (cell surface proteins on activated microglia), and the interleukin-6 receptor (IL-6R), among others19-25. In the nervous system, gene array profiles from brains of normal, healthy older adults also showed increased expression of inflammatory genes, including CD33, IL-6R, and CR126, and a recently published study indicated persistent activation of innate immunity in brain for months after a systemic inflammatory exposure27. Finally, individuals who come from long-lived populations that are enriched in healthy centenarians (i.e., they exhibit a prolonged health span) have lower levels of inflammatory markers throughout their life. Thus, multiple lines of evidence indicate that inappropriate and sustained activation of inflammation occurs during aging across species.

On a molecular level, NFB is the key gateway nuclear transcription factor controlling expression of many chronic inflammatory mediators, including IL-628,29. IL-6 in turn activates a host of inflammatory actions, including induction and activation of NADPH oxidase (Nox), a multimeric enzyme complex first described as the respiratory burst oxidase in neutrophils. Nox complexes produce large amounts of oxidants, predominantly superoxide30,31, and Nox isoforms are now known to be expressed widely, including in muscle, heart, kidney, retina, peripheral nerves and brain. Because of its near-ubiquitous distribution, systemic activation of Nox(s) can provide wide-spread injury chronic injury across organ systems during aging, producing gradual functional decline. Taken together, there is strong evidence that preventing inappropriate activation of inflammation in nagging might have broad beneficial effects and enhance healthy aging.

Current anti-inflammatory drugs do not effectively target inflammation in aging. An ever-increasing number of anti-inflammatory drugs have been developed to treat conditions as diverse as arthritis, asthma, cancer and colitis. In addition to older anti-inflammatory agents and steroids, newer anti-TNFα, anti-IL-6, and IL-IL-1 therapies are in now clinically approved. These might be attractive as anti-inflammatory interventions in aging, at a minimum to test the idea that anti-inflammatory treatments might improve the health span of aging individuals.

However, it has been difficult to bring these agents into aging research to address this question. These drugs and immunotherapies have been developed to be highly selective and do not produce broad anti-inflammatory effects. Many of these agents produce adverse reactions, especially with sustained treatments32,33, few cross the blood-brain barrier34-36, and few have been tested long-term in older patients, the populations that should benefit from such an intervention. The studies proposed here with C3, therefore, may serve two purposes: to test efficacy of C3 itself and to provide support for future studies on the potential of anti-inflammatory strategies for maintaining health in aging (as opposed to treating a specific disease).

Published and Preliminary Data Supporting this Application.

Background on C3 carboxyfullerene. C3 is a first-in-class anti-inflammatory compound which represents 22 years of careful, systematic development. C3 is a member of the malonic acid C60 fullerene family of compounds. These C60 derivatives possess truly unique properties which reflect both the singular chemical properties of fullerenes (sometimes called exotic carbon chemistry) of the spherical all-carbon C60 “soccer ball”, and the position of chemical groups added to the ball. Unlike the parent C60 molecule, C3 and other carboxyfullerenes are highly water soluble (Figure 1). The carboxyfullerene family act as catalytic superoxide dismutase mimetics, and as such, are nearly 10,000 times more potent than classic anti-oxidants such as vitamin E or vitamin C. C3 is member of a family of six fullerene-based compounds that possess these properties, but C3, for several reasons, has merged as the lead compound. Its molecular mechanism of action is similar to the native superoxide dismutase enzyme, which converts (dismutes) two superoxide anions to one oxygen and two water molecules. This catalytic activity sets C3 apart from other agents which are used as “antioxidants”, in that it can eliminate thousands of superoxide radicals per molecule of C3, in contrast to classic anti-oxidants are “one and done”- one antioxidant molecule is consumed while eliminating one radical molecule. In addition, these compounds are much more effective in the presence of inflammation, and therefore do not interfere with the normal role of oxidants in protecting against infection, or intracellular signaling. Finally, they exhibit uptake into brain, a feature that has been difficult to achieve with essentially all current anti-oxidant agents.

C3 has demonstrated therapeutic efficacy in a number of animal models of nervous system disease, but most relevant to a discussion of its translational potential, C3 showed therapeutic efficacy in a non-human primate model of Parkinson’s disease. That study was designed as a placebo-controlled, double-blind study in which C3 was administered after injury to the dopaminergic neurons was already in process. In several other in vivo models of CNS injury and disease, C3 has demonstrated efficacy as well; these studies have been published in Science, J. Neurosci., Ann. Neurology, and PNAS among others. The C3 molecule exhibits desirable pharmacokinetics and tissue distribution, with a plasma half-life of 8 hours, and clearance through both renal and hepatic routes. It was shown to cross the blood brain barrier not only by its efficacy in the primate PD study, but by documenting uptake of radiolabeled C3 into brain. Blood studies carried out on the monkeys did not show evidence of adverse effects on hematologic, hepatic or renal parameters after 2 months of treatment, and EKGs and autopsy results on the monkeys at the end of the trial found no difference between the placebo and C3 treated groups. Of note, a study in mice which were administered C3 orally for up to 3 years showed no adverse health effects, and a significant increase in lifespan in the C3-treateed mice. The current proposal seeks to more thoroughly characterize this longevity effect, to determine in a systematic manner the domains in which C3 also enhances heathy aging.

In our original studies, published in 2006 and 200817,37, we administered C3, a small-molecule synthetic enzyme superoxide dismutase (SOD) mimetic to wild-type (i.e., non-transgenic, non-senescence accelerated) mice starting at middle age. Chronic treatment not only reduced age-associated oxidative stress and mitochondrial radical production, but significantly extended lifespan. Treated mice also exhibited improved performance on the Morris water maze learning and memory task. This is to our knowledge the first demonstration that an administered antioxidant with mitochondrial activity and nervous system penetration not only increases lifespan, but rescues age-related cognitive impairment in mammals. SOD mimetics with such characteristics may provide unique complements to genetic strategies to study the contribution of oxidative processes to nervous system aging.



Figure 1 Structure and properties of carboxyfullerene C3.

Structure of C3 using (A) space-filling and (B) stick models showing addition of the three malonic acid groups to the C60 sphere. The synthesis of C3 from its precursor, C60, involves multiple steps, which takes C60, which is completely insoluble in water (C), to the final C3 product, which is highly water soluble, clear red solution, with solubility up to 420 mM in water.

Figure 2 Kaplan-Meyer Survival Curve from Quick et al. (2008). In this study, mice began low-dose (only 1 mg/kg/day) C3 treatment at middle age (12 mos.). Despite this, there was a 15% increase in both mean (p=0.004) and maximal lifespan (p=0.02). In the current proposal, we believe starting treatment in young adulthood, and using a dose-escalation paradigm may enhance longevity effects.

Carboxyfullerene C3 produced neuroprotection post-injury in parkinsonian nonhuman primates38. We evaluated the efficacy of C3 to salvage nigrostriatal neuronal function after MPTP exposure in nonhuman primates. This tested C3, as a first-in-class functionalized water-soluble fullerene in a highly translational neurodegeneration model. Macaque fascicularis monkeys were used in a double-blind, placebo-controlled study design. MPTP-lesioned primates were given systemic C3 (n = 8) or placebo (n = 7) for two months starting one week after MPTP. Outcomes included in vivo behavioral measures of motor parkinsonism using a validated non-human primate rating scale, kinematic analyses of peak upper extremity velocity, PET imaging of 6-[18F]fluorodopa (FD, reflects dopa decarboxylase) and [11C]dihydrotetrabenazine (DTBZ; reflects vesicular monoamine transporter type 2), as well as ex vivo quantification of striatal dopamine (DA) and stereologic counts of tyrosine hydroxylase (TH) immunostained neurons in substantia nigra. After two months, C3 treated monkeys had greater striatal FD and DTBZ uptake (Figure 3 A, B), significantly improved parkinsonian motor ratings (Figure 3 C), and higher striatal dopamine levels. None of the C3 treated animals developed any toxicity39. Systemic treatment with C3 reduced striatal injury and improved motor function despite administration after the MPTP injury process had begun. These data strongly support further development of C3 as a promising therapeutic age-related neurodegenerative conditions.

Figure 3 PET imaging of DTBZ and FD, and dynamic functional assessment of Parkinson symptoms for C3-treated and control animals. Representative coronal PET images are shown from two control primates (A; monkeys M1 and M2) and two C3-treated monkeys (B; monkeys M3 and M4) just prior to intracarotid MPTP (top panels) and at the end of two months of treatment with placebo or C3 (bottom panels). Images of DTBZ (left panels) and FD (right panels) are shown. Note the bilateral uptake of each tracer pre-MPTP, showing the tear- drop-shaped substantia nigra bilaterally for all four animals. However, at the end of the study, there is significantly less uptake of both tracers on the lesioned side in placebo-treated animals compared to C3-treated monkeys, or conversely, there is preservation of DTBZ and FD in C3-treated animals. (C) Animals were rated for Parkinsonian deficits using an 18-point system which assesses the severity of parkinsonian symptoms (0= no symptoms; 18= most severely affected). Data are the mean and standard deviation of scores for all animals in each arm. Note the similarity of scores shortly after MPTP treatment, but prior to starting infusion of C3 or placebo, and the progressively different scores attained with longer infusions of C3. Filled circles, C3-treated animals; open circles, control animals.

Experimental Plan

Synthesis, purification and Preparation of the C3 carboxyfullerene. The carboxyfullerene compound, C3, is prepared as described40,41. C3 synthesis requires a two-step process beginning with esterification and followed by conversion to an acid. The C3 ester [e,e,e-tris (dimethylmalonyl) -fullerene] was prepared by reacting of C60 with dimethyl bromomalalonate and 1, 8-diazabicyclo (5.4.0) undec-7-ene (DBU) and the C3 ester was purified by silica gel column chromatography. Alkaline (methanolic NaOH) hydrolysis of C3 ester provided C3 acid which was further purified by C18 reverse phase column chromatography.

All lots are tested by HPLC with diode array detection, which allows contaminants to be detected at levels less than 0.5%. Lots are also analyzed by LC-Mass Spectroscopy, and by NMR.

Mouse in vivo studies: long-term treatment with the carboxyfullerene, C3. Wild-type, healthy C57BL6 mice bred from founders purchased from Jackson Labs will start treatment at 4 months of age, after they are fully mature. This age corresponds roughly to a 25 year-old human. In-house breeding allows mice to start treatment right at 4 months of age, and eliminate the substantial stress of being shipped. Three groups of mice will be enrolled, with 50 mice per group, equal numbers of males and females, and all mice will receive C3 in the drinking water. The 3 groups are: C3 (3 mg/kg/day), C3 (10 mg/kg/day), controls, who receive dilute red food coloring (Durkee’s) in the water to color-match the C3 solutions. This is done so that all observers remain blind to the experimental group throughout the study.

As shown in the schematic below, mice will be maintained on the drug or control solutions throughout life, with solutions changed weekly. At 12 mos. and then at 22 mos. of age, mice will undergo behavioral testing, including motor performance (rotorod) and cognitive behavioral testing. Multiple studies detect subtle behavioral deficits, especially in learning and memory, in 12-month old animals that become much more pronounced by 20+ months of age. We expect that C3 treatment at both doses will blunt or prevent this decline in performance.

To allow reliable autopsies (necropsies) to be performed, 30 mice (15 male, 15 female) from each treatment group will be perfused just after the second round of behavioral testing is completes (shown as yellow rectangles on the schematic). This age precedes the age at which aged C57BL6 mice begin to die, yet is associated with development of a broad range of pathologies, such as cataracts, cancers, and glomerulonephritis. These comprehensive autopsies will provide a comprehensive snapshot across multiple organs of beneficial effects of C3. The specific organs and tissues that will be examined are detailed more fully below, and in the letter provided by the Veterinary Pathologist, Dr. Boyd

In support of our continuing investigation and characterization of e,e,e-fullerene(60)-63-tris malonic acid ( C3 ) we optimized the conditions for obtaining mass spectra. The best mass spectra were obtained when solution containing a weak organic acid added to aqueous methanol (1:1) in positive mode under electrospray C3 was sprayed from a ionization (ESI). In positive ion mode the spectra are simple with minimal fragmentation and the molecular ion region increases linearly with analyte concentration over the range studied40.

Construction of Kaplan-Meyer Survival Curves, and Statistical Analysis of Survival. The date of death will be recorded by an investigator blind to treatment, and Kaplan-Meyer survival curves, similar to Figure 2 above will be constructed. The study sample for the survival analysis will contain a total of 150 mice (75 females, 75 males). 50 mice will be treated with C3 at 3 mg/kg/day, 50 mice will receive C3 at 10 mg/kg/day, with 50 mice will receive color-matched control solution. The survival distributions for the C3 treated mice and the control mice will be estimated by the nonparametric Kaplan-Meier Product-Limit estimator and compared by a log rank test [1]. In our previous study using 1 mg/kg/day C3, treated mice had a significantly lower rate of death over time (log-rank test statistic=11.4, p=0.0007) compared to the control mice. For comparison purposes, the mean lifespan for female C57BL6 mice is 753 ± 23 days and for male B6 mice is 797 ± 23 days42.

Under the assumption of proportional hazards between the treated mice group and the control groups which will be verified by a test through the incorporation of time-dependent covariates [2], the estimated hazard ratio of death between the C3 treated mice and the control mice will be calculated, to provide the 95% confidence interval range. We will further confirm statistical effects on survival by the analysis based on another Cox proportional hazards model [1], which controls for the gender effect by treating it as strata. We have used these statistical approaches previously to calculate both censored and uncensored survival in aging mice.

Studies to Assess Motor Function. The standard mouse rotorod “mouse treadmill” test will be used to assess motor performance. In this test, mice are placed on a rotating cylinder with grooves, and mice run forward to stay on the cylinder as it rotates. Short-distance falls from the rotorod to a touch-sensitive pad are recorded as the length pf time spent on the rotorod before the fall. After training, three consecutive attempts to stay on the rotating cylinder are measured and summed. This test has been highly sensitive to impaired muscle strength and stamina in many models, including aging mice. Statistics will use ANOVA with the Tukey’s post-hoc test, comparing the 3 treatment groups. We will determine whether there are intra-group difference between males and females, given this is a strength task, and if differences are observed, we will then use a 2-way ANOVA where sex is included as one factor. P will be set to 0.05.

Behavioral studies to assess preservation of cognitive function: Clearly, brain aging and diseases of the nervous system have a critical impact on healthy aging. In fact, older adults report that their single greatest health concern is fear of developing a dementia such as Alzheimer’s Disease. Any intervention that seeks to preserve health span, therefore, will need to help protect the brain from age-related disease states. In that context, a significant role for inflammation and downstream free radical production in cognitive aging has been reported in multiple studies. Results from genetically modified animals showing that decreasing superoxide by either overexpression of superoxide dismutases or disruption of Nox subunit assembly affects long-term potentiation43. However, overproduction of reactive oxygen species (ROS), as in inflammation or aging, also adversely affects cognition. Studies in organisms ranging from yeast to man indicate that aging is associated with enhanced oxidative stress and accumulation of oxidized cellular components44. Two dominant sources of ROS production in brain during aging have been proposed45. Mitochondria are one source of enhanced ROS. Neuronal superoxide production is elevated in several brain regions including hippocampus and cortex in aged mice, and mitochondria contributed to this increase37,46. However, results over the last several years have suggested that non-mitochondrial sources are important as well17,37. We and others have shown that the NADPH oxidase family (Nox complexes) are significant sources of ROS in aging brain of superoxide47.

In our original study on C3 effects on aging17,37, C3-treated mice performed better on several behavioral tasks, including the Morris water maze, which tests spatial learning and memory. That study started treatment in mid-life, however, and did not test cognition broadly. To allow a more comprehensive assessment of cognitive benefits of treatment with C3, we will perform a sequential battery of well-established cognitive tasks twice during the trial. Male and female C57BL6 mice will be tested at 12 months of age (corresponding to roughly age 50 in humans) and at 22 months of age (the age at which significant cognitive deficits occur, but prior to attrition of mice). Three domains will be tested sequentially, with the test period listed beside each task.

1. Activity and Anxiety (week 1). These measures of activity in a novel test chamber are important controls for interpretation of later tasks.
a. Locomotor activity and Open field analysis assess overall motor activity, and anxiety as time spent by the mouse along the edge vs center of the box (less time in center reflects greater anxiety). Performance on this test is modified by anxiolytic drugs such as benzodiazepines and anti-depressant drugs such as SSRIs. Activity is measured in 30 X 30 cm arenas, housed within sound-attenuating chambers, by the breaking of infrared beams across a 30-60 min. test session (MedAssociates).


b. Elevated zero maze, is another standard test for anxiety, which tests fear of entering a new, brightly lit
open area as latency to move from a safe dark compartment within a 5 minute test period. Video monitoring and automated tracking software allows analysis of time and distance travelled within each area of the maze as well as latency to enter each zone (AnyMaze).

2. Aggression and social dominance (week 2)
a. Dominance tube test.
Two mice are place at either end of a cleaned, clear 3 cm diameter plexiglass tube, and allowed to enter and approach the center of the tube. Aggressive mice demonstrate dominance in the tube, by squinting, stiffening their bodies and tails, by failure to respond to overtures of the other mouse, and by dominance scent. The non-dominant mouse backs out of the tube, while the aggressive mouse remains. This test has a high correlation with aggressive behavior elsewhere, for example attacking cage-mates, but does not allow the aggressor to actually attack the other mouse. Dominance relationships within group-housed animals can be reliably determined using this task even in the absence of any overt aggression
48. The test continues for 5 minutes or until one mouse exits the tube. A video system is mounted beside the tube so that the mice do not see the observer.

Mice will be tested against naïve wild-type mice to establish whether wins are greater than chance (50%) in any of the genotypes tested. A secondary measure of social dominance will be established by noting whisker trimming in mice within the home cage. Dominant mice typically trim the whiskers of the other mice in the cage.48

a. Resident intruder/dyadic aggression. Two mice are placed in a clean cage, and the time measured until an aggressive attack is made. Non-aggressive mice will sniff and groom, whereas aggressive mice will chase or attack the other resident. Mice will remain together for 5 minutes, or until an attack is made and they are then separated. The latency to attack is a standard measure of aggression, and correlates well with observed aggressive behavior in other tests. C3-treated mice will all be tested against control treated mice.

b. Further determination of aggressive behavior can be made using an alternate version of resident intruder in which test mice will be single housed for up to one week before a sex-matched wild-type mouse is placed in the cage. Behaviors are recorded and monitored as above for a number of aggressive behaviors. Single housing mice increases territorial behavior and may lead to a greater array of behavioral exhibitions of aggressiveness. Since single housing can increase aggression and territorial behavior, particularly in males, there is increased likelihood of fighting if mice are subsequently re-housed with previous cage mates. Thus, this task will be conducted at the end of the test battery, or in a separate set of mice, as appropriate.

3. Depression/apathy-like behavior (week 3)

a. Forced Swim Test (FST). This test, which measures the percentage of time spent floating in the water versus actively swimming as an attempt to escape, is a validated measure of a depression phenotype, and has been shown to respond to several classes of anti-depressants. Mice remain in the apparatus (a clear plexiglass cylinder with room temperature water) for 6 minutes and sessions are recorded for subsequent scoring of time spent immobile (floating) either by hand or using automated software (Ethovision, Noldus). A greater degree of immobility, particularly in earlier parts of the session correlates with a depressive-phenotype.

b. Novelty suppressed feeding, (hyponeophagia) is a conflict-based test which measures the latency (in seconds) for a hungry mouse to feed when placed in a novel, anxiogenic, environment, with shorter latency indicative of less anxiety. Mice are deprived of food up to 24 hours prior to testing. When placed in the arena they face a choice of approaching and consuming a piece of food in the center of a brightly lit, novel open arena or staying to the side and avoiding the center of this anxiogenic environment. Latency to eat (defined as the amount of time it takes for the animal to enter the center of the arena and bite the food pellet with use of forepaws while sitting on its haunches) is measured. Animals show a distribution of performance in this test, where some animals show a clear latency to eat and others do not, therefore providing a platform for determining whether inflammation predicts a greater anxiety phenotype (longer latency). This strategy was recently used to predict the response of mice to fluoxitene treatment. That study induced an anxiety phenotype in mice by chronic steroid treatment, and then looked at whether a proteomic profile from peripheral blood cells predicted response to drug on the NSF. They found a high correlation between a constellation of proteomic changes, and individual treatment response. These results, first confirmed as has been previously shown, that NSF performance responds to anti-depressant and anxiolytic treatments, and also give face validity to our plan to test whether C3-treatement will affect test performance. We predict that C3-treated mice will be less impaired on each of these task, and that.C3 treated mice will spend greater time in the dark areas of the EZM, and close to the walls of the activity chambers. Aggression, which develops in many older adults, often even in mild cognitive impairment, we believe will be reduced by C3 treatment, manifesting as C3 treated mice are less likely to win bouts in the dominance tube task, or initiate more aggressive behaviors in the dyadic aggression task. Finally, we have shown in a previous pilot that C3 reduces depressive behavior in aging mice; here we will determine whether C3-treated mice show less signs of depression, i.e. they will spend less time floating in the FST and have shorter latencies to approach the food in the NSF task.

4. Testing timeline.

The tests progress from least stressful to most stressful, and allows for at least 3 days’ rest between tests to minimize the effects of a prior test on the next test results. All testing will be conducted in the facilities of the Vanderbilt Mouse Behavior Core (see details in letter of support and facilities statement).

5. Statistical approach and number of animals to be tested. We calculate the need for 10 animals per group (SigmaStat) to detect a 15% difference between groups with a power of >0.80 for ANOVA comparisons of 3 groups with an empiric SD of 8. If C3 treatment improves behavior, then we should see a positive relationship between C3 treatment, and individual performance on a behavioral task, and speculate we will observe a dose- response effect with higher C3 dosing providing enhanced improvement.

Necropsy Studies to evaluate effects on known age-related pathologies. Naturally aging mice develop many of the important changes observed in aging humans. These include cataracts, kidney and liver fibrosis, a number of types of cancer including lung cancer, loss of muscle mass (sarcopenia), and cognitive decline, among others. Pilot data suggests that chronic treatment with C3 reduced many of these processes, but the number of mice which came to necropsy was limited. Here, we will do a fully-powered necropsy study on mice at 22 months of age, to look at all of these age-related pathologic changes. If a majority of them are impacted by C3 treatment, this would lend strong support for C3 providing not only longer lifespan, but importantly, health span.

Immunostaining of tissues, including brain, for markers of inflammation. Mice will be anesthetized with isoflurane, and transcardially perfused with ice-cold phosphate buffered saline (PBS) for 1 minute, followed by perfusion with cold 4% paraformaldehyde (PFA) in 10 μM PBS for 5 minutes. Whole brains are removed and post-fixed in 4% PFA at 4°C overnight, switched to 2% PFA for an additional 24 hours, then sliced on a Vibratome to generate 50 μm sections which are maintained in 30% sucrose, 30% ethylene glycol, 1% PVP-40 at -20°C until they are ready to be processed for immunostaining. Floating sections are then blocked, immunostained with primary antibody, washed, labelled with fluorescent secondary antibody, and mounted in Vectashield mounting medium on glass slides for imaging. All analysis is performed by individuals blind to treatment information. Antibodies to be used include (Vendor, CAT#, Dilution): rabbit anti-p62 (Sigma, P0067, 1:4K), mouse mab anti-parkin (Abcam, ab77924, 1:1K), rabbit anti-pink1 (Abcam, ab23707, 1:5K with TSA), rabbit mab anti-lamp2b (Abcam, ab125068, 1:5K with TSA), and rabbit anti-LC3B (Sigma, L7543, 1:1 to 2K). Immunostained sections are imaged on a Zeiss LSM 880 2-photon confocal system. Slides are stored in the dark when not being imaged. Immunofluorescence for a given autophagy marker will be quantified by the Zeiss Zen Blue analytical software or Image J. We will define lysosome failure as an increase in p62, and lamp2 deposits in the presence of increased levels of LC3b-II, all by immunostaining. We will further look for other undegraded proteins as evidence for a general defect in lysosome degradation of cargo. All animal studies were approved by the Animal Care Program at Vanderbilt University, and are in accordance with the PHS Guide for the Care and Use of Laboratory Animals, USDA Regulations, and the AVMA Panel on Euthanasia.

Summary. This study will determine the maximal health and longevity benefits in aging that can be provided by long-term treatment with C3, It will also systematically examine several outcomes that are highly relevant to successful, healthy aging. In addition to survival, full autopsy (necropsy) evaluations will be performed, looking at cancer prevalence, organ damage and fibrosis, and evidence of inflammatory changes. Functional outcomes that will be assessed include a battery of behavioral test, especially focused on memory, learning, and neuropsychiatric changes. Muscle health and motor performance will be measured, as well. Outcomes from this study will not only provide a comprehensive analysis of C3, but will indicate whether inflammation could be a viable target in aging to maintain function and health.



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