Influence of aging on chondroitin sulfate proteoglycan expression and neural stem/progenitor cells in rat brain and improving effects of a herbal medicine, yokukansan

There is evidence of structural and functional deterioration in the brain, including the prefrontal cortex (PFC) and hippocampus, during the normal aging process in animals and humans. Extracellular matrix-associated glycoproteins, such as chondroitin sulfate proteoglycans (CSPGs), are involved in not only maintaining the structures and functions of adult neurons, but also regulating the proliferation, migration, and neurite outgrowth of neural stem cells in the brain. On the other hand, a herbal medicine, yokukansan (YKS), is used in a variety of clinical situations for treating symptoms associated with age-related neurodegenerative disorders such as Alzheimer's disease, but its pharmacological properties have not been fully understood. The present study was designed to clarify the influence of aging and the improving effects of YKS on the expression of aggrecan, a major molecule of CSPGs, and on the proliferation and migration of neural stem/progenitor cells identified by bromodeoxyuridine (BrdU) incorporation in the PFC and hippocampus including the dentate gyrus. Aged rats (24 months old) showed a significant increase in aggrecan expression throughout the PFC and in the hippocampus particularly in the CA3 subfield, but not the dentate gyrus compared to young rats (5 months old), evaluated by the immunohistochemical method. YKS treatment decreased the age-related increase in aggrecan expression as well as normal expression in young rats. Aged rats also showed a decreased number of BrdU-labeled cells in the PFC and hippocampus, and these decreases were improved by YKS treatment, which also increased the numbers in young rats. These results suggest that aging influences the microenvironment for adult and immature neurons in the brain, which may affect the proliferation and migration of neural stem/progenitor cells, and YKS has pharmacological potency for these age-related events. These findings help to understand the physiology and pathology of the aged brain and provide an anti-aging strategy for the brain.     

Up-regulation of alpha1D Ca2+ channel subunit mRNA expression in the hippocampus of aged F344 rats

There is growing evidence that alterations in calcium (Ca2+) homeostasis may play a role in processes of brain aging and neurodegeneration. There also is evidence that some of the altered Ca2+ homeostasis in hippocampal neurons may arise from an increased density of L-type voltage sensitive Ca2+ channels (L-VSCC). In the present studies, we tested the possibility that previously observed increases in functional L-VSCC with aging might be related to up-regulated gene/mRNA expression for Ca2+ channel subunits. A significant aging-related increase in mRNA content for the alpha1D subunit of the L-type VSCC was observed in hippocampus of aged F344 rats (25 months old) relative to young (4 months old) and middle-aged animals (13 months old), as assessed by both in situ hybridization analyses (densitometry and grain density) and ribonuclease protection assay (RPA). In RPA analyses, the alpha1C subunit mRNA also showed a significant increase in 25-month-old rats. No age changes were seen in mRNA for the beta1b subunit of VSCC or for GAPDH, a standard control. The clearest increases in alpha1D mRNA expression were observed in subfield CA1, with little or no change seen in dentate gyrus. Although these results alone do not demonstrate that mRNA/gene expression changes contribute directly to changes in functional Ca2+ channels, they clearly fulfill an important prediction of that hypothesis. Therefore, these studies may have important implications for the role of gene expression in aging-dependent alterations in brain Ca2+ homeostasis.     

Regulation of iron-related molecules in the rat hippocampus: sex- and age-associated differences

Iron accumulation, especially that of free oxidized ferrous iron, has been shown to induce tissue oxidative damage and contribute to brain aging and the development of neurodegenerative disease. Here we examine whether sex and advanced age affect the expression of iron-related molecules that participate in regulating free iron levels (heme oxygenase 1 (HO1), iron-regulatory protein 1 (IRP1), and ferritin heavy chain (FTH)) and whether changes in the expression of these molecules are associated with differences in the expression of alpha-synuclein (ASN) which is thought to be a critical regulator in the pathogenesis of neurodegeneration. Using a well-established aging animal model, we demonstrate that the expression of HO1, FTH, and IRP1 mRNAs is higher in the female hippocampus than that observed in male Fischer 344/NNiaHSD x Brown Norway/BiNia (F344BN) rats, regardless of age group. Consistent with these sex-associated alterations in iron-related regulators, the expression of ASN mRNA and protein in the female hippocampus was lower than that found in male rats. These results suggest a sex-dependent difference in regulating the expression of molecules involved in iron metabolism and neurodegeneration. A similar finding in humans, if present, may help to shed light on why sex may affect the incidence of neurodegenerative disorders.     

Stereological Investigation of the Age‐Related Changes of the Myelinated Fibers in the Hippocampus of Male Rats

The decline of hippocampus‐dependent learning and memory during normal aging is not associated with neuron death and synapse loss. Until now, age‐related changes in the myelinated fibers of the hippocampus have not been investigated. Therefore, in this study, the myelinated fibers in the hippocampi of young (6 months), middle‐aged (18 months), and old‐aged (28 months) male Sprague‐Dawley rats were studied with transmission electron microscope and stereological methods, following spatial learning tests in a Morris water maze. The results showed that hippocampus‐dependent spatial learning was impaired in old‐aged rats but that the total volume, length, and mean diameter of the myelinated fibers in the hippocampus, as well as the hippocampal volume, remained constant during the normal aging process. Our results suggest that the age‐related decline in hippocampus‐dependent spatial learning is not attributable to myelinated fiber changes in the hippocampus and that other, undetermined factors are responsible. Anat Rec, 297:1490–1497, 2014. © 2014 Wiley Periodicals, Inc.     

Aging affects transcranial magnetic modulation of hippocampal evoked potentials

Transcranial magnetic stimulation (TMS) is being proposed as a method of choice for the treatment of clinical depression, yet its action in the brain is still not well understood. In previous studies we found that TMS has a long-term effect on reactivity of the hippocampus to perforant path stimulation. Since the efficacy of antidepressants is highly age-dependent, we studied possible age-related effects of TMS on hippocampal evoked responses. Young adult (3 months), aging (10 months) and aged (24-26 months) awake rats were subjected to daily TMS for one week, followed by measurements of several parameters of reactivity to perforant path stimulation in the anesthetized rat. TMS did not affect responses of the hippocampus to single perforant path stimulation, but reduced drastically paired-pulse and frequency dependent depression in the young and aging but not the old rats. Likewise, TMS increased LTP expression in the young but not the old rats, and reduced the efficacy of serotonin modulation of reactivity of the hippocampus, in the young but not the old rats. Thus, long term effects of chronic TMS on local GABAergic inhibition are highly age dependent.     

Aging-related alterations in the expression and distribution of GluR2 and PICK1 in the rat hippocampus

Deficit in synaptic plasticity in the hippocampus frequently occurs during normal aging. Although the protein level and calcium permeability of AMPARs alter with aging, the alteration of AMPARs and their regulatory proteins during aging are far from understanding. Dynamics of GluR2 subunit are dependent on the function of protein interacting with Cα kinase 1 (PICK1), PKCα and calcineurin (CaN). Here, we firstly show that the expression of PICK1 and CaN B decreased significantly in the hippocampus of old rats compared to that of young and adult rats. The decrease was accompanied by a reduction of GluR2 and PKCα and an increase in CaN A. Next, we found that in young and adult rats, the distribution of PICK1 and GluR2 diffused in the cytoplasm of hippocampal neurons, but closely around perinuclear in the hippocampal neurons of old rats. These results suggest that the expression of GluR2, PICK1, PKCα and CaN B significant decreased in the hippocampus and these alterations may lead to altered distribution of GluR2 and PICK1 during aging.     

老年大鼠血液白细胞基因表达谱的生物信息学分析

目的了解老年大鼠血液白细胞基因表达水平的变化。方法将雄性Wister大鼠分为老年组（18月龄）和年轻组（4月龄），用illumina大鼠全基因组芯片研究其分子水平的变化。结果生物信息学分析显示．造成老年组和年轻组显著生物学差异的基因功能及所属基因是：28条凋亡基因和22条炎性基因上调，9条涉及氧与活性氧中间物代谢的基因下调。结论差异基因功能导致老年大鼠和年轻大鼠的生物学差异。     

Increased expression of heat shock proteins in rat brain during aging: relationship with mitochondrial function and glutathione redox state

It is generally recognized that lipid peroxides play an important role in the pathogenesis of several diseases and that sulfhydryl groups are critically involved in cellular defense against endogenous or exogenous oxidants. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Oxidative damage plays a crucial role in the brain aging process and induction of Hsps is critically utilized by brain cells in the repair process following various pathogenic insults. In the present study, we investigated, in rats 6, 12, and 28 months old, the role of heat shock expression on aging-induced changes in mitochondrial and antioxidant redox status. In the brain expression of Hsp72 and Hsc70 increased with age up to 28 months; at this age the maximum induction was observed in the hippocampus and substantia nigra followed by cerebellum, cortex, septum and striatum. Hsps induction was associated with significant changes in glutathione (GSH) redox state and HNE levels. Interestingly, a significant positive correlation between decrease in GSH and increase in Hsp72 was observed in all brain regions examined during aging. Analysis of mitochondrial complexes showed a progressive decrease of Complex I activity and mRNA expression in the hippocampus and a significant decrease of Complex I and IV activities in the substantia nigra and septum. Our results sustain a role for GSH redox state in Hsp expression. Increase of Hsp expression promotes the functional recovery of oxidatively damaged proteins and protects cells from progressive age-related cell damage. Conceivably, heat shock signal pathway by increasing cellular stress resistance may represent a crucial mechanism of defence against free radical-induced damage occurring in aging brain and in neurodegenerative disorders.     

Up-regulation of α1D Ca channel subunit mRNA expression in the hippocampus of aged F344 rats

There is growing evidence that alterations in calcium (Ca²⁺) homeostasis may play a role in processes of brain aging and neurodegeneration. There also is evidence that some of the altered Ca²⁺ homeostasis in hippocampal neurons may arise from an increased density of L-type voltage sensitive Ca²⁺ channels (L-VSCC). In the present studies, we tested the possibility that previously observed increases in functional L-VSCC with aging might be related to up-regulated gene/mRNA expression for Ca²⁺ channel subunits. A significant aging-related increase in mRNA content for the α_1D subunit of the L-type VSCC was observed in hippocampus of aged F344 rats (25 months old) relative to young (4 months old) and middle-aged animals (13 months old), as assessed by both in situ hybridization analyses (densitometry and grain density) and ribonuclease protection assay (RPA). In RPA analyses, the α_1C subunit mRNA also showed a significant increase in 25-month-old rats. No age changes were seen in mRNA for the β_1b subunit of VSCC or for GAPDH, a standard control. The clearest increases in α_1D mRNA expression were observed in subfield CA1, with little or no change seen in dentate gyrus. Although these results alone do not demonstrate that mRNA/gene expression changes contribute directly to changes in functional Ca²⁺ channels, they clearly fulfill an important prediction of that hypothesis. Therefore, these studies may have important implications for the role of gene expression in aging-dependent alterations in brain Ca²⁺ homeostasis.     

The length of hippocampal cholinergic fibers is reduced in the aging brain

Cholinergic deficits occur in the aged hippocampus and they are significant in Alzheimer's disease. Using stereological and biochemical approaches, we characterized the cholinergic septohippocampal pathway in old (24 months) and young adult (3 months) rats. The total length of choline acetyltransferase (ChAT)-positive fibers in the dorsal hippocampus was significantly decreased by 32% with aging (F((1,9))=20.94, p=0.0014), along with the levels of synaptophysin, a presynaptic marker. No significant changes were detected in ChAT activity or in the amounts of ChAT protein, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin related kinase receptor (Trk) A, TrkB, or p75 neurotrophin receptor (p75(NTR)) in the aged dorsal hippocampus. The number and size of ChAT-positive neurons and the levels of ChAT activity, NGF and BDNF were not statistically different in the septum of aged and young adult rats. This study suggests that substantial synaptic loss and cholinergic axonal degeneration occurs during aging and reinforces the importance of therapies that can protect axons and promote their growth in order to restore cholinergic neurotransmission.     

Dopamine neurotoxicity: age-dependent behavioral and histological effects

The oxidative stress (OS) theory has implicated the involvement of reactive oxygen species (ROS) in both aging and age-dependent neurodegenerative diseases. The dopaminergic system is particularly vulnerable to ROS, and dopamine (DA) itself can be an endogenous source of ROS. The present study evaluated the hypothesis that DA-induced toxicity is age-dependent, and tested the behavioral and histological correlates of DA neurotoxicity in aging. Young (6 months) and middle-aged (15 months) rats were chronically treated with DA in the substantia nigra (SN, 1 micromol/2 microl vehicle per side/day/5 days) and were subsequently examined for changes in motor function and histology. The neurotoxic effect of DA treatment was an age-dependent effect, as middle-aged animals that received DA infusions in the SN were more impaired than their age-matched controls, especially on tasks that involved greater sensory-motor coordination, whereas young animals that received DA behaved similarly to their age-matched controls. The behavioral effects noted were accompanied by a loss of the tyrosine hydroxylase phenotype in substantia nigra. However, selective neurodegeneration was not noted in the SN of the treated animals, nor was a selective iron deposition noted at the site of injection. These results suggest that a neurochemical deficit and not cell loss per se within the nigrostriatal system underlies the motor behavioral deficits observed in the middle-aged rats.     

Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice

The brain is susceptible to oxidative stress, which is associated with age-related brain dysfunction, because of its high content of peroxidizable unsaturated fatty acids, high oxygen consumption per unit weight, high content of key components for oxidative damage, and the relative scarcity of antioxidant defense systems. Protein oxidation, which results in functional disruption, is not random but appears to be associated with increased oxidation in specific proteins. By using a proteomics approach, we have compared the protein levels and specific protein carbonyl levels, an index of oxidative damage in the brains of old mice, to these parameters in the brains of young mice and have identified specific proteins that are altered as a function of aging. We show here that the expression levels of dihydropyrimidinase-like 2 (DRP2), alpha-enolase (ENO1), dynamin-1 (DNM1), and lactate dehydrogenase 2 (LDH2) were significantly increased in the brains of old versus young mice; the expression levels of three unidentified proteins were significantly decreased. The specific carbonyl levels of beta-actin (ACTB), glutamine synthase (GS), and neurofilament 66 (NF-66) as well as a novel protein were significantly increased, indicating protein oxidation, in the brains of old versus young mice. These results were validated by immunochemistry. In addition, enzyme activity assays demonstrated that oxidation was associated with decreased GS activity, while the activity of lactate dehydrogenase was unchanged in spite of an up-regulation of LDH2 levels. Several of the up-regulated and oxidized proteins in the brains of old mice identified in this report are known to be oxidized in neurodegenerative diseases as well, suggesting that these proteins may be particularly susceptible to processes associated with neurodegeneration. Our results establish an initial basis for understanding protein alterations that may lead to age-related cellular dysfunction in the brain.     

Age-specific methylation of high-mobility-group proteins of the rat liver and its modulation by spermine and sodium butyrate

Liver slices from young (20 weeks) and old (117 weeks) rat were incubated with [methyl-¹⁴C]methionine in the absence or presence of spermine or sodium butyrate. The high-mobility-group (HMG) non-histone proteins were extracted from the liver with perchloric acid and separated by acid-urea polyacrylamide slab gel electrophoressi. Methylation of HMG proteins decreased drastically in old rats. Whereas spermine inhibited the methylation of total HMG proteins in young rats, it had no effect in old age. On the contrary, sodium butyrate did not change the incorporation of methyl groups into total HMG proteins of young rats, but inhibited that of old rats. Particularly, the incorporation of [¹⁴C]methyl groups into HMG 2 was enhanced but into other HMGs it was reduced by both effectors in young and old age. Such discrepancies in the methylation of HMG proteins and their differential modulation by spermine and butyrate might affect the higher-order organization of chromatin and consequential destabilize the expression of genes during aging.     

Decreased expression of ErbB4 and tyrosine hydroxylase mRNA and protein in the ventral midbrain of aged rats

Decreased availability or efficacy of neurotrophic factors may underlie an increased susceptibility of mesencephalic dopaminergic cells to age-related degeneration. Neuregulins (NRGs) are pleotrophic growth factors for many cell types, including mesencephalic dopamine cells in culture and in vivo. The functional NRG receptor ErbB4 is expressed by virtually all midbrain dopamine neurons. To determine if levels of the NRG receptor are maintained during aging in the dopaminergic ventral mesencephalon, expression of ErbB4 mRNA and protein was examined in young (3 months), middle-aged (18 months), and old (24–25 months) Brown Norway/Fischer 344 F1 rats. ErbB4 mRNA levels in the substantia nigra pars compacta (SNpc), but not the adjacent ventral tegmental area (VTA) or subtantia nigra pars lateralis (SNl), were significantly reduced in the middle-aged and old animals when compared to young rats. Protein expression of ErbB4 in the ventral midbrain was significantly decreased in the old rats when compared to the young rats. Expression of tyrosine hydroxylase (TH) mRNA levels was significantly reduced in the old rats when compared to young animals in the SNpc, but not in the VTA or SNI. TH protein levels in the ventral midbrain were also decreased in the old animals when compared to the young animals. These data demonstrate a progressive decline of ErbB4 expression, coinciding with a loss of the dopamine-synthesizing enzyme TH, in the ventral midbrain of aged rats, particularly in the SNpc. These findings may implicate a role for diminished NRG/ErbB4 trophic support in dopamine-related neurodegenerative disorders of aging such as Parkinson's disease.     

Age-related toxicity to lactate, glutamate, and beta-amyloid in cultured adult neurons

The age-related susceptibility of the brain to neurodegenerative disease may be inherent in the susceptibility of individual neurons to various stressors. Neurons were isolated from embryonic, young- and old-aged rat hippocampus, cultured in serum-free medium and exposed to lactic acid, glutamate or beta-amyloid. Yields of isolated adult cells were 1 million cells/hippocampus, 12,000 cells/mg tissue, independent of age. For lactic acidosis, there was a non-significant 10% increment in killing of neuron-like cells from old rats compared to young. For glutamate, there was a 5-10% increment in killing of neuron-like cells from old rats compared to young rats and embryonic neurons. For cells exposed to the toxic fragment of beta-amyloid, A beta (25-35), toxicity was age, dose and time-dependent. Maximum toxicity in cells treated for 1 day with 25 microM A beta (25-35) was 16%, 24%, and 33% for embryonic, young and old cells. Similar results were found for A beta (1-40) (LD50 = 2 microM). These results suggest that aging imparts to individual cells an increased susceptibility to toxic substances relevant to neurodegenerative diseases.     

Proteomic analysis of aging brain in SAMP10 mouse: A model of age-related cerebral degeneration

Senescence-accelerated mouse prone 10 (SAMP10) strain is a model of age-related neurodegeneration in the limbic forebrain. To investigate changes in protein expression profiles involved in neurodegeneration, we performed two-dimensional fluorescence difference gel electrophoresis and compared protein expression in the limbic and non-limbic forebrains of SAMP10 and control mice at various ages. Among protein spots in which patterns of aging in expression in the limbic forebrain differed between SAMP10 and control, we identified three proteins by mass spectrometry: pyridoxal phosphate phosphatase (PLPP), collapsin response mediator protein 2 (CRMP-2) and α-internexin. Expression of PLPP was increased in the limbic forebrain of 3-month-old SAMP10 mice. Levels of CRMP-2 and phosphorylated α-internexin were increased in the limbic forebrain of SAMP10 mice at age 8 months and remained high until 14 months. Western blot revealed elevation in the level of phosphorylated CRMP-2 and the ratio of phosphorylation of α-internexin. Immunohistochemistry revealed that α-internexin was chiefly distributed in axons. Aging in SAMP10 mice was associated with abnormality of PLPP, CRMP-2 and α-internexin, all of which are known to be involved in brain cytoskeleton formation and associated with acute and chronic neurodegenerative conditions. These proteins are promising targets for further investigation of the mechanisms underlying brain aging.