Dysfunction of astrocytes in senescence-accelerated mice SAMP8 reduces their neuroprotective capacity

Early onset increases in oxidative stress and tau pathology are present in the brain of senescence-accelerated mice prone (SAMP8). Astrocytes play an essential role, both in determining the brain's susceptibility to oxidative damage and in protecting neurons. In this study, we examine changes in tau phosphorylation, oxidative stress and glutamate uptake in primary cultures of cortical astrocytes from neonatal SAMP8 mice and senescence-accelerated-resistant mice (SAMR1). We demonstrated an enhancement of abnormally phosphorylated tau in Ser(199) and Ser(396) in SAMP8 astrocytes compared with that of SAMR1 control mice. Gsk3beta and Cdk5 kinase activity, which regulate tau phosphorylation, was also increased in SAMP8 astrocytes. Inhibition of Gsk3beta by lithium or Cdk5 by roscovitine reduced tau phosphorylation at Ser(396). Moreover, we detected an increase in radical superoxide generation, which may be responsible for the corresponding increase in lipoperoxidation and protein oxidation. We also observed a reduced mitochondrial membrane potential in SAMP8 mouse astrocytes. Glutamate uptake in astrocytes is a critical neuroprotective mechanism. SAMP8 astrocytes showed a decreased glutamate uptake compared with those of SAMR1 controls. Interestingly, survival of SAMP8 or SAMR1 neurons cocultured with SAMP8 astrocytes was significantly reduced. Our results indicate that alterations in astrocyte cultures from SAMP8 mice are similar to those detected in whole brains of SAMP8 mice at 1-5 months. Moreover, our findings suggest that this in vitro preparation is suitable for studying the molecular and cellular processes underlying early aging in this murine model. In addition, our study supports the contention that astrocytes play a key role in neurodegeneration during the aging process.     

Neuronal polarization is impaired in mice lacking RhoE expression

Proper development of neuronal networks relies on the polarization of the neurons, thus the establishment of two compartments, axons and dendrites, whose formation depends on cytoskeletal rearrangements. Rnd proteins are regulators of actin organization and they are important players in several aspects of brain development as neurite formation, axon guidance and neuron migration. We have recently demonstrated that mice lacking RhoE/Rnd3 expression die shortly after birth and have neuromotor impairment and neuromuscular alterations, indicating an abnormal development of the nervous system. In this study, we have further investigated the specific role played by RhoE in several aspects of neuronal development by using hippocampal neuron cultures. Our findings show that neurons from a mice lacking RhoE expression exhibit a decrease in the number and the total length of the neurites. We also show that RhoE-deficient neurons display a reduction in axon outgrowth and a delay in the process of neuronal polarization. In addition, our results suggest an involvement of the RHOA/ROCK/LIMK/COFILIN signaling pathway in the neuronal alterations induced by the lack of RhoE. These findings support our previous report revealing the important role of RhoE in the normal development of the nervous system and may provide novel therapeutic targets in neurodegenerative disorders.     

Proteomic analysis of the reactive phenotype of astrocytes following endothelin-1 exposure

Reactive gliosis is an invariant feature of the pathology of central nervous system (CNS) injury and a major determinant of neuronal survival and regeneration. To begin to understand the alterations in astrocyte protein expression that drive glial changes that occur following injury, we used an established model system (endothelin-1 stimulation of hypertrophy) and proteomic analysis to define a discrete set of differentially expressed proteins and post-translational modifications that occur as the astrocytes change from a quiescent to a reactive state. This orchestrated set of changes included proteins involved in cytoskeletal reorganization (caldesmon, calponin, alpha B-crystallin, stathmin, collapsing response mediator protein-2), cell adhesion (vinculin, galectin-1), signal transduction (RACK-1) and astrocyte differentiation (glutamine synthetase). Using proteomic analysis to understand what drives astrocyte expression of these functionally divergent molecules may offer insight into the mechanisms by which astrocytes can exhibit both pro-regenerative and anti-regenerative activities following CNS injury.     

Differential expression of the liver proteome in senescence accelerated mice

The senescence-accelerated mouse (SAM) is a useful animal model to study aging or age-associated disorders due to its inherited aging phenotype. To investigate proteins involved in the aging process in liver, we compared the young (4- or 20-week old) and the aged group (50-week-old) of SAMP8 (short life span) and SAMR1 (control) mice, and identified 85 differentially expressed distinct proteins comprising antioxidation, glucose/amino acid metabolism, signal transduction and cell cycle systems using proteomics tools. For the antioxidation system, the aged SAMP8 mice showed a large increase in glutathione peroxidase and decreases in glutathione-S-transferase and peroxiredoxin, ranging from 2.5- to 5-fold, suggesting lower detoxification potentials for oxidants in the aged SAMP8 liver. Similarly, levels of key glycolytic enzymes decreased greatly in the aged SAMP8 compared to SAMR1, indicating a disturbance in glucose homeostasis that may be closely related to the typical deficits in learning and memory of the aged SAMP8. Protein profiles of amino acid metabolic enzymes suggest that accumulation of glutamine and glutamate in tissues of the aged SAMP8 may be due to hyperexpression of ornithine aminotransferase and/or glutamate dehydrogenase. Decreases in levels of proteins involved in signal transduction/apoptosis (e.g., cathepsin B) in the aged SAMP8 may support the previously proposed negative relationship between apoptosis and aging. However, the changes described above were not markedly seen in the young group of both strains. For cell cycle systems, levels of selenium binding protein increased about four-fold with age in SAMP8. Yet, almost no change occurred in either the young or the aged SAMR1, which may explain problems associated with cell proliferation and tissue regeneration in the aged SAMP8. In conclusion, composite profiles of key proteins involved in age-related processes enable assessment of accelerated senescence and the appearance of senescence-related pathologies in the aged SAMP8.     

Regional differences in PACAP transport across the blood-brain barrier in mice: a possible influence of strain,amyloid beta protein,and age

The blood–brain barrier (BBB) controls the exchange of peptides and regulatory proteins between the central nervous system (CNS) and the blood. Transport across the BBB of such regulatory substances is altered in animal models of Alzheimer’s disease. These alterations could lead to cognitive impairments or diminish their therapeutic potential. Here, we measured the transport rate of radioactively labeled pituitary adenylate cyclase-activating polypeptide (PACAP) from blood into whole brain and into 11 brain regions in three groups of mice: young (2 months old) ICR, young (2 months old) SAMP8, and aged (12 months old) SAMP8 mice. The SAMP8 is a strain which develops impaired learning and memory with aging that correlates with an age-related increase in brain levels of amyloid β protein (AβP). PACAP is a powerful neurotrophin that may have a therapeutic role in neurodegenerative diseases. We found that I-PACAP crossed the BBB fastest at the hypothalamus and the hippocampus in all three groups. Slower transport rates into the whole brain, the olfactory bulb, the hypothalamus, and the hippocampus for aged SAMP8 mice was likely related to differences both from strain and expression of AβP with aging.     

Viral complementation allows HIV-1 replication without integration

Previous studies have reported that various inbred SAM mouse strains differ markedly with regard to a variety of parameters, such as capacity for learning and memory, life spans and brain histopathology. A potential cause of differences seen in these strains may be based on the fact that some strains have a high concentration of infectious murine leukemia virus (MuLV) in the brain, whereas other strains have little or no virus. To elucidate the effect of a higher titer of endogenous retrovirus in astroglial cells of the brain, we established astroglial cell lines from SAMR1 and SAMP8 mice, which are, respectively, resistant and prone to deficit in learning and memory and shortened life span. MuLV-negative astroglial cell lines established from ICR mice served as controls. Comparison of these cell lines showed differences in: 1) levels of the capsid antigen CAgag in both cell lysates and culture media, 2) expression of genomic retroelements, 3) the number of virus particles, 4) titer of infectious virus, 5) morphology, 6) replication rate of cells in culture and final cell concentrations, 7) expression pattern of proinflammatory cytokine genes. The results show that the expression of MuLV is much higher in SAMP8 than SAMR1 astrocyte cultures and that there are physiological differences in astroglia from the 2 strains. These results raise the possibility that the distinct physiological differences between SAMP8 and SAMR1 are a function of activation of endogenous retrovirus.     

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial creatine kinase, lactate dehydrogenase B, and dihydropyrimidinase-like protein 2 in process associated with learning and memory of SAMP8 mice.     

Trimethylamine‐N‐oxide promotes brain aging and cognitive impairment in mice

Gut microbiota can influence the aging process and may modulate aging‐related changes in cognitive function. Trimethylamine‐N‐oxide (TMAO), a metabolite of intestinal flora, has been shown to be closely associated with cardiovascular disease and other diseases. However, the relationship between TMAO and aging, especially brain aging, has not been fully elucidated. To explore the relationship between TMAO and brain aging, we analysed the plasma levels of TMAO in both humans and mice and administered exogenous TMAO to 24‐week‐old senescence‐accelerated prone mouse strain 8 (SAMP8) and age‐matched senescence‐accelerated mouse resistant 1 (SAMR1) mice for 16 weeks. We found that the plasma levels of TMAO increased in both the elderly and the aged mice. Compared with SAMR1‐control mice, SAMP8‐control mice exhibited a brain aging phenotype characterized by more senescent cells in the hippocampal CA3 region and cognitive dysfunction. Surprisingly, TMAO treatment increased the number of senescent cells, which were primarily neurons, and enhanced the mitochondrial impairments and superoxide production. Moreover, we observed that TMAO treatment increased synaptic damage and reduced the expression levels of synaptic plasticity‐related proteins by inhibiting the mTOR signalling pathway, which induces and aggravates aging‐related cognitive dysfunction in SAMR1 and SAMP8 mice, respectively. Our findings suggested that TMAO could induce brain aging and age‐related cognitive dysfunction in SAMR1 mice and aggravate the cerebral aging process of SAMP8 mice, which might provide new insight into the effects of intestinal microbiota on the brain aging process and help to delay senescence by regulating intestinal flora metabolites.     

Curcumin prevents mitochondrial dysfunction in the brain of the senescence-accelerated mouse-prone 8

The aging brain suffers mitochondrial dysfunction and a reduced availability of energy in the form of ATP, which in turn may cause or promote the decline in cognitive, sensory, and motor function observed with advancing age. There is a need for animal models that display some of the pathological features of human brain aging in order to study their prevention by e.g. dietary factors. We thus investigated the suitability of the fast-aging senescence-accelerated mouse-prone 8 (SAMP8) strain and its normally aging control senescence-accelerated mouse-resistant 1 (SAMR1) as a model for the age-dependent changes in mitochondrial function in the brain. To this end, 2-months old male SAMR1 (n = 10) and SAMP8 mice (n = 7) were fed a Western type diet (control groups) for 5 months and one group of SAMP8 mice (n = 6) was fed an identical diet fortified with 500 mg curcumin per kg. Dissociated brain cells and brain tissue homogenates were analyzed for malondialdehyde, heme oxygenase-1 mRNA, mitochondrial membrane potential (MMP), ATP concentrations, protein levels of mitochondrial marker proteins for mitochondrial membranes (TIMM, TOMM), the mitochondrial permeability transition pore (ANT1, VDAC1, TSPO), respiration complexes, and fission and fusion (Fis, Opa1, Mfn1, Drp1). Dissociated brain cells isolated from SAMP8 mice showed significantly reduced MMP and ATP levels, probably due to significantly diminished complex V protein expression, and increased expression of TSPO. Fission and fusion marker proteins indicate enhanced mitochondrial fission in brains of SAMP8 mice. Treatment of SAMP8 mice with curcumin improved MMP and ATP and restored mitochondrial fusion, probably by up-regulating nuclear factor PGC1α protein expression. In conclusion, SAMP8 compared to SAMR1 mice are a suitable model to study age-dependent changes in mitochondrial function and curcumin emerges as a promising nutraceutical for the prevention of neurodegenerative diseases that are accompanied or caused by mitochondrial dysfunction.     

TAT‐mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression

Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT‐mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)‐TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP‐TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum‐containing medium; (2) monocultures grown in serum‐free medium; (3) cocultures with neurons in serum‐free medium. The efficiency of GFP‐TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP‐TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT‐mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT‐mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell‐type and phenotype‐dependence of TAT‐mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs. Biotechnol. Bioeng. 2009; 104: 10–19 © 2009 Wiley Periodicals, Inc.     

Proteome analysis of primary neurons and astrocytes from rat cerebellum

Neurons and astrocytes are predominant cell types in brain and have distinguished morphological and functional features. Although several proteomics studies were carried out on the brain, work on individual brain cells is limited. Generating individual proteomes of neurons and astrocytes, however, is mandatory to assign protein expression to cell types rather than to tissues. We aimed to provide maps of rat primary neurons and astrocytes using two-dimensional gel electrophoresis with subsequent in-gel digestion, followed by MALDI-TOF/TOF. 428 protein spots corresponding to 226 individual proteins in neurons and 406 protein spots representing 228 proteins in astrocytes were unambiguously identified. Proteome data include proteins from several cascades differentially expressed in neurons and astrocytes, and specific expressional patterns of antioxidant, signaling, chaperone, cytoskeleton, nucleic acid binding, proteasomal, and metabolic proteins are demonstrated. We herein present a reference database of primary rat primary neuron and astrocyte proteomes and provide an analytical tool for these structures. The concomitant expressional patterns of several protein classes are given and potential neuronal and astrocytic marker candidates are presented.     

A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Down's syndrome

Background

Down''s syndrome (DS) is the most common genetic cause of mental retardation. Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology. However, the mechanisms involved in DS spine alterations are not known. In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons. We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion. In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology.

Methodology/Principal Findings

Using a human astrocyte/rat hippocampal neuron coculture, we found that DS astrocytes are directly involved in the development of spine malformations and reduced synaptic density. We also show that thrombospondin 1 (TSP-1), an astrocyte-secreted protein, possesses a potent modulatory effect on spine number and morphology, and that both DS brains and DS astrocytes exhibit marked deficits in TSP-1 protein expression. Depletion of TSP-1 from normal astrocytes resulted in dramatic changes in spine morphology, while restoration of TSP-1 levels prevented DS astrocyte-mediated spine and synaptic alterations. Astrocyte cultures derived from TSP-1 KO mice exhibited similar deficits to support spine formation and structure than DS astrocytes.

Conclusions/Significance

These results indicate that human astrocytes promote spine and synapse formation, identify astrocyte dysfunction as a significant factor of spine and synaptic pathology in the DS brain, and provide a mechanistic rationale for the exploration of TSP-1-based therapies to treat spine and synaptic pathology in DS and other neurological conditions.     

GFAP-Deficient Astrocytes Are Capable of Stellationin VitroWhen Cocultured with Neurons and Exhibit a Reduced Amount of Intermediate Filaments and an Increased Cell Saturation Density

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein predominantly expressed in cells of astroglial origin. To allow for the study of the biological functions of GFAP we have previously generated GFAP-negative mice by gene targeting [Peknyet al.(1995)EMBO J.14, 1590–1598]. Astrocytes in culture, similar to reactive astrocytesin vivo,express three intermediate filament proteins: GFAP, vimentin, and nestin. Using primary astrocyte-enriched cultures from GFAP-negative mice, we now report on the effect of GFAP absence on (i) the synthesis of other intermediate filament proteins in astrocytes, (ii) intermediate filament formation, (iii) astrocyte process formation (stellation) in response to neurons in mixed cerebellar astrocyte/neuron cultures, and (iv) saturation cell densityin vitro.GFAP−/− astrocytes were found to produce both nestin and vimentin. At the ultrastructural level, the amount of intermediate filaments as revealed by transmission electron microscopy was reduced in GFAP−/− astrocytes compared to that in GFAP+/+ astrocytes. GFAP−/− astrocytes retained the ability to form processes in response to neurons in mixed astrocyte/neuron cultures from the cerebellum. GFAP−/− astrocyte-enriched primary cultures exhibited an increased final cell saturation density. The latter leads us to speculate that the loss of GFAP expression observed focally in a proportion of human malignant gliomas may reflect tumor progression toward a more rapidly growing and malignant phenotype.     

Collapsin response mediator proteins of neonatal rat brain interact with chondroitin sulfate

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix.     

Glial glutamate transporters: New actors in brain signaling

Glutamate, the main excitatory amino acid in the vertebrate brain, is critically involved in most of the physiological functions of the central nervous system. It has traditionally been assumed that glutamate triggers a wide array of signaling cascades through the activation of specific membrane receptors. The extracellular levels are tightly regulated to prevent neurotoxic insults. Electrogenic Na(+)-dependent glial glutamate transporters remove the bulk of the neurotransmitter from the synaptic cleft. An exquisitely ordered coupling between glutamatergic neurons and surrounding glia cells is fundamental for excitatory transmission. The glutamate/glutamine and astrocyte/neuron lactate shuttles provide the biochemical framework of this compulsory association. In this context, recent advances show that glial glutamate transporters act as signal transducers that regulate the expression of proteins involved in their compartmentalization with neurons in the so-called tripartite synapse.     

The level of butyrylcholinesterase activity increases and the content of the mRNA remains unaffected in brain of senescence-accelerated mouse SAMP8

Looking at cholinesterases (ChEs) changes in age-related mental impairment, the expression of ChEs in brain of senescence accelerated-resistant (SAMR1) and senescence accelerated-prone (SAMP8) mice was studied. Acetylcholinesterase (AChE) activity was unmodified and BuChE activity increased twofold in SAMP8 brain. SAMR1 brain contained many AChE-T mRNAs, less BuChE and PRiMA mRNAs and scant AChE-R and AChE-H mRNAs. Their content unchanged in SAMP8 brain. Amphiphilic (G(4)(A)) and hydrophilic (G(4)(H)) AChE and BuChE tetramers, besides amphiphilic dimers (G(2)(A)) and monomers (G(1)(A)) were identified in SAMR1 brain and their distribution was little modified in SAMP8 brain. Blood plasma does not seem to provide the excess of BuChE activity in SAMP8 brain; it probably arises from glial cell changes owing to astrocytosis.     

Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes: IMPLICATIONS FOR DISEASE SPREADING AND MOTOR NEURON PATHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS*

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.     

Changes in membrane fatty acids and delta-9 desaturase in senescence accelerated (SAMP8) mouse hippocampus with aging

Senescence accelerated mice (SAMP8) exhibit age induced impairments such as loss of memory and learning disabilities by the age of 8-10 months. Analysis of hippocampus of SAMP8 mice revealed that delta 9-desaturase (delta9desaturase) activity reduced up to 44-50% with age. Correspondingly, levels of unsaturated fatty acids are also lowered in the aged animals approximately to the same levels. RNase protection assay showed that delta9specific message decreased similarly with age. As such a decrease is known to cause alterations in membrane fluidity and affect cellular signaling pathways, these results suggest that lowering of delta9gene expression may be partly involved in age induced impairments.     

Expression of the actin stress fiber-associated protein CLP36 in the human placenta

Differentiation processes in the trophoblast comprise polarization, cell fusion and migration. All these processes involve dramatic reorganizations of cytoskeletal proteins such as intermediate filaments or actin. Due to very restricted knowledge on cytoskeletal changes in trophoblast, we analyzed the protein expression of an actin stress fiber-associated protein, the carboxy-terminal LIM domain protein (CLP36). CLP36 belongs to the enigma family of proteins, binds to α-actinin and is involved in the cytoskeletal reorganization and signal transduction of a variety of cells. CLP36 protein was found to be exclusively expressed in the cytotrophoblast layer. Colocalization of CLP36 with Mib-1 revealed that CLP36 protein expression is restricted to proliferative and early post-proliferative trophoblast cells. Blockage of syncytial fusion by culture of villous explants in the presence of caspase 8 inhibitors further supported this notion since CLP36 was only found in the basal and proliferative layer of the multilayered cytotrophoblast. We present evidence for the exclusive protein expression of CLP36 in proliferative and early post-proliferative trophoblast cells. Pathological pregnancy syndromes such as preeclampsia are driven by alterations of trophoblast differentiation and turnover, where it needs to be elucidated whether CLP36 is involved in these alterations.     

The Hippocampal Proteomic Analysis of Senescence-Accelerated Mouse: Implications of Uchl3 and Mitofilin in Cognitive Disorder and Mitochondria Dysfunction in SAMP8

Senescence-accelerated mouse prone 8 (SAMP8) is considered as a useful animal model for age-related learning and memory impairments. Hippocampus, a critical brain region associated with cognitive decline during normal aging and various neurodegenerative diseases, appeared a series of abnormalities in SAMP8. To investigate the molecular mechanisms underlying age-related cognitive disorders, we used 2-DE coupled with MALDI TOF/TOF MS to analyze the differential protein expression of the hippocampus of SAMP8 at 6-month-old compared with the age-matched SAM/resistant 1 (SAMR1) which shows normal aging process. Two proteins were found to be markedly changed in SAMP8 as compared to SAMR1: ubiquitin carboxyl-terminal hydrolase L3 (Uchl3), implicating in cytosolic proteolysis of oxidatively damaged proteins, was down-regulated while mitofilin, a vital protein for normal mitochondria function, exhibited four isoforms with a consistent basic shift of isoelectric point among the soluble hippocampal proteins in SAMP8 compared with SAMR1. The alterations were confirmed by Western blotting analysis. The analysis of their expression changes may shed light on the mechanisms of learning and memory deficits and mitochondrial dysfunction as observed in SAMP8.