Johan Lundkvist

Improved Recovery and Delayed Cytokine Induction after Closed Head Injury in Mice with Central Overexpression of the Secreted Isoform of the Interleukin-1 Receptor Antagonist

The acute inflammatory response following traumatic brain injury (TBI) has been shown to play an important role in the development of secondary tissue damage. The proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNFalpha), are induced early after brain injury and have been implicated in the delayed damage. The IL-1 receptor antagonist (IL-1ra) has been shown to modulate the proinflammatory cytokine cascade by blocking the binding of IL-1 to its signaling receptor. In this study, we investigated the effect of transgenic overexpression of IL-1ra on the cytokine expression and neurological damage in a closed head injury (CHI) model of TBI. The neurological recovery, as analyzed by neurological severity score (NSS), was significantly higher in transgenic mice overexpressing the human secreted form of IL-1ra in astrocytes, directed by the murine glial fibrillary acidic protein promoter, as compared to wild-type mice. Analysis of tissue levels of cytokines by ELISA showed increased levels of TNFalpha in the cerebral cortex from the wild type mice 1 h after injury. After 4 h significant increases in the levels of IL-1beta and IL-6 were observed in the wild type mice. In the transgenic mice, on the other hand, no effect on TNFalpha levels was observed and no significant increases in IL-1beta and IL-6 levels could be detected until 6 h after injury. Thus, it can be concluded that blockage of IL-1 signaling by elevated levels of IL-1ra has a neuroprotective effect, in agreement with previous reports, and that central overexpression of IL-1ra results in delayed proinflammatory cytokine induction and improved neurological recovery after traumatic brain injury.

A non peptidic corticotropin releasing factor receptor antagonist attenuates fever and exhibits anxiolytic-like activity.

The multiple actions of corticotropin-releasing factor (CRF) on neuroendocrine and behavioural functions can now be examined using new, high affinity, non peptidic antagonists which exhibit central activity upon systemic application. We have shown that compound CP 154,526 (butyl-ethyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo [2,3-d]pyrimidin-4-yl]amine) displaces [125I][Tyr0]CRF from rat hippocampal CRF receptors (IC50 = 0.5 nM) and from pituitary CRF receptors (IC50 = 0.04 nM). The same compound inhibits in a concentration-dependent manner the ovine CRF (0.1 microM)-stimulated adenylate cyclase activity in membranes of a mouse pituitary adenoma cell line, AtT20, with an IC50 value of 50 nM. Systemic application of the CRF receptor antagonist (0.16 mg/kg i.p.) blocked recombinant human interleukin-1 beta 5 micrograms/kg i.p.) induced fever in rats. The CRF receptor antagonist CP 154,526 (1 mg/kg i.p.) also exhibited signs of anxiolytic-like activity in the elevated plus-maze test in rats.

A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling

Notch receptors are single transmembrane receptors that contain a large number of epidermal growth factor-like repeats (EGF repeats) in their extracellular domains. Mutations in the EGF repeats of the human Notch 3 receptor lead to the vascular dementia disease Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). The vast majority of CADASIL mutations are missense mutations removing or inserting cysteine residues in the EGF repeats, but it is not yet clear whether these mutations primarily affect receptor trafficking, maturation, and/or signaling. To address this issue, we have generated and analyzed stable cell lines expressing either wild-type murine Notch 3 (mNotch 3) or the mutant mNotch 3R142C, which corresponds to the prevalent CADASIL form of Notch 3, Notch 3R141C in humans. We find that a lower proportion of mNotch 3R142C is expressed in the site 1-cleaved configuration, and that reduced amounts of mNotch 3R142C appear at the cell surface, as compared with wild-type mNotch 3. This observation is accompanied by a higher propensity for mNotch 3R142C to form intracellular aggregates, which may be a result of increased accumulation or slowed transport in the secretory pathway. In contrast to the impaired cell surface expression, mNotch 3R142C signals equally well in response to Delta 1 and Jagged 1 as wild-type mNotch 3. Taken together, these data suggest that trafficking and localization rather than signaling of mNotch 3 are affected in mNotch 3R142C.

Notch and the birth of glial cells

Notch receptors play crucial roles in many cellular differentiation programs. In addition to a more classical role for Notch in keeping cells in an undifferentiated state, a recent paper has provided clear evidence that Notch signaling is a powerful means of turning adult CNS precursor cells into astrocytes. This work, combined with other reports from the developing CNS, retina and the PNS, demonstrates a role for Notch in gliogenesis. These findings can also be linked to novel insights into the function of proneural basic helix-loop-helix proteins in astrocytic differentiation.

Expression of the intermediate filament nestin in gastrointestinal stromal tumors and interstitial cells of Cajal.

It has recently been proposed that gastrointestinal stromal tumors (GISTs) originate from stem cells that differentiate toward a phenotype of interstitial cells of Cajal (ICCs). Nestin is a newly identified intermediate filament protein, and is predominantly expressed in immature cells, such as neuroectodermal stem cells and skeletal muscle progenitor cells, and tumors originating from these cells. In this study, we examined, using immunohistochemistry, the nestin expression in GISTs and ICCs to clarify the origin of GISTs. Strong immunoreactivity for nestin was observed in all 18 GISTs, and its expression was confirmed by Western blot and Northern blot analyses. In contrast, three leiomyomas and a schwannoma that developed in the gastrointestinal tract showed no apparent immunoreactivity for nestin. Among 17 mesenchymal tumors (seven leiomyosarcomas, five malignant peripheral nerve sheath tumors, and five fibrosarcomas) that occurred in sites other than the gastrointestinal tract, only two malignant peripheral nerve sheath tumors were moderately immunoreactive for nestin. Furthermore, with fluorescence double immunostaining of the normal small intestine, nestin expression was demonstrated in ICCs. These results show that nestin may be a useful marker for diagnosis of GISTs, and support the current hypothesis that GISTs are tumors of stem cells that differentiate toward an ICC phenotype.

The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity.

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-J kappa. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. In this report we explore the molecular basis for this functional difference between Notch 1 and Notch 3 IC. We find that Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-J kappa on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-J kappa in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-J kappa. We show that two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators. Second, we identify a novel important region in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 ICs ability to activate and for Notch 3 ICs ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors.

Interleukin-1 receptor accessory protein interacts with the type II interleukin-1 receptor

Stably transfected HEK‐293 cells express on their surface the murine type II IL‐1 receptor (mIL‐1RII) as demonstrated by FACS analysis using the mAb 4E2, however binding of [125I]‐hrIL‐1β to these cells is nearly absent. Saturable high affinity binding of [125I]‐hrIL‐1β is observed when the murine IL‐1 receptor accessory protein (mIL‐1RAcP) is coexpressed with mIL‐1RII. Binding of [125I]‐hrIL‐1β to mIL‐1RII‐mIL‐1RAcP complex can be inhibited either with antibodies to mIL‐1RII (mAb 4E2), or by antibodies to mIL‐1RAcP (mAb 4C5). The number of high affinity binding sites in cells stably transfected with the cDNA for mIL‐1RII is dependent on the dose of cDNA for mIL‐1RAcP used to transfect the cells. The high affinity complex between mIL‐1RII and mIL‐1RAcP is not preformed by interaction between the intracellular domains of these two transmembrane proteins, rather it appears to require the extracellular portions of mIL‐1RII and mIL‐1RAcP and the presence of a ligand. We suggest that in addition to its earlier described decoy receptor role, IL‐1RII may modulate the responsiveness of cells to IL‐1 by binding the IL‐1RAcP in unproductive/non‐signalling complexes and thus reducing the number of signalling IL‐1RI‐IL‐1RAcP‐agonist complexes when IL‐1 is bound.

Acute-phase responses in transgenic mice with CNS overexpression of IL-1 receptor antagonist

The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1α and IL-1β by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to ∼50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1β (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1β (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 μg/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 μg/kg ip; 1 and 6 h after injection) IL-1β and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1β and IL-6 plasma levels.

Notch receptor cleavage depends on but is not directly executed by presenilins

Notch receptors undergo three distinct proteolytic cleavages during maturation and activation. The third cleavage occurs within the plasma membrane and results in the release and translocation of the intracellular domain into the nucleus to execute Notch signaling. This so-called γ-secretase cleavage is under the control of presenilins, but it is not known whether presenilins themselves carry out the cleavage or whether they act by means of yet-unidentified γ-secretase(s). In this article, we show that Notch intracellular cleavage in intact cells completely depends on presenilins. In contrast, partial purification of the Notch cleavage activity reveals an activity, which is present only in protein extracts from presenilin-containing cells, and which does not comigrate with presenilin. This finding provides evidence for the existence of a specific Notch-processing activity, which is physically distinct from presenilins. We conclude from these experiments that presenilins are critically required for Notch intracellular cleavage but are not themselves directly mediating the cleavage.

BACE1 inhibition induces a specific cerebrospinal fluid β-amyloid pattern that identifies drug effects in the central nervous system.

BACE1 is a key enzyme for amyloid-β (Aβ) production, and an attractive therapeutic target in Alzheimers disease (AD). Here we report that BACE1 inhibitors have distinct effects on neuronal Aβ metabolism, inducing a unique pattern of secreted Aβ peptides, analyzed in cell media from amyloid precursor protein (APP) transfected cells and in cerebrospinal fluid (CSF) from dogs by immunoprecipitation-mass spectrometry, using several different BACE1 inhibitors. Besides the expected reductions in Aβ1-40 and Aβ1-42, treatment also changed the relative levels of several other Aβ isoforms. In particular Aβ1-34 decreased, while Aβ5-40 increased, and these changes were more sensitive to BACE1 inhibition than the changes in Aβ1-40 and Aβ1-42. The effects on Aβ5-40 indicate the presence of a BACE1 independent pathway of APP degradation. The described CSF Aβ pattern may be used as a pharmacodynamic fingerprint to detect biochemical effects of BACE1-therapies in clinical trials, which might accelerate development of novel therapies.