Hippocampal Extracellular Matrix Levels and Stochasticity in Synaptic Protein Expression Increase with Age and Are Associated with Age-dependent Cognitive Decline

Age-related cognitive decline is a serious health concern in our aging society. Decreased cognitive function observed during healthy brain aging is most likely caused by changes in brain connectivity and synaptic dysfunction in particular brain regions. Here we show that aged C57BL/6J wild-type mice have hippocampus-dependent spatial memory impairments. To identify the molecular mechanisms that are relevant to these memory deficits, we investigated the temporal profile of mouse hippocampal synaptic proteome changes at 20, 40, 50, 60, 70, 80, 90, and 100 weeks of age. Extracellular matrix proteins were the only group of proteins that showed robust and progressive up-regulation over time. This was confirmed by immunoblotting and histochemical analysis, which indicated that the increased levels of hippocampal extracellular matrix might limit synaptic plasticity as a potential cause of age-related cognitive decline. In addition, we observed that stochasticity in synaptic protein expression increased with age, in particular for proteins that were previously linked with various neurodegenerative diseases, whereas low variance in expression was observed for proteins that play a basal role in neuronal function and synaptic neurotransmission. Together, our findings show that both specific changes and increased variance in synaptic protein expression are associated with aging and may underlie reduced synaptic plasticity and impaired cognitive performance in old age.As the proportion of aged individuals in our population continues to grow, we are faced with an increase in age-related health problems. Brain aging invariably leads to functional decline and impairments in cognitive function and motor skills, which can seriously affect quality of life. A better understanding of the neurobiological mechanisms underlying age-related cognitive decline is crucial to facilitate maintenance of cognitive health in the elderly and to reveal potential causes of highly prevalent age-related forms of dementia, in particular Alzheimer disease, in which cognitive decline is severely impaired by yet unknown mechanisms.Several studies showed that normal brain aging is associated with subtle morphological and functional alterations in specific neuronal circuits (1, 2) and that reduced cognitive function with increasing age is likely due to synaptic dysfunction (3). Increasing evidence supports the idea that alterations in hippocampal activity are correlated with deficits in learning and memory in healthy aging humans (4, 5). In addition, rodent models of healthy aging demonstrate strong correlations between impaired performance in learning and memory tests and disturbed hippocampal network activity (6, 7). Electrophysiological studies provide additional evidence that age-related disturbances in the hippocampus involve changes in the principal cellular features of learning and memory, synaptic long-term potentiation and long-term depression (8, 9). Together, these observations suggest that a decline in hippocampal synaptic efficacy and plasticity plays a critical role in age-dependent cognitive impairment.Aging is also the primary risk factor for Alzheimer disease, which clinically manifests as severe and accelerated age-dependent cognitive decline (10). Genetic causes of familial early-onset Alzheimer disease all point to a key role in disease etiology for increased brain levels of the protein amyloid-β (11). Familial Alzheimer disease, however, is rare, and it is likely that increased amyloid-β levels in sporadic Alzheimer disease result from age-dependent and/or genetically determined alterations in the expression of other genes or proteins (12, 13). Thus, the identification of molecular mechanisms of normal brain aging might also contribute to our understanding of cognitive decline under pathological conditions, in particular in Alzheimer disease.Although the exact mechanisms underlying brain aging remain to be fully determined, they likely include changes at the molecular, cellular, and neuronal-network levels. In particular, characterization of alterations in the molecular composition and dynamics of hippocampal synapses could potentially reveal important aspects of the underlying mechanisms of brain aging. Age-related changes in global hippocampal gene and protein expression have been investigated previously (14, 15), but these studies were not geared to identify the specific synaptic molecular substrates of brain aging. Here, we made use of iTRAQ¹ technology and high-coverage mass spectrometry to study the effects of aging on the proteomic composition of mouse hippocampal synaptosomes. We investigated the synaptic proteomes of individual mice at 20, 40, 50, 60, 70, 80, 90, and 100 weeks of age. Our findings show that both specific changes and increased variance in synaptic protein expression are associated with age-related cognitive decline.     

Gene Regulation in Primates Evolves under Tissue-Specific Selection Pressures

Regulatory changes have long been hypothesized to play an important role in primate evolution. To identify adaptive regulatory changes in humans, we performed a genome-wide survey for genes in which regulation has likely evolved under natural selection. To do so, we used a multi-species microarray to measure gene expression levels in livers, kidneys, and hearts from six humans, chimpanzees, and rhesus macaques. This comparative gene expression data allowed us to identify a large number of genes, as well as specific pathways, whose inter-species expression profiles are consistent with the action of stabilizing or directional selection on gene regulation. Among the latter set, we found an enrichment of genes involved in metabolic pathways, consistent with the hypothesis that shifts in diet underlie many regulatory adaptations in humans. In addition, we found evidence for tissue-specific selection pressures, as well as lower rates of protein evolution for genes in which regulation evolves under natural selection. These observations are consistent with the notion that adaptive circumscribed changes in gene regulation have fewer deleterious pleiotropic effects compared with changes at the protein sequence level.     

Synaptic proteome changes in a DNA repair deficient ercc1 mouse model of accelerated aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline.     

Necroptosis inhibition counteracts neurodegeneration,memory decline,and key hallmarks of aging,promoting brain rejuvenation

Age is the main risk factor for the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction, and cognitive disabilities in humans, primates, rodents, and invertebrates. Necroptosis mediates degeneration of injured axons, but whether necroptosis triggers neurodegeneration and cognitive impairment along aging is unknown. Here, we show that the loss of the necroptotic effector Mlkl was sufficient to delay age-associated axonal degeneration and neuroinflammation, protecting against decreased synaptic transmission and memory decline in aged mice. Moreover, short-term pharmacologic inhibition of necroptosis targeting RIPK3 in aged mice, reverted structural and functional hippocampal impairment, both at the electrophysiological and behavioral level. Finally, a quantitative proteomic analysis revealed that necroptosis inhibition leads to an overall improvement of the aged hippocampal proteome, including a subclass of molecular biofunctions associated with brain rejuvenation, such as long-term potentiation and synaptic plasticity. Our results demonstrate that necroptosis contributes to age-dependent brain degeneration, disturbing hippocampal neuronal connectivity, and cognitive function. Therefore, necroptosis inhibition constitutes a potential geroprotective strategy to treat age-related disabilities associated with memory impairment and cognitive decline.     

Time-course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging

The study of aging is critical for a better understanding of many age-related diseases. The free radical theory of aging, one of the prominent aging hypotheses, holds that during aging, increasing reactive oxygen species in mitochondria causes mutations in the mitochondrial DNA and damages mitochondrial components, resulting in senescence. Understanding a mitochondrial gene expression profile and its relationship to mitochondrial function becomes an important step in understanding aging. The objective of the present study was to determine mRNA expression of mitochondrial-encoded genes in brain slices from C57BL6 mice at four ages (2, 12, 18, and 24 months) and to determine how these altered mitochondrial genes influence age-related changes, including oxidative damage and cytochrome c in apoptosis. Using northern blot analysis, in situ hybridization, and immunofluorescence analyses, we analyzed changes in the expression of mitochondrial RNA encoding the mitochondrial genes, oxidative damage marker, 8-hydroxyguanosine (8-OHG), and cytochrome c in brain slices from the cortex of C57BL6 mice at each of the four ages. Our northern blot analysis revealed an increased expression of mitochondrial-encoded genes in complexes I, III, IV, and V of the respiratory chain in 12- and 18-month-old C57BL6 mice compared to 2-month-old mice, suggesting a compensatory mechanism that allows the production of proteins involved in the electron transport chain. In contrast to the up-regulation of mitochondrial genes in 12- and 18-month-old C57BL6 mice, mRNA expression in 24-month-old C57BL6 mice was decreased, suggesting that compensation maintained by the up-regulated genes cannot be sustained and that the down-regulation of expression results in the later stage of aging. Our in situ hybridization analyses of mitochondrial genes from the hippocampus and the cortex revealed that mitochondrial genes were over-expressed, suggesting that these brain areas are critical for mitochondrial functions. Our immunofluorescence analysis of 8-OHG and cytochrome c revealed increased 8-OHG and cytochrome c in 12-month-old C57BL6 mice, suggesting that age-related mitochondrial oxidative damage and apoptosis are associated with mitochondrial dysfunction. Our double-labeling analysis of in situ hybridization of ATPase 6 and our immunofluorescence analysis of 8-OHG suggest that specific neuronal populations undergo oxidative damage. Further, double-labeling analysis of in situ hybridization of ATPase 6 and immunofluorescence analysis of cytochrome c suggest cytochrome c release is related to mitochondrial dysfunction in the aging C57BL6 mouse brain. This study also suggests that these mitochondrial gene expression changes may relate to the role of mitochondrial dysfunction, oxidative damage, and cytochrome c in aging and in age-related diseases such as Alzheimer's disease and Parkinson's disease.     

The aging brain: protein phosphorylation as a target of changes in neuronal function.

There is evidence that senescence affects neurotransmission at different levels. In particular, this review summarizes the studies on age-dependent modifications in protein phosphorylation, which represents the final pathway in the action of transmitters and hormones at neuronal level. Cyclic AMP-dependent protein kinase and protein kinase C have been reported to be modified during aging in various cerebral areas; the changes may involve either enzyme activity or substrate availability. These findings can be related to the alterations in neurotransmitter function and synaptic efficiency observed in the senescent brain. The activity of the other types of protein kinases (tyrosine-, cGMP-, calcium/calmodulin-dependent) during aging needs to be explored. An emerging point is the role of protein phosphorylation in the transfer of membrane signals to the nucleus, for the activation or disactivation of specific genes responsible for long-term neuronal events. Along this view, alterations in protein kinase pathway during senescence would ultimately affect gene expression, resulting in long term modifications of cell function. The reviewed literature opens the perspective of restoring some of the deficits associated with senescence by modulating protein phosphorylation pathway.     

Gene expression profile of oxidant stress and neurodegeneration in transgenic mice deficient in alpha-tocopherol transfer protein

Alpha-tocopherol transfer protein (TTP) regulates the retention and secretion of alpha-tocopherol (alpha-T) by the liver. Deletion of the TTP gene (Ttpa) in mice results in systemic deficiency of alpha-T and neurological dysfunctions described in patients with mutated Ttpa. We have explored genome-wide changes in mRNAs from brain cortex and liver of Ttpa-deficient (Ttpa(-/-)) mice and wild-type (Ttpa(+/+)) mice. Selective inductions of genes regulated by antioxidant response elements were detected in Ttpa(-/-) livers compared to Ttpa(+/+) livers, suggesting increased oxidant stress in Ttpa(-/-) livers. The activation of cell proliferation pathways in Ttpa(-/-) livers was indicated by the induction of genes that encode growth factor-binding proteins, mitogen-activated protein kinase kinase 3, and apoptosis inhibitor 6. The induction of synuclein-alpha and repression of synuclein-beta genes was detected in Ttpa(-/-) cortex. This may predispose Ttpa(-/-) cortex to increased formation of synuclein-alpha aggregates and Lewy body, often associated with oxidant stress. Cortex of Ttpa(-/-) mice revealed repression of genes encoding synaptic proteins, protein kinase C family members, and myelin proteins. A 13-fold decrease in the expression of retinoic acid receptor-related orphan receptor-alpha mRNA predicts staggerer-like phenotype (ataxia and deficits of motor coordination) of Ttpa(-/-) mice. The repression of specific genes that determine synaptic plasticity and neuronal development may account for suppressed electrophysiological activities of cortex and impaired behavior in Ttpa(-/-) mice.     

mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction

Sarcopenia is an age-associated loss of skeletal muscle mass and strength that increases the risk of disability. Calorie restriction (CR), the consumption of fewer calories while maintaining adequate nutrition, mitigates sarcopenia and many other age-related diseases. To identify potential mechanisms by which CR preserves skeletal muscle integrity during aging, we used mRNA-Seq for deep characterization of gene regulation and mRNA abundance in skeletal muscle of old mice compared with old mice subjected to CR. mRNA-Seq revealed complex CR-associated changes in expression of mRNA isoforms, many of which occur without a change in total message abundance and thus would not be detected by methods other than mRNA-Seq. Functional annotation of differentially expressed genes reveals CR-associated upregulation of pathways involved in energy metabolism and lipid biosynthesis, and downregulation of pathways mediating protein breakdown and oxidative stress, consistent with earlier microarray-based studies. CR-associated changes not noted in previous studies involved downregulation of genes controlling actin cytoskeletal structures and muscle development. These CR-associated changes reflect generally healthier muscle, consistent with CR's mitigation of sarcopenia. mRNA-Seq generates a rich picture of the changes in gene expression associated with CR, and may facilitate identification of genes that are primary mediators of CR's effects.     

Selective age-related changes in the PKC-sensitive, calmodulin-binding protein, neurogranin, in the mouse brain

Brain ageing is associated with a dysregulation of intracellular calcium (Ca(2+)) homeostasis which leads to deficits in Ca(2+)-dependent signalling pathways and altered neuronal functions. Given the crucial role of neurogranin/RC3 (Ng) in the post-synaptic regulation of Ca(2+) and calmodulin levels, age-dependent changes in the levels of Ng mRNA and protein expression were analysed in 3, 12, 24 and 31-month-old mouse brains. Ageing produced significant decreases in Ng mRNA expression in the dorsal hippocampal subfields, retrosplenial and primary motor cortices, whereas no reliable changes were seen in any other cortical regions examined. Western blot indicated that Ng protein expression was also down-regulated in the ageing mouse brain. Analysis of Ng immunoreactivity in both hippocampal CA1 and retrosplenial areas indicated that Ng protein in aged mice decreased predominantly in the dendritic segments of pyramidal neurones. These data suggest that age-related changes of post-synaptic Ng in selected brain areas, and particularly in hippocampus, may contribute to altered Ca(2+)/calmodulin-signalling pathways and to region-specific impairments of synaptic plasticity and cognitive decline.     

Magnetic resonance spectroscopy: an in vivo tool for monitoring cerebral injury in SIV-infected macaques

The purpose of our study was to demonstrate the feasibility of using in vivo proton Magnetic Resonance Spectroscopy (MRS) to monitor the brain manifestations of SIV infection in the macaque model of AIDS. Previous spectroscopy work on macaque brain tissue and in vivo work in humans is reviewed to provide the motivation and context for this study. We collected 34 MRS data sets on 14 uninfected rhesus macaques. From this data, we demonstrate that we are capable of detecting changes similar to those observed in human MRS studies for most metabolites using less than 10 animals. The juvenile macaques utilized in this study demonstrate age-related changes in the levels of N-acetyl aspartate (NAA), a neuronal marker. The quantity and distribution of neurochemicals in the macaque are found to be slightly, but significantly, different than in the human.     

Global expression profiles from C57BL/6J and DBA/2J mouse lungs to determine aging-related genes.

This study identified gene expression profiles that provided evidence for genomic mechanisms underlying the pathophysiology of aging lung. Aging lungs from C57BL/6 (B6) and DBA/2 (D2) mouse strains differ in physiology and morphometry. Lungs were harvested from B6 mice at 2, 18, and 26 mo and from D2 mice at 2 and 18 mo of age. Purified RNA was subjected to oligonucleotide microarray analyses, and differential expression analyses were performed for comparison of various data sets. A significant majority of differentially expressed genes were upregulated with aging in both strains. Aging D2 lungs uniquely exhibited upregulation in stress-response genes including xenobiotic detoxification cascades. In contrast, aging B6 lungs showed downregulation of heat shock-response genes. Age-dependent downregulation of genes common to both B6 and D2 strains included several collagen genes (e.g., Col1a1 and Col3a1). There was a greater elastin gene (Eln) expression in D2 mice at 2 mo, and Eln was uniquely downregulated with age in this strain. The matrix metalloproteinase 14 gene (Mmp14), critical to alveolar structural integrity, was also downregulated with aging in D2 mice only. Several polymorphisms in the regulatory and untranslated regions of Mmp14 were identified between strains, suggesting that variation in Mmp14 gene regulation contributes to accelerated aging of lungs in D2 mice. In summary, lungs of B6 and D2 mice age with variable rates at the gene expression level, and these quantifiable genomic differences provide a template for understanding the variability in age-dependent changes in lung structure and function.     

MicroRNA expression and regulation in human, chimpanzee, and macaque brains

Among other factors, changes in gene expression on the human evolutionary lineage have been suggested to play an important role in the establishment of human-specific phenotypes. However, the molecular mechanisms underlying these expression changes are largely unknown. Here, we have explored the role of microRNA (miRNA) in the regulation of gene expression divergence among adult humans, chimpanzees, and rhesus macaques, in two brain regions: prefrontal cortex and cerebellum. Using a combination of high-throughput sequencing, miRNA microarrays, and Q-PCR, we have shown that up to 11% of the 325 expressed miRNA diverged significantly between humans and chimpanzees and up to 31% between humans and macaques. Measuring mRNA and protein expression in human and chimpanzee brains, we found a significant inverse relationship between the miRNA and the target genes expression divergence, explaining 2%-4% of mRNA and 4%-6% of protein expression differences. Notably, miRNA showing human-specific expression localize in neurons and target genes that are involved in neural functions. Enrichment in neural functions, as well as miRNA-driven regulation on the human evolutionary lineage, was further confirmed by experimental validation of predicted miRNA targets in two neuroblastoma cell lines. Finally, we identified a signature of positive selection in the upstream region of one of the five miRNA with human-specific expression, miR-34c-5p. This suggests that miR-34c-5p expression change took place after the split of the human and the Neanderthal lineages and had adaptive significance. Taken together these results indicate that changes in miRNA expression might have contributed to evolution of human cognitive functions.     

Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila

Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. Here we demonstrate that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity.     

Changes in the structural complexity of the aged brain

Structural changes of neurons in the brain during aging are complex and not well understood. Neurons have significant homeostatic control of essential brain functions, including synaptic excitability, gene expression, and metabolic regulation. Any deviations from the norm can have severe consequences as seen in aging and injury. In this review, we present some of the structural adaptations that neurons undergo throughout normal and pathological aging and discuss their effects on electrophysiological properties and cognition. During aging, it is evident that neurons undergo morphological changes such as a reduction in the complexity of dendrite arborization and dendritic length. Spine numbers are also decreased, and because spines are the major sites for excitatory synapses, changes in their numbers could reflect a change in synaptic densities. This idea has been supported by studies that demonstrate a decrease in the overall frequency of spontaneous glutamate receptor-mediated excitatory responses, as well as a decrease in the levels of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and N-methyl-d-aspartate receptor expression. Other properties such as gamma-aminobutyric acid A receptor-mediated inhibitory responses and action potential firing rates are both significantly increased with age. These findings suggest that age-related neuronal dysfunction, which must underlie observed decline in cognitive function, probably involves a host of other subtle changes within the cortex that could include alterations in receptors, loss of dendrites, and spines and myelin dystrophy, as well as the alterations in synaptic transmission. Together these multiple alterations in the brain may constitute the substrate for age-related loss of cognitive function.