Hippocampal SPARC regulates depression-related behavior.

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein highly expressed during development, reorganization and tissue repair. In the central nervous system, glial cells express SPARC during development and in neurogenic regions of the adult brain. Astrocytes control the glutamate receptor levels in the developing hippocampus through SPARC secretion. To further characterize the role of SPARC in the brain, we analyzed the hippocampal-dependent adult behavior of SPARC KO mice. We found that SPARC KO mice show increased levels of anxiety-related behaviors and reduced levels of depression-related behaviors. The antidepressant-like phenotype could be rescued by adenoviral vector-mediated expression of SPARC in the adult hippocampus, but anxiety-related behavior persisted in these mice. To identify the cellular mechanisms underlying these behavioral alterations, we analyzed neuronal activity and neurogenesis in the dentate gyrus (DG). SPARC KO mice have increased levels of neuronal activity, evidenced as more neurons that express c-Fos after a footshock. SPARC also affects cell proliferation in the subgranular zone of the DG, although it does not affect maturation and survival of new neurons. SPARC expression in the adult DG does not revert the proliferation phenotype in KO mice, but our results suggest a role of SPARC in limiting the survival of new neurons in the DG. This work suggests that SPARC could affect anxiety-related behavior by modulating neuronal activity, and that depression-related behavior is dependent upon the adult expression of SPARC, which affects adult brain function by mechanisms that need to be elucidated.

Neuroprotective and neurodegenerative effects of the chronic expression of tumor necrosis factor α in the nigrostriatal dopaminergic circuit of adult mice.

Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine, has been implicated in both neuronal death and survival in Parkinson's disease (PD). The substantia nigra (SN), a CNS region affected in PD, is particularly susceptible to inflammatory insults and possesses the highest density of microglial cells, but the effects of inflammation and in particular TNF-α on neuronal survival in this region remains controversial. Using adenoviral vectors, the CRE/loxP system and hypomorphic mice, we achieved chronic expression of two levels of TNF-α in the SN of adult mice. Chronic low expression of TNF-α levels reduced the nigrostriatal neurodegeneration mediated by intrastriatal 6-hydroxydopamine administration. Protective effects of low TNF-α level could be mediated by TNF-R1, GDNF, and IGF-1 in the SN and SOD activity in the striatum (ST). On the contrary, chronic expression of high levels of TNF-α induced progressive neuronal loss (63% at 20 days and 75% at 100 days). This effect was accompanied by gliosis and an inflammatory infiltrate composed almost exclusively by monocytes/macrophages. The finding that chronic high TNF-α had a slow and progressive neurodegenerative effect in the SN provides an animal model of PD mediated by the chronic expression of a single cytokine. In addition, it supports the view that cytokines are not detrimental or beneficial by themselves, i.e., their level and time of expression among other factors can determine its final effect on CNS damage or protection. These data support the view that new anti-parkinsonian treatments based on anti-inflammatory therapies should consider these dual effects of cytokines on their design.

Knocking-down the NMDAR1 subunit in a limited amount of neurons in the rat hippocampus impairs learning.

Amplicon vectors derived from herpes simplex virus type 1 were built to modify NMDA receptors by expressing antisense RNA for the essential NR1 subunit. Their ability to modify endogenous levels of NR1 was tested in cultures of rat embryo neocortical neurons. We studied behaviour and tested for expression in adult rats injected with those vectors into the dorsal hippocampus to find out which cells and how many appear involved in memory formation. Rats injected with vectors expressing NR1 antisense performed significantly worse than control rats in an inhibitory avoidance task. Immunohistochemistry was performed in brain slices from the same animals. The transduced cells represented 6-7% of pyramidal neurons in CA1, showing that a single gene knockdown of NR1 in a small number of neurons significantly impaired memory formation. Perhaps neurons undergoing synaptic plasticity are more susceptible to NR1 knockdown, and hence NMDAR are particularly required in those neurons undergoing synaptic plasticity during learning, or perhaps, and more likely, there is not a high level of redundancy in the hippocampal circuits involved, leading to the idea that a certain level of NR1 expression/availability appears necessary for memory formation in most of CA1 pyramidal neurons.

Interleukin-6: a cytokine to forget.

It is known that proinflammatory cytokines such as interleukin-6 (IL-6) are expressed in the central nervous system (CNS) during disease conditions and affect several brain functions including memory and learning. In contrast to these effects observed during pathological conditions, here we describe a physiological function of IL-6 in the "healthy" brain in synaptic plasticity and memory consolidation. During long-term potentiation (LTP) in vitro and in freely moving rats, IL-6 gene expression in the hippocampus was substantially increased. This increase was long lasting, specific to potentiation, and was prevented by inhibition of N-methyl-D-aspartate receptors with (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Blockade of endogenous IL-6 by application of a neutralizing anti-IL-6 antibody 90 min after tetanus caused a remarkable prolongation of LTP. Consistently, blockade of endogenous IL-6, 90 min after hippocampus-dependent spatial alternation learning resulted in a significant improvement of long-term memory. In view of the suggested role of LTP in memory formation, these data implicate IL-6 in the mechanisms controlling the kinetics and amount of information storage.

Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.

IL-1beta is one of a family of proinflammatory cytokines thought to be involved in many acute and chronic diseases. Although it is considered to participate in wound repair, no major role has been attributed to IL-1beta in tissue fibrosis. We used adenoviral gene transfer to transiently overexpress IL-1beta in rat lungs after intratracheal administration. The high expression of IL-1beta in the first week after injection was accompanied by local increase of the proinflammatory cytokines IL-6 and TNF-alpha and a vigorous acute inflammatory tissue response with evidence of tissue injury. The profibrotic cytokines PDGF and TGF-beta1 were increased in lung fluid samples 1 week after peak expression of IL-1beta. Although PDGF returned to baseline in the third week, TGF-beta1 showed increased concentrations in bronchoalveolar lavage fluid for up to 60 days. This was associated with severe progressive tissue fibrosis in the lung, as shown by the presence of myofibroblasts, fibroblast foci, and significant extracellular accumulations of collagen and fibronectin. These data directly demonstrate how acute tissue injury in the lung, initiated by a highly proinflammatory cytokine, IL-1beta, converts to progressive fibrotic changes. IL-1beta should be considered a valid target for therapeutic intervention in diseases associated with fibrosis and tissue remodeling.

Bias in estimations of DNA content by competitive polymerase chain reaction.

Competitive PCR is a highly sensitive method for specific DNA quantification. Despite the lack of studies related to the accuracy of the method it has been widely used. Here we present a simulation model for competitive PCR, which takes into account the efficiency decay as a linear relationship of the total product yield. The model helped us to study the kind and magnitude of errors that arise from quantitative and semiquantitative competitive PCR protocols and to find ways to minimize them. The simulation data suggest that differences in amplification efficiency between target and standard templates induce stronger biases in quantitative than in semiquantitative competitive PCR. Quantitative competitive PCR can only be used when both efficiencies are equal. In contrast, semiquantitative competitive PCR can be used even when the target is amplified with a higher efficiency than the standard, since under such conditions the method tends to underestimate the differences in initial DNA content. These predictions have been confirmed with experimental data and show that the estimation of the amplification efficiencies is a prerequisite for the use of quantitative and semiquantitative competitive PCR. A simple method for this estimation is also presented.

Not all peripheral immune stimuli that activate the HPA axis induce proinflammatory cytokine gene expression in the hypothalamus.

Administration of low doses of lipopolysaccharide (LPS) that do not disrupt the blood-brain barrier (BBB) results in the expression of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) in the hypothalamus in parallel to stimulation of the hypothalamus-pituitary-adrenal (HPA) axis. This endocrine response is triggered by peripheral cytokines, and we recently obtained evidence that brain-borne IL-1 contributes to its maintenance. LPS preferentially stimulates cells of the macrophage lineage and B lymphocytes. The possibility that primarily stimulation of other types of peripheral immune cells also results in the expression of proinflammatory cytokines in the brain and in the activation of the HPA axis was investigated. Our results showed that, in contrast to LPS, administration of the superantigen staphylococcal enterotoxin B (SEB), which stimulates T cells by binding to appropriate V beta domains of the T-cell receptor, did not result in induction of IL-1 beta, IL-6, and TNF alpha expression in the hypothalamus. Furthermore, although IL-2 transcripts in the spleen were highly increased, expression of this gene was not detected in the brain. However, as with LPS, SEB administration also results in elevated levels of glucocorticoids in blood. Therefore, our data suggest that increased expression of proinflammatory cytokines in the brain is not a necessary step in the stimulation of the HPA axis by SEB.

A neuromodulatory role of interleukin-1beta in the hippocampus.

It is widely accepted that interleukin-1beta (IL-1beta), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we show in vivo and in vitro that IL-1beta gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with the N-methyl-D-aspartate (NMDA) receptor antagonist, (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1beta in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.

Induction of cytokine transcripts in the central nervous system and pituitary following peripheral administration of endotoxin to mice.

The regional distribution and inducibility of cytokines in the normal brain is still a matter of controversy. As an attempt to clarify this issue, we studied the constitutive and induced expression of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma mRNAs in the brain, pituitary, and spleen of mice using qualitative and semiquantitative reverse-transcription polymerase chain reaction. The contribution of nonbrain cells to the cytokine transcripts detected was considered. With the exception of IFN-gamma mRNA, transcripts for the other cytokines were found to be constitutively present in the brain. Following i.p. injection of lipopolysaccharide (LPS) at a dose below those described to disrupt the blood-brain barrier (BBB), cytokine mRNA expression was increased in the spleen, the pituitary, and the brain. In the brain, the onset of transcription varied from 45 min (IL-1beta, TNF-alpha) to 4 hr (IFN-gamma), and the peak of mRNA accumulation was observed at different times depending on the cytokine and the brain region studied. IL-1 and IL-6 were highly expressed in the hypothalamus and hippocampus, while TNF-alpha expression was more marked in the thalamus-striatum. The cortex was the region in which cytokines were less inducible. The inducible expression of cytokine mRNAs in the brain was paralleled by stimulation of the hypothalamus-pituitary-adrenal axis. These results show the capacity of brain cells to synthesize different cytokine mRNAs in vivo and define the kinetics of their expression in several brain areas and in the periphery in parallel to the activation of a neuroendocrine pathway by endotoxin.

Inhibition of tumor necrosis factor-alpha action within the CNS markedly reduces the plasma adrenocorticotropin response to peripheral local inflammation in rats.

The present study tested the hypothesis that the cytokine tumor necrosis factor-alpha (TNF-alpha) is an important CNS mediator of the hypothalamo-pituitary-adrenal (HPA) axis response to local inflammation in the rat. Recombinant murine TNF-alpha administered directly into the cerebroventricles of normal rats produced a dose-dependent increase in plasma adrenocorticotropin (ACTH) concentration. Local inflammation induced by the intramuscular injection of turpentine (50 microl/100 gm body weight) also produced an increase in plasma ACTH, peaking at 160-200 pg/ml at 7.5 hr after injection (compared with 10-30 pg/ml in controls). Intracerebroventricular pretreatment with either 5 microl of anti-TNF-alpha antiserum or 1-50 microg of soluble TNF receptor construct (rhTNFR:Fc) reduced the peak of the ACTH response to local inflammation by 62-72%. In contrast, intravenous treatment with the same doses of anti-TNF-alpha or rhTNFR:Fc had no significant effect on the ACTH response to local inflammation. Although these data indicated an action of TNF-alpha specifically within the brain, no increase in brain TNF-alpha protein (measured by bioassay) or mRNA (assessed using either in situ hybridization histochemical or semi-quantitative RT-PCR procedures) was demonstrable during the onset or peak of HPA activation produced by local inflammation. Furthermore, increased passage of TNF-alpha from blood to brain seems unlikely, because inflammation did not affect plasma TNF-alpha biological activity. Collectively these data demonstrate that TNF-alpha action within the brain is critical to the elaboration of the HPA axis response to local inflammation in the rat, but they indicate that increases in cerebral TNF-alpha synthesis are not a necessary accompaniment.

A functional GTP-binding motif is necessary for antiviral activity of Mx proteins.

Mx proteins are interferon-induced GTPases that inhibit the multiplication of certain negative-stranded RNA viruses. However, it has been unclear whether GTPase activity is necessary for antiviral function. Here, we have introduced mutations into the tripartite GTP-binding consensus elements of the human MxA and mouse Mx1 proteins. The invariant lysine residue of the first consensus motif, which interacts with the beta- and gamma-phosphates of bound GTP in other GTPases, was deleted or replaced by methionine or alanine. These Mx mutants and appropriate controls were then tested for antiviral activity, GTP-binding capacity, and GTPase activity. We found a direct correlation between the GTP-binding capacities and GTP hydrolysis activities of the purified Mx mutants in vitro and their antiviral activities in transfected 3T3 cells, demonstrating that a functional GTP-binding motif is necessary for virus inhibition. Our results, thus, firmly establish antiviral activity as a novel function of a GTPase, emphasizing the enormous functional diversity of GTPase superfamily members.

Mx proteins: GTPases with antiviral activity.

Mx proteins are synthesized in interferon-treated vertebrate cells. They have attracted much attention because some of them can block the multiplication of influenza A virus and certain other negative-stranded RNA viruses. Recently, Mx proteins have been shown to be GTPases with significant homology to dynamins and yeast VPS1, enzymes involved in intracellular protein trafficking. Several biochemical properties of dynamin and VPS1 are similar to those of Mx, promoting new speculation about how Mx proteins might interfere with virus multiplication.

Mx proteins: GTPases involved in the interferon-induced antiviral state.

Mx proteins have molecular masses between 70 and 80 kDa and their synthesis is tightly regulated by interferons in mammalian and non-mammalian vertebrates. Some Mx proteins function as intracellular mediators of the interferon-induced antiviral state. When suitable cDNA constructs were constitutively expressed in mouse 3T3 cells the mouse nuclear Mx1 protein conferred selective resistance to influenza virus. The human cytoplasmic MxA protein conferred resistance to influenza virus and vesicular stomatitis virus but not to other viruses. Mx1 blocks influenza virus mRNA synthesis within the nucleus of infected cells. Mx1 presumably interacts with the influenza virus polymerase subunit PB2, because overexpression of PB2 titrates out the Mx1 block. MxA does not inhibit mRNA synthesis of influenza virus; it inhibits a subsequent cytoplasmic viral multiplication step. A possible target is the transport of newly synthesized influenza virus polymerase proteins back to the nucleus. Inhibition by MxA of vesicular stomatitis virus, which replicates in the cytoplasm, is at the transcriptional level. Parts of the N-terminal halves of all known Mx proteins are highly conserved. They contain the typical GTP-binding motif and show significant homology to other members of a new family of GTPases that includes rat dynamin, Drosophila Shibire and the yeast proteins Vps1/Spo15 and Mgm1. Purified Mx1 and MxA proteins possess GTPase activity. The GTP/GDP conversion rates are about 40 per min, and Km values about 700 microM. Mx1 and MxA variants with mutations in the GTP-binding sequences that violate the consensus are unable to confer virus resistance in vivo or to hydrolyse GTP in vitro, suggesting that GTPase activity is necessary for antiviral activity of Mx proteins. We hypothesize that the antivirally active Mx proteins (directly or indirectly) bind to polymerase proteins of susceptible viruses, thereby abolishing normal viral polymerase function. Interaction of Mx with viral targets is probably a GTP-dependent process.

Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily.

A partial cDNA was isolated that encoded a protein kinase, termed rac (related to the A and C kinases). This cDNA was subsequently used to screen libraries derived from the human cell lines MCF-7 and WI38 and led to the isolation of full-length cDNA clones. DNA sequence analysis identified an open reading frame of 1440 base pairs encoding a protein of 480 amino acids (Mr, 55,716). This result was supported by the synthesis of a Mr 58,000 protein in an in vitro translation system that used RNA transcribed from cloned cDNAs with SP6 RNA polymerase. The predicted protein contains consensus sequences characteristic of a protein kinase catalytic domain and shows 73% and 68% similarity to protein kinase C and the cAMP-dependent protein kinase, respectively. Northern (RNA) analysis revealed a single mRNA transcript of 3.2 kilobases that varied up to 300-fold between different cell lines. Specific antisera directed towards the carboxyl terminal of the rac protein kinase were prepared and used to identify that phosphorylated several substrates in immunoprecipitates prepared with the rac-specific antisera.