A major obstacle for preclinical testing of Alzheimer's disease (AD) therapies is the availability of translationally relevant AD models. Critical for the validation of such models is the application of the same approaches and techniques used for the neuropathological characterization of AD. Deposition of amyloid-β 42 (Aβ42) plaques and neurofibrillary tangles containing phospho-Tau (pTau) are the pathognomonic features of AD. In the neuropathologic evaluation of AD, immunohistochemistry (IHC) is the current standard method for detection of Aβ42 and pTau. Although IHC is indispensable for determining the distribution of AD pathology, it is of rather limited use for assessment of the quantity of AD pathology. We have recently developed Luminex-based assays for the quantitative assessment of Aβ42 and pTau in AD brains. These assays are based on the same antibodies that are used for the IHC-based diagnosis of AD neuropathologic change. Here we report the application and extension of such quantitative AD neuropathology assays to commonly used genetically engineered AD models and to animals that develop AD neuropathologic change as they age naturally. We believe that identifying AD models that have Aβ42 or pTau levels comparable to those observed in AD will greatly improve the ability to develop AD therapies. Abbreviations: Alzheimer's disease (AD); amyloid β 42 (Aβ42); phospho-Tau (pTau); immunohistochemistry (IHC).
Geroscience is a multidisciplinary field that examines the relationship between biological aging and agerelated diseases [1]. Seven processes discussed by the trans-NIH Geroscience Interest Group Summit that contribute to biological aging included macromolecular damage, epigenetic changes, inflammation, adaptation to stress, impairments to proteostasis, stem cell regeneration, and metabolism [2]. These processes are highly integrated with one another such that targeting them as a group may be an effective approach to developing therapies to prevent or delay age-related disease. Alzheimer’s disease (AD) is an age-related disease and is expected to increase with the number of elderly individuals rapidly rising in both developed and developing countries. Efforts to find diseasemodifying treatments have met with limited success possibly because they have focused on identifying a specific pathogenic mechanism targeted by a specific drug. AD is a complex disease involving numerous mechanisms in line with processes of biological aging. Therefore, a geroscience approach to successfully treating AD is a logical concept that unfortunately has not yet been widely accepted by the neuroscience community. It is now time to explore preclinical studies in AD animal models to begin screening different drug combinations that target multiple aging-related processes for effect on AD dementia and neuropathology. A major challenge for preclinical drug testing is the selection of an AD animal model. A model is needed that shows amyloid (A) β and tau neuropathology, inflammation, oxidative stress, neuronal degeneration, and neurovascular deficits in an aging background. Currently available models are transgenic mice expressing amyloid precursor protein (APP) and presenilin mutations found in patients with early onset of AD. These mouse models are useful, but develop lesions at an early age, and none represent all the mechanisms representative of human AD. Ideally, the model should be easily manipulated so that dementia and neuropathology can be induced in an old-age animal as well as middle age, and young age to compare disease progression in different aging backgrounds. The animal of choice for large-scale drug testing is the mouse, but the rat could also be considered. There are advantages and disadvantages for both but our lab has extensive experience with aging mice so the aging mouse will be the prototype animal for this discussion. Aging in mice is in many ways similar to aging in people, so the geroscience concept is applicable, as depicted in Figure 1. The two hall-mark molecular pathologic changes of AD are accumulation of amyloid β 42 peptides (Aβ42) and paired helical filament (PHF)-tau (τ). Aβ42 pathology is not evenly distributed, but systematically localized to certain parts of the brain, following a prototypical sequence in which the regions are hierarchically involved: cortical Aβ42 deposits, followed by involvement of allocortical regions, involvement of subcortical forebrain regions and striatum, deposits in the brainstem and finally cerebellar Aβ42-deposition. The distribution of Aβ42 across the brain is part of the current NIA-AA consensus criteria for the neuropathologic diagnosis of AD [3]. To allow control over the extent of Aβ42 expression and better alignment with the clinical distribution of Aβ42, an ADmodel that is based on adeno-associated virus (AAV) mediated Aβ42 expression was developed and validated in adult rats [4]. This model overcomes the shortcomings of current transgenic models of AD pathology because it allows induction of pathology at a disease-relevant age and restricts pathology to injected brain regions selected based on their relevance to AD. While genetic data clearly support mutations in the APP gene as sufficient to produce AD, and extensive experimental evidence points to the neurotoxicity of Aβ peptides, it is now appreciated that cerebral cortical accumulation of fibrillar Aβ occurs in virtually all older adults and reaches maximal concentration in the preclinical or prodromal stages of AD prior to clinical expression of dementia. In contrast, AD dementia is closely associated with the extension of PHF-τ beyond mesial temporal lobe structures. The distribution of PHF-τ pathology in AD also follows a prototypical sequence with hierarchical involvement of the following regions: transentorhinal cortical layer, entorhinal layer and hippocampus, and isocortical PHF-τ containing tangles. Similar to Aβ42, the distribution pattern of PHF-τ is part of the current NIA-AA consensus criteria for the neuropathologic diagnosis of AD [3]. Therefore, we also include the formation of PHF-τ in AAV-based models of AD. Although there are no known associations between mutations in the gene encoding τ (MAPT) and AD, there are strong associations between mutations in MAPT and hereditary frontotemporal PATHOBIOLOGY OF AGING & AGE-RELATED DISEASES 2019, VOL. 9, 1616994 https://doi.org/10.1080/20010001.2019.1616994
Insulin resistance is a metabolic disorder that is highly prevalent in older populations. Mice expressing a truncated X-ray repair cross-complementing protein 1 (XRCC1tp) have normal repair of single-stranded breaks (SSBs) but are sensitive to alkylating agents. XRCC1tp mice thus provide a model to study perturbations in physiological function, such as metabolism, in the presence of normal DNA repair but attenuated XRCC1 activity. XRCC1tp male mice at six months of age fed a diet high in fat (lard) and sugar (sucrose) (HFSD) for three months showed a significant delay in glucose clearance, indicative of insulin resistance. These mice also had a decrease in respiratory exchange ratio, suggesting a change in the way fats and carbohydrates are used as a fuel source. Mechanisms for these observations are of interest, since there is a suggestion that XRCC1 is involved in glucoregulatory pathways, and XRCC1tp mice would provide an excellent model to pursue these studies in an age-related manner.
Aging intervention studies are hampered by the lack of predictive measures for determination of individuals at risk of age-associated chronic disease. Assessment of physical resilience could be informative in this regard, especially for age-related diseases such as cancer. Voluntary wheel running is a mildly stressful physical activity that is easily quantifiable in the mouse but has not been studied as a predictor of resistance to tumor invasiveness with increasing age. Male C57BL/6 mice in cohorts of 4, 12, 20, and 28 months of age were allowed access to a slanted in-cage running wheel for 3 days. Three months later, mice were injected subcutaneously with B16 melanoma tumor cells and followed for two weeks before harvesting. No relation was observed between running distance and tumor burden in the 4-month age group. The 12-month age group showed a trend, and the 20- and 28-month age groups showed a negative correlation (P < 0.05) between running distance and tumor burden. Mice in the 20-month age group that ran longer distances had lower tumor invasive scores compared to mice in the same age group that ran shorter distances. In conclusion, short term exercise capability could be a marker for resilience to cancer, and possibly other age-related disease conditions, in mice.
Metformin has beneficial effects on several age-related diseases (e.g., diabetes, obesity, cancer) and extends lifespan in nematodes and mice. Acarbose, an FDA-approved agent for treating type 2 diabetes, prevents breakdown of complex carbohydrates. Both compounds have been suggested as potential anti-aging interventions and acarbose has been shown to extend mouse longevity by the Intervention Testing Program (ITP). One potential next step is to assess the effect of these interventions on healthspan and lifespan in non-human primates. The common marmoset (Callithrix jacchus) is a small new world monkey with a relatively short life span and small size, both valuable for the translation potential of this nonhuman primate species for the study of aging and chronic disease. However, the dosing and assessment of potential side effects of either metformin or acarbose in this species have yet to be assessed. This study evaluated the pharmacokinetics of two dosage levels each of metformin or acarbose (given separately) in two small groups of young marmosets (n = 5/group) treated for 24 h to define the pharmacokinetics of each drug. The ability to rapidly and reliably dose socially housed marmosets with an oral form of acarbose or metformin that is well tolerated indicates that this species is a reliable model for testing acarbose and metformin in a safe and efficient way in a long-term intervention.
Hematologic analysis is an efficient and valuable tool for real-time health monitoring and immune analysis in mouse aging studies. However, many frequently used blood sampling techniques in mice are incompatible with continuous monitoring with increasing age, as they may involve anesthesia, cause severe stress, or require a high volume of blood. This technical report describes a convenient relatively noninvasive procedure for counting white blood cells in C57Bl/6 mice by an optimized tail blood collection method followed by Wright-Giemsa and Turk staining. This technique can be performed on unanesthetized mice in less than 1 min with minimal stress using only a few microliters of blood. White blood cell analysis can include but is not limited to total and differential white blood cell count and cell morphology. The low blood volume collected is ideal for aging mice in longer-term experiments requiring regular and continuous monitoring.
Rapamycin is an immunosuppressive and anti-cancer drug recently shown to enhance healthy aging in animal models. Regular physical exercise is associated with healthy aging and reduced risk of age-related diseases, such as cancer. In order to test the combined effect of these approaches, mice with 4T1 breast cancer were fed rapamycin at 14 ppm and allowed access to voluntary running wheels. After 17 days of treatment, mice fed the rapamycin diet that ran showed a significant increase in tumor burden compared with mice that did not run (P = 0.017). Not only does this have implications for young breast cancer patients, but suggests that combining rapamycin and exercise as an anti-aging strategy at a young age might be contraindicated.
We are pleased to announce that Pathobiology of Aging and Age-related Diseases is now the official journal of the Geropathology Research Network (GRN). In this capacity, the GRN will use the journal to publish workshop and conference proceedings, as well as technical articles related to pathological assessment of age-related lesions in rodents, other laboratory animals, pet dogs and cats, other domestic animals, nonhuman primates, and humans. The GRN has an active working committee, designated as the Geropathology Grading Committee, focusing on the establishment of guidelines for grading severity of lesions in tissues collected from mice in genetic or pharmacological aging intervention studies. In addition, Lesion of the Month and Geropathology in practice sections will be added to the journal. It is anticipated that these additions will be of interest, and help generate comments and feedback, as well as provide an increased awareness of the critical role geropathology plays in aging research, especially in preclinical studies, but in clinical studies as well. In addition to an active lesion grading committee, the GRN has an active molecular pathology working group focusing on identifying and characterizing secretory proteins associated with aging and age-related diseases, especially neurological. The group, designated as the Geropathology Secretome Committee, will be contributing technical as well as scientific articles for publication. Lastly, as the result of extensive tissue collection from rodents in aging studies in the last several years, the GRN has established a Geropathology Rodent Tissue Bank. This is just now getting started and will be ready for requests in the coming months from the scientific community. The journal will publish links for the tissue bank database, and periodic updates for issues of interest. We look forward to these relevant and interesting contributions. PATHOBIOLOGY OF AGING & AGE-RELATED DISEASES 2019, VOL. 9, 1593786 https://doi.org/10.1080/20010001.2019.1593786
We examined the effects of continuous overexpression of thioredoxin (Trx) 1 on aging in Trx1 transgenic mice [Tg(TXN)(+/0)]. This study was conducted to test whether increased thioredoxin expression over the lifespan in mice would alter aging and age-related pathology because our previous study demonstrated that Tg(act-TXN)(+/0) mice had no significant maximum life extension, possibly due to the use of actin as a promoter, which may have resulted in loss of Trx1 overexpression during aging. To test this hypothesis, we generated new Trx1 transgenic mice using a fragment of the human genome containing the TXN gene with an endogenous promoter to ensure continuous overexpression of Trx1 throughout the lifespan. Universal overexpression of Trx1 was observed, and Trx1 overexpression was maintained during aging (up to 22-24months old) in the Tg(TXN)(+/0) mice. The levels of Trx1 are significantly higher (approximately 4 to 31 fold) in all of the tissues examined in the Tg(TXN)(+/0) mice compared to the wild-type (WT) littermates. The overexpression of Trx1 did not cause any changes in the levels of Trx2, glutaredoxin, glutathione, or other major antioxidant enzymes. The survival study demonstrated that male Tg(TXN)(+/0) mice slightly extended the earlier part of the lifespan compared to WT littermates, but no significant life extension was observed over the lifespan. The cross-sectional pathological analysis (22-25months old) showed that Tg(TXN)(+/0) mice had a significantly higher severity of lymphoma and more tumor burden than WT mice, which was associated with the suppression of the apoptosis signal-regulating kinase 1 (ASK1) pathway. Our findings suggest that the increased levels of Trx1 over the lifespan in Tg(TXN)(+/0) mice showed some beneficial effects (slight extension of lifespan) in the earlier part of life but had no significant effects on median or maximum lifespans, and increased Trx1 levels enhanced tumor development in old mice.
The prevalence of Alzheimer's disease (AD) is expected to dramatically increase in older people worldwide. Efforts to find disease-modifying treatments have been largely unsuccessful because of the focus on disease-specific pathogenesis, and lack of animal models to study AD in the context of aging and age-related co-morbidities. The geroscience approach to studying AD would suggest that modulation of aging per se would be a useful strategy, but a mammalian model system that combines both aging and AD is not available. One approach to study old age and AD is to utilize murine models of progeroid syndrome, which can provide a number of advantages not only for basic aging biology but also for preclinical drug testing. A progeria background, such as the Ercc1 mutant mouse (Ercc1-/Δ ), provides an aging component not seen in current murine models of AD that lack age-related co-morbidities typical of AD patients. Ercc1-/Δ mice experience the same types of stochastic endogenous DNA damage as WT mice, but accumulate lesions faster due to impaired DNA repair, which accelerates the normal aging process by 6-fold. These mice do not show frank AD pathology but represent a predisposed or hypersensitive environment for AD pathology, where pathogenic elements of AD can be introduced, either by crossing with well-established AD transgenic mouse lines, or transcranial stereotaxic delivery directly into the brain. Since Ercc1-/Δ mice age five to six times faster than WT mice, very rapid characterization and testing of therapeutic interventions is possible. Studies are urgently needed to capitalize on the highly informative potential of this novel AD mouse model.
Scanning acoustic microscopy (SAM) can assess tissue stiffness by calculating the speed of sound (SOS) through tissues. SOS increases as tissue stiffness increases. Sensitivity to protease digestion depends on protein type, concentration, and modification. We analyzed the SOS images of formalin-fixed paraffin-embedded skin sections from elderly, young, diabetic, and nondiabetic subjects, as well as chronic and acute wounds. SAM provided high-resolution histology similar to LM and revealed characteristic SOS alteration following pepsin treatment. SOS values of dermis samples from elderly subjects (especially females) were lower than those of younger adults, which was indicative of age-related dermal softening and loosening. SOS values of elderly females were lower than those of younger females and elderly males. Dermal SOS showed a positive correlation with epidermal thickness. SOS values of epidermis of elderly subjects were higher than those of younger adults and showed a rapid decline 0.5h after protease digestion. Reticular dermis of diabetic patients exhibited greater pepsin resistance than that of nondiabetic patients. Chronic wounds exhibited greater SOS values and pepsin resistance than acute wounds. SOS variation with aging, diabetes mellitus, and wound fibrosis reflected histological and mechanical changes associated with senescence and disease duration. Epidermal thickness reflects age-related changes in dermal stiffness.
Photoreceptors and their supporting retinal pigment epithelium constitute the key functional parts of the retina. Here, a study was undertaken to show how aging and lifestyle factors affect the photoreceptor layer and the retinal pigment epithelium and Bruch's membrane complex (RPE-BM) in vivo in a healthy Danish population using spectral-domain optical coherence tomography. This was a cross-sectional study of healthy humans aged ≥50 years. All participants were interviewed for medical history and lifestyle factors. Maculae of all participants were scanned using spectral-domain optical coherence tomography. The thickness of the photoreceptor layer and the RPE-BM was measured on one eye from each participant. In 150 eyes of 150 participants, it was found that aging was associated with a decrease in the thickness of the photoreceptor layer (-0.143 μm/year, P = 0.031) and an increase in the thickness of the RPE-BM layer (0.100 μm/year, P = 0.029) at the foveal minimum. Regarding lifestyle factors, alcohol intake or BMI were not associated with any significant trend, but physical inactivity and smoking had effects on the photoreceptor layer (decreased thickness) and the RPE-BM layer (increased thickness) indicating an accelerated aging process of the macula. Taken together, aging affects photoreceptors and the RPE-BM, and these aging trends are accelerated in smokers and the physically inactive.
Considerable evidence suggests that oxidative stress plays a role in the pathogenesis of Parkinson's disease (PD), the most prevalent neurodegenerative movement disorder. Reduced expression of aldehyde dehydrogenase-1 (ALDH1) and glutathione peroxidase-1 (GPX1), enzymes that function to detoxify aldehydes and hydroxyl radicals, respectively, has been reported in the substantia nigra of patients who died with PD. To determine whether deficiency in these two genes contributes to the pathogenesis of PD, mice were generated with homozygous null mutations of both Aldh1a1 (the murine homolog of ALDH1) and Gpx1 genes [knockout (KO) mice]. At 6 and 18 months of age, KO mice showed a significantly decreased latency to fall in the automated accelerating rotarod test and increased time to complete the pole test opamine levels were not altered; however, the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and the DOPAC/dopamine ratio were significantly reduced at 18 months of age. Proteins adducted with 4-hydroxynonenal, the end-product of lipid peroxidation, were increased in the. midbrain and striatum of KO mice at 6 and 18 months. In conclusion, dual mutations in Gpx1 and Aldh1a1 genes are associated with motor deficits and increased lipid peroxidation in adult mice.
Pulmonary inflammation and the development of spontaneous lung tumors are important age-related lesions in mice. Therefore, gross and histological examination of the respiratory system is a critical component of geropathology research studies for translating surrogate endpoints to clinical aging studies. Collection, trimming, and processing of lung tissue from aged mice require a high-quality sequential process since aged mice are irreplaceable resource-intensive animal models. This protocol provides a basic technique that provides excellent sections for histological evaluation of the respiratory system of old mice suitable for most research applications. The points of emphasis are infusing the lungs at necropsy with formalin through the airways to prevent atelectasis artifacts that can preclude accurate histological evaluation, and embedding of anatomically oriented in toto lung lobes to allow for complete and thorough evaluation of all lung regions.
Investigations into pharmaceutical intervention of pancreatic and hepatic dysfunction associated with metabolic disturbances have received relatively little attention. The aim of this study was to investigate the protective effects of exendin-4 in mice receiving D-galactose, a reducing sugar that triggers ROS production and inflammatory mediators affecting the pancreas and liver. Exendin-4 is an United States Food and Drug Administration (FDA) approved glucagon-like peptide that increases insulin dependent glycogen synthesis and glucose uptake. Male NMRI mice (20-25 g), 3 months of age, were randomly divided into 6 groups of 12 mice each: control, exendin-4 (1 nmol/kg), exendin-4 (10 nmol/kg), D-galactose, D-galactose + exendin-4 (1 nmol/kg) and D-galactose + exendin-4 (10 nmol/kg). D-galactose (500 mg/kg) was given daily by oral gavage for 6 weeks. During the last 10 days, exendin-4 (1 and 10 nmol/kg) was injected intraperitoneally daily. Glucose, insulin, insulin resistance, lipid profiles, and hepatic enzyme levels significantly increased in the D-galactose group (p < 0.05), along with a significant decrease in superoxide dismutase activity and pancreatic islet insulin secretion (p < 0.05). Exendin-4 decreased D-galactose-induced increases in serum glucose and insulin, insulin resistance, lipid profiles, and hepatic enzymes, and improved pancreatic islet insulin secretion and antioxidant defense status. The results show that exendin-4 can prevent complications in mice with compromised pancreatic and hepatic function. Long term administration of D-galactose in mice may be a useful model to study insulin resistance, metabolic syndrome, and aging.
Physical resilience is the ability of an organism to respond to stressors that acutely disrupt normal physiological homeostasis. By definition, resilience decreases with increasing age, while frailty, defined as a decline in tissue function, increases with increasing age. Assessment of resilience could therefore be an informative early paradigm to predict healthy aging compared to frailty, which measures late life dysfunction. Parameters for resilience in the laboratory mouse are not yet well defined, and no single standardized stress test exists. Since aging involves multiple genetic pathways, integrative responses involving multiple tissues, organs, and activities need to be measured to reveal the overall resilience status, suggesting a battery of stress tests, rather than a single all-encompassing one, would be most informative. Three simple, reliable, and inexpensive stressors are described in this review that could be used as a panel to determine levels of resilience. Brief cold water immersion allows a recovery time to normothermia as an indicator of resilience to hypothermia, i.e. the quicker the return to normal body temperature, the more robust the resilience. Sleep deprivation (SD) impairs remote memory in aged mice, and has detrimental effects on glucose metabolism. Cyclophosphamide (CYP) targets white blood cells, especially myeloid cells resulting in neutropenia with a rebound neutrophilia in an age-dependent manner. Thus a strong neutrophilic response indicates resilience. In conclusion, resilience promises to be an especially useful measurement of biological age, i.e. how fast a particular organ or tissue ages. The three stressors, cold, SD, and CYP, are applicable to human medicine and aging because they represent clinically relevant stress conditions that have effects in an age-dependent manner. They are thus an attractive perturbation for resilience testing in mice to measure the effectiveness of interventions that target basic aging processes.
Background Inhibition of mechanistic target of rapamycin (mTOR) has emerged as a viable means to lengthen lifespan and healthspan in mice, although it is still unclear whether these benefits will extend to other mammalian species. We previously reported results from a pilot experiment wherein common marmosets (Callithrix jacchus) were treated orally with rapamycin to reduce mTOR signaling in vivo in line with previous reports in mice and humans. Further, long-term treatment did not significantly alter body weight, daily activity, blood lipid concentrations, or glucose metabolism in this cohort. Methods In this study, we report on the molecular consequences of rapamycin treatment in marmosets on mechanisms that regulate protein homeostasis (proteostasis) in vivo. There is growing appreciation for the role of proteostasis in longevity and for the role that mTOR plays in regulating this process. Tissue samples of liver and skeletal muscle from marmosets in our pilot cohort were assessed for expression and activity of components of the ubiquitin-proteasome system, macroautophagy, and protein chaperones. Results Rapamycin treatment was associated with increased expression of PSMB5, a core subunit of the 20S proteasome, but not PSMB8 which is involved in the formation of the immunoproteasome, in the skeletal muscle and liver. Surprisingly, proteasome activity measured in these tissues was not affected by rapamycin. Rapamycin treatment was associated with an increased expression of mitochondria-targeted protein chaperones in skeletal muscle, but not liver. Finally, autophagy was increased in skeletal muscle and adipose, but not liver, from rapamycin-treated marmosets. Conclusions Overall, these data show tissue-specific upregulation of some, but not all, components of the proteostasis network in common marmosets treated with a pharmaceutical inhibitor of mTOR.
