Fibroblast growth factor 2 (FGF-2) differentially regulates connexin (cx) 43 expression and function in astroglial cells from distinct brain regions

Fibroblast growth factor (FGF)-2 is a peptide growth factor that promotes the generation, differentiation, and survival of neurons and glial cells. In the CNS, astroglial cells are coupled in a region-specific manner by gap junctions consisting of connexin 43 (cx43). In the present study we have investigated effects of FGF-2 and of other growth factors on the expression and function of cx43 in astroglial cells cultured from telencephalic cortex, striatum, and mesencephalon of newborn rats. Confluent cultures were maintained for two days in low serum, and then exposed to FGF-2 (10 ng/ml) for 48 h. FGF-2 caused a reduction of cx43-protein, -mRNA, and intercellular communication revealed by dye spreading. These changes occurred in cortical and striatal cells, but not in mesencephalic astroglial cells. Effects of FGF-2 were time- and concentration-dependent, with a minimal effective dose of 1 ng/ml FGF-2, and an onset of effects after 6 h of incubation. The reduction of coupling by FGF-2 was transient, since in cortical and striatal cultures coupling recovered to control levels 48 h after removal of the growth factor. Like FGF-2, transforming growth factor-β3 (TGF-β3) decreased coupling of cortical and striatal, but not mesencephalic astroglial cells. Astroglial cells from all brain regions showed a slight FGF-mediated increase in 5-bromo-2′-desoxy-uridine (BrdU) incorporation, which was abolished upon co-treatment with TGF-β3. However, TGF-β3 did not interfere with the repression of cx43-function by FGF-2. Epidermal growth factor (EGF) that has been demonstrated to influence coupling in other cell types had no effect on dye spreading but significantly increased BrdU incorporation. Our results reveal a novel function of FGF-2 on cultured astroglial cells which may be relevant to the regulation of astroglial cell connectivity in vivo. GLIA 22:19–30, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of astrocyte GFAP expression by TGF-β1 and FGF-2

Astrocytes play a critical role in the development of the CNS and its response to injury and disease. A key indicator of astrocyte activation is the increased accumulation of intermediate filaments composed of glial fibrillary acidic protein (GFAP). Treatment of astrocytes in vitro with transforming growth factor-β1 (TGF-β1) produced little morphological change, but resulted in a significant increase in GFAP mRNA and protein. Treatment with basic fibroblast growth factor (FGF-2) produced a dramatic change from a polygonal to a stellate morphology, and resulted in a significant decrease in GFAP mRNA and protein. FGF-2 also inhibited the TGF-β1-mediated increase in GFAP mRNA and protein. Cycloheximide did not block the effects of TGF-β1 or FGF-2 on GFAP mRNA levels, but blocked the inhibitory effects of FGF-2 on the TGF-β1-mediated increase in GFAP expression. All effects of FGF-2 were blocked by co-incubation with 5′-methylthioadenosine, a specific inhibitor of FGF-2-induced tyrosine kinase activity and FGF receptor (FGFR) autophosphorylation. We also examined astrocyte expression of FGFR, and demonstrate the presence of FGFR 1 and 2, and lower levels of FGFR 3. Our results demonstrate that TGF-β1 and FGF-2 cause differential effects on the astrocyte cytoskeleton and morphology, suggesting an uncoupling of process outgrowth from GFAP synthesis. GLIA 22:202–210, 1998. © 1998 Wiley-Liss, Inc.     

Differentiation of serum-free mouse embryo cells into astrocytes is accompanied by induction of glutamine synthetase activity

Serum-free mouse embryo (SFME) cells derived in a defined serum-free medium have been cultured for more than 200 generations and display properties of neural progenitor cells. SFME cells express the neuroepithelial stem cell marker nestin in defined serumfree medium. Exposure of SFME cells to transforming growth factor beta (TGF-β) or serum decreases nestin expression and induces the astrocyte marker glial fibrillary acidic protein, suggesting that SFME cells differentiate into astrocytes upon exposure to TGF-β or serum. We examined the expression by SFME cells of the functional central nervous system (CNS) astrocyte marker glutamine synthetase (GS). GS activity is induced in SFME cells upon exposure to TGF-β or serum. The induction of GS activity was dose- and time-dependent and was reversible. Retinoic acid, hydrocortisone, and dibutyryl cyclic AMP also induced GS expression. The induction of GS activity was accompanied by an increase in the level of GS mRNA and protein. This work provides further evidence that SFME cells represent neural progenitor cells which differentiate into functional astrocytes upon exposure to TGF-β or serum. © 1995 Wiley-Liss, Inc.     

Downregulation of spinal astrocytic connexin43 leads to upregulation of interleukin‐6 and cyclooxygenase‐2 and mechanical hypersensitivity in mice

Connexin43 (Cx43), involved in intercellular signaling, is expressed in spinal dorsal horn astrocytes and crucial in the maintenance of neuropathic pain. Downregulation of spinal astrocytic Cx43 in mice enhances glutamatergic neurotransmission by decreasing glutamate transporter GLT‐1 expression, resulting in cutaneous hypersensitivity. Decreased expression of astrocytic Cx43 could lead to altered expression of other nociceptive molecules. Transfection of Cx43‐targeting siRNA in cultured spinal astrocytes increased expression of the pronociceptive cytokine interleukin‐6 (IL‐6) and the prostaglandin synthesizing enzyme cyclooxygenase‐2 (COX‐2). Increased expression of IL‐6 and COX‐2 was due to decreased Cx43 expression rather than due to diminished Cx43 channel function. In mice, downregulation of spinal Cx43 expression by intrathecal treatment with Cx43‐targeting siRNA increased IL‐6 and COX‐2 expression and induced hind paw mechanical hypersensitivity. Cx43 siRNA‐induced mechanical hypersensitivity was attenuated by intrathecal treatment with anti‐IL‐6 neutralizing antibody and intraperitoneal treatment of selective COX‐2 inhibitor celecoxib, demonstrating that these molecules play a role in nociceptive processing following Cx43 downregulation. Restoring spinal Cx43 by intrathecal injection of an adenovirus vector expressing Cx43 in mice with a partial sciatic nerve ligation reduced spinal IL‐6 and COX‐2 expression. Suppression of glycogen synthase kinase‐3β (GSK‐3β), a serine/threonine protein kinase, prevented upregulation of IL‐6 and COX‐2 expression induced by Cx43 downregulation in both cultured astrocytes and in mouse spinal dorsal horn. Inhibition of spinal GSK‐3β also ameliorated Cx43 siRNA‐induced mechanical hypersensitivity. The current findings indicate that downregulation of spinal astrocytic Cx43 leads to changes in spinal expression of pronociceptive molecules underlying the maintenance of pain following nerve injury.     

Induction of connexin43 and gap junctional communication in PC12 cells overexpressing the carboxy terminal region of amyloid precursor protein

Previous studies have shown that PC12 cells overexpressing β/A4 amyloid peptide display altered morphology characterized by pronounced membrane ruffling and extensive intercellular appositions. Having observed other cell types in which these features accompany increased connexin43 (Cx43) production and gap junctional communication, we examined Cx43 in normal and β/A4-transfected PC12 cells. Studies of two β/A4-transfected PC12 clones revealed an induction of Cx43 expression by Western blotting, intracellular and plasma membrane-associated Cx43 in some cells of cultures processed by immunofluorescence, dye-transfer between some cells microinjected with Lucifer Yellow, and gap junctions between cells examined by EM. Normal and vector-transfected PC12 cells exhibited none of these properties. Increased immunofluorescence in some clusters of β/A4-transfected cells was also observed with a monoclonal antibody against connexin32. The results suggest that β/A4 amyloid peptide may cause aberrant intercellular communication and gap junction formation through induction or increased expression of connexins in cells that are not normally coupled or only poorly coupled by gap junctions. © 1996 Wiley-Liss, Inc.     

Transforming growth factor-β1 inhibits the proliferation of rat astrocytes induced by serum and growth factors

A number of cytokines and growth factors may affect astrocyte proliferation and functions. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine which exerts multiple effects on growth and differentiation of different cell types. TGF-β1 is present in low amounts in the normal brain. TGF-β1 gene expression, however, is increased in the central nervous system (CNS) in several pathological conditions. In this study we examined the in vitro effects of TGF-β1 on the proliferative response of rat astrocytes to serum and growth factors. Astrocyte cultures were established from the cerebellum and cortex of newborn Lewis rats. The proliferative response of these cultures to serum and growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), IGF-2, interleukin 1 (IL-1)] was studied by [³H]-thymidine incorporation test in the presence or absence of TGF-β1. TGF-β1 significantly inhibited the proliferative response of astrocyte cultures to both autologous and heterologous serum. In addition, a strong inhibition of bFGF-, EGF-, and PDGF-induced proliferation was observed. The effect of TGF-β1 on the proliferative response to IL-1 was less evident but still significant. No effect was observed when TGF-β1 was added to IGF-1 and IGF-2 stimulated cultures. These data confirm previous reports showing a down-regulating activity of TGF-β on astrocyte proliferation and suggest that this cytokine may play physiological and pharmacological roles in the regulation of reactive astrocytosis in the CNS. © 1995 Wiley-Liss, Inc.     

Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: A role for transforming growth factor β

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1β (IL-1β) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1β expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1β was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B₄-isolectin and an antibody against GFAP. Production of IL-1β and NO was determined by measurement of IL-1β and nitrite concentrations in cell lysates and the culture medium, respectively. TGFβ, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGFβ. The potential effect of TGFβ was studied by using immunoneutralizing antibodies against TGFβ1 and TGFβ2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGFβ antibodies (3 μg/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1β was not affected. The antibodies against TGFβ1 and TGFβ2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGFβ1 and TGFβ2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGFβ on NO production by microglial cells (possibly) by activation of TGFβ or by increasing the sensitivity of microglial cells for TGFβ. GLIA 19:190–198, 1997. © 1997 Wiley-Liss, Inc.     

Antiparkinsonian effects of the novel D3/D2 dopamine receptor agonist, S32504, in MPTP-lesioned marmosets: Mediation by D2, not D3, dopamine receptors.

L-dopa remains the most common treatment for Parkinson's disease. However, there is considerable interest in D3/D2 receptor agonists such as the novel agent S32504, since they exert antiparkinsonian properties in the absence of dyskinesia. An important question concerns the roles of D2 vs. D3 receptors, an issue we addressed with the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned nonhuman primate model of Parkinson's disease. In L-dopa-primed animals, S32504 (0.16-2.5 mg/kg p.o.) dose-dependently enhanced locomotor activity. This action was abolished by the D2 antagonist, L741,626 (2.5 mg/kg), but potentiated by the D3 antagonist, S33084 (0.63 mg/kg). Both antagonists were inactive alone. In drug-naive animals, a maximally effective dose of S32504 (2.5 mg/kg p.o.) displayed pronounced antiparkinsonian properties from the third day of administration, and its actions were expressed rapidly and durably. Thus, on day 33, antiparkinsonian properties of S32504 were apparent within 5 minutes and present for > 4 hours. Moreover, they were associated with neither wearing off nor significant dyskinesia. In conclusion, the novel D3/D2 agonist S32504 may offer advantages over L-dopa in the treatment of newly diagnosed parkinsonian patients. Its actions are expressed primarily by activation of D2, not D3, receptors.     

Conserved "PAL" sequence in presenilins is essential for gamma-secretase activity, but not required for formation or stabilization of gamma-secretase complexes

Generation of A beta from the beta-amyloid precursor protein (APP) requires a series of proteolytic processes, including an intramembranous cleavage catalyzed by an aspartyl protease, gamma-secretase. Two aspartates in presenilins (PS) are required for gamma-secretase activity (D257 and D385 of PS1), suggesting that PS may be part of this protease. Little is known concerning the importance of other sequences in PS for activity. We introduced point mutations (P433L, A434D, L435R) into a completely conserved region C-terminal to transmembrane domain eight of PS1. The P433L mutation abolished PS1 endoproteolysis as well as gamma-secretase cleavage of APP and Notch in PS1/2 K/O cells. In HEK cells, expression of PS1/P433L reduced A beta production and caused accumulation of APP C-terminal stubs. When the P433L mutation was introduced into the non-cleavable Delta exon 9 (Delta E9) variant of PS1, it abolished gamma-secretase cleavage of APP and Notch. The P433L holoprotein is stable and incorporated into the high molecular weight gamma-secretase complex, arguing that P433 is not necessary for formation or stabilization of the gamma-secretase complex. Other non-conservative mutations in the invariant P(433)A(434)L(435) sequence also result in a phenotype that is indistinguishable from the aspartate mutants, suggesting a direct involvement of this sequence in gamma-secretase activity.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

Long‐term oral intake of aluminium or zinc does not accelerate Alzheimer pathology in AβPP and AβPP/tau transgenic mice

Whether or not the oral intake of metals such as aluminium (Al) and zinc (Zn) is a risk for Alzheimer's disease (AD) has been a matter of controversy. Lack of AD pathology in patients with Al encephalopathy indicates Al does not cause AD. On the other hand, some epidemiological studies have suggested high Al increases the occurrence of AD. Our purpose is to test if high Al in drinking water is a risk factor for AD. We administered Al and Zn in drinking water to Tg2576, a transgenic mouse model for amyloid β‐protein (Aβ) deposition with the Aβ precursor protein (AβPP) mutations (K670N/M671L), and Tg2576/tau(P301L), a model for Aβ and tau deposition. Deionized water was given to the control Tg2576 and Tg2576/tau. After administration for 4–10 months of approximately 100 mg/kg body weight Al or Zn per day, we were not able to find by quantitative immunohistochemical analyses differences in the deposition of Aβ and tau between the treated and untreated groups. Nor did the Al or Zn treatment affect the amount of soluble Aβ and Aβ*56, an Aβ oligomer, measured by ELISA or immunoblot. The oral intake of excess Al or Zn does not accelerate AD pathology in the transgenic mouse models for Aβ and tau accumulation. Such results do not seem to support the notion that excessive oral intake of Al or Zn is a risk factor for AD.     

Soybean isoflavone ameliorates β‐amyloid 1‐42‐induced learning and memory deficit in rats by protecting synaptic structure and function

This research aims to investigate whether soybean isoflavone (SIF) could alleviate the learning and memory deficit induced by β‐amyloid peptides 1‐42 (Aβ1‐42) by protecting the synapses of rats. Adult male Wistar rats were randomly allocated to the following groups: (1) control group; (2) Aβ1‐42 group; (3) SIF group; (4) SIF + Aβ1‐42 group (SIF pretreatment group) according to body weight. The 80 mg/kg/day of SIF was administered orally by gavage to the rats in SIF and SIF+Aβ1‐42 groups. Aβ1‐42 was injected into the lateral cerebral ventricle of rats in Aβ1‐42 and SIF+Aβ1‐42 groups. The ability of learning and memory, ultramicrostructure of hippocampal synapses, and expression of synaptic related proteins were investigated. The Morris water maze results showed the escape latency and total distance were decreased in the rats of SIF pretreatment group compared to the rats in Aβ1‐42 group. Furthermore, SIF pretreatment could alleviate the synaptic structural damage and antagonize the down‐regulation expressions of below proteins induced by Aβ1‐42: (1) mRNA and protein of the synaptophysin and postsynaptic density protein 95 (PSD‐95); (2) protein of calmodulin (CaM), Ca²⁺/calmodulin‐dependent protein kinase II (CaMK II), and cAMP response element binding protein (CREB); (3) phosphorylation levels of CaMK II and CREB (pCAMK II, pCREB). These results suggested that SIF pretreatment could ameliorate the impairment of learning and memory ability in rats induced by Aβ1‐42, and its mechanism might be associated with the protection of synaptic plasticity by improving the synaptic structure and regulating the synaptic related proteins. Synapse 67:856–864, 2013 . © 2013 Wiley Periodicals, Inc.     

Multiple sclerosis: myelin basic protein induced mRNA expression of proinflammatory cytokines in mononuclear cells is suppressed by interferon-β 1b in vitro

Beta-interferon (IFN-β) is a promising treatment in multiple sclerosis (MS), reducing the exacerbation rate and MRI lesion burden, as well as the disease progression in relapsing-remitting MS. IFN-β was originally defined by its antiviral effects, but the interest has recently been focused on its immunomodulatory properties. Myelin basic protein (MBP) is one of several autoantigens considered to be the target for autoaggressive immune responses, which eventually might lead to the development of MS. To study in-vitro effects of IFN-β1b on MBP induced cytokine expression, mRNA for the Th1 cytokines IFN-γ and TNF-α, the Th2 related IL-4 and IL-6, the cytolytic perforin and the immune response downregulating TGF-β was measured with in situ hybridization after culture of blood mononuclear cells (MNC) in the presence and absence of MBP. Numbers of cells expressing IFN-γ, TNF-α, perforin and IL-4 mRNA were significantly suppressed after culture with 10 U/ml IFN-β1b. No such effect was seen on MBP induced IL-6 or TGF-β mRNA expression. These observations suggest that one of the major effects of IFN-β1b is the induction of a shift in the cytokine mRNA profile towards a more immunosuppressive pattern. In parallel in vitro tests, the control substance dexametasone (40 μg/ml) reduced the numbers of cells expressing mRNA for all cytokines under study with the exception of TGF-β, to an extent equal to or even more pronounced than IFN-β1b.     

Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, β-endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area

The role of corticotropin-releasing factor (CRF) and opiocortin neuronal systems and a possible functional relationship between the two in the control of luteinizing hormone-releasing hormone (LH-RH) activity in the medial preoptic area (MPOA) for the regulation of lordosis behaviour were assessed in ovariectomised oestrogen-progesterone-treated female rats. Lordosis behaviour (assessed as the lordosis quotient) triggered by male mounting was significantly inhibited by either CRF or β-endorphin infused into the MPOA in animals treated with normal doses of oestradiol benzoate (OEP) (5 μg) and progesterone (500 μg). Saline-treated animals exhibited high levels of lordosis. The inhibition of lordosis produced by either CRF or β-endorphin could be reversed by LH-RH microinfusions into the MPOA. While naloxone pretreatment of the MPOA site prevented the inhibitory effects of β-endorphin, neither the opiate antagonist nor anti-β-endorphin-γ-globulin (even in high concentrations) infused into the MPOA was effective in completely preventing the inhibition of lordosis produced by CRF. These findings suggest that the inhibition of LH-RH neuronal activity and lordosis behaviour by CRF may be due to a direct action and may not be the result of activation of β-endorphin release. The possibility that the two peptidergic systems may act in a synergistic fashion is supported by the data showing that combined CRF-β-endorphin treatment in the MPOA completely abolished lordosis. This is further supported by the finding that CRF totally abolished lordosis in animals pretreated with anti-corticotropin (ACTH-γ-globulin although this result could suggest that CRF could preferentially stimulate the release of ACTH in the MPOA. Conversely, naloxone, anti-β-endorphin-γ-globulin, anti-CRF-γ-globulin and ACTH infused into the MPOA produced high levels of lordosis in female rats normally showing low levels of lordosis by treatment with low doses of OEB (2 μg) and normal doses of progesterone (500 μg). In each case the facilitation could be blocked by pretreatment of the MPOA site with a potent antagonist analogue of LH-RH. The results indicate the key role of LH-RH in the action of endogenous CRF, β-endorphin and ACTH in the regulation of lordosis behaviour. Each of these substances may act directly on the LH-RH neurone. The postulated presynaptic relationship between CRF and β-endorphin neuronal systems that seem to exist in the mediobasal hypothalamus and the central gray may be less predominant in the MPOA. The results, however, provide clear further evidence for the potent effects of CRF and opiocortin peptides in the regulation of LH-RH and reproduction.     

Translocation of protein kinase C-betaII in astrocytes requires organized actin cytoskeleton and is not accompanied by synchronous RACK1 relocation

Protein kinase C (PKC)-betaII is the most abundant PKC isoform in astrocytes. Upon activation, this isoform of PKC translocates from the cytosol to the plasma membrane (PM). In this study, we investigated in astrocytes the modality of PKC-betaII translocation as far as the participation of the receptor for activated C kinase-1 (RACK1) and the requirement for intact cytoskeleton in the process. In astrocytes, Western blots and immunocytochemistry coupled to confocal microscopic quantitative analysis showed that after 5 min of phorbol-12-myristate-13-acetate (PMA) exposure, native PKC-betaII, but not PKC-betaI, is relocated efficiently from the cytosol to the PM. Translocation of PKC-betaII was not associated with synchronous RACK1 relocation. Furthermore, the quantity of PM-associated PKC-betaII that co-immunoprecipitated with PM-bound RACK1 increased following PMA exposure, indicating a post activation binding of the two proteins in the PM. Because RACK1 and PKC-betaII relocation seemed not to be synchronous, we hypothesized that an intermediate interaction with the cytoskeleton was taking place. In fact, we were able to show that pharmacological disruption of actin-based cytoskeleton greatly deranged PKC-betaII translocation to the PM. The requirement for intact actin cytoskeleton was specific, because depolymerization of tubulin had no effect on the ability of the kinase to translocate to the PM. These results indicate that in astrocytes, RACK1 and PKC-betaII synchronous relocation is not essential for relocation of PKC-betaII to the PM. In addition, we show for the first time that the integrity of the actin cytoskeleton plays a specific role in PKC-betaII movements in these cells. We hypothesize that in glial cells, rapidly occurring changes of actin cytoskeleton arrangement may be involved in the fast reprogramming of PKC targeting to specific PM location to phosphorylate substrates in different cellular locations.     

Regionally different immunoreactivity for Smurf2 and pSmad2/3 in TDP‐43‐positive inclusions of amyotrophic lateral sclerosis

M. Nakamura, S. Kaneko, R. Wate, S. Asayama, Y. Nakamura, K. Fujita, H. Ito and H. Kusaka (2013) Neuropathology and Applied Neurobiology 39, 144–156 Regionally different immunoreactivity for Smurf2 and pSmad2/3 in TDP‐43‐positive inclusions of amyotrophic lateral sclerosis Aims: Smad ubiquitination regulatory factor‐2 (Smurf2), an E3 ubiquitin ligase, can interact with Smad proteins and promote their ubiquitin‐dependent degradation, thereby controlling the cellular levels of these signalling mediators. We previously reported that phosphorylated Smad2/3 (pSmad2/3) was sequestered in transactive response DNA‐binding protein‐43 (TDP‐43) inclusions in the spinal cord of patients with amyotrophic lateral sclerosis (ALS). Recent biochemical and immunohistochemical studies on spinal cord and brain of ALS patients demonstrated that the composition of the TDP‐43 inclusions is regionally distinct, suggesting different underlying pathogenic processes. We aimed to elucidate regional differences in pathomechanisms and composition of TDP‐43 inclusions in relation to pSmad2/3 and Smurf2. Methods: The spinal cord and brain tissues of 13 sporadic ALS (SALS) patients were investigated using immunohistochemical analysis. Results: TDP‐43‐positive inclusions in lower motor neurones of SALS patients were immunopositive for Smurf2 and pSmad2/3. Multiple immunofluorescence staining for Smurf2, pSmad2/3, TDP‐43 and ubiquitin revealed co‐localization of these four proteins within the inclusions in lower motor neurones of SALS patients. Furthermore, the loss of nuclear pSmad2/3 immunoreactivity was observed in cells bearing TDP‐43 inclusions. In contrast, TDP‐43‐positive inclusions in the extramotor neurones in the brain of SALS patients were noticeably negative for Smurf2 and pSmad2/3. In addition, pSmad2/3 immunoreactivity was preserved in the nuclei of inclusion‐bearing cells. Conclusions: This regional difference in the expression of Smurf2 and pSmad2/3 within TDP‐43‐positive inclusions might be one of the pathomechanisms underlying the loss of lower motor neurones and comparatively spared cortical neurones seen in ALS.     

Female Sex Hormones Influence the Febrile Response Induced by Lipopolysaccharide,Cytokines and Prostaglandins but not by Interleukin‐1β in Rats

There are differences in the immune response, and particularly fever, between males and females. In the present study, we investigated how the febrile responses induced by lipopolysaccharide (LPS) and different endogenous pyrogens were affected by female gonadal hormones. The febrile response to i.p. injection of LPS (50 μg/kg) was 40% lower in female rats compared to male or ovariectomised (OVX) female rats. Accordingly, oestrogen replacement in OVX animals reduced LPS‐induced fever. Treatment with the prostaglandin synthesis inhibitor indomethacin (2 mg/kg, i.p. 30 min before) reduced the febrile response induced by LPS in both OVX (88%) and sham‐operated (71%) rats. In line with the enhanced fever in OVX rats, there was increased expression of cyclooxygenase‐2 (COX‐2) in the hypothalamus and elevated levels of prostaglandin E₂ (PGE₂). In addition, OVX rats were hyper‐responsive to PGE₂ injected i.c.v. By contrast to the enhanced fever in response to LPS and PGE₂, the febrile response induced by i.c.v. injection of interleukin (IL)‐1β was unaffected by ovariectomy, whereas the responses induced by tumour necrosis factor (TNF)‐α and macrophage inflammatory protein (MIP)‐1α were completely abrogated. These results suggest that the mediators involved in the febrile response in females are similar to males, although the reduction of female hormones may decrease the responsiveness of some mediators such as TNF‐α and MIP‐1α. Compensatory mechanisms may be activated in females after ovariectomy such as an augmented synthesis of COX‐2 and PGE₂.     

The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel

Background: During intramembrane proteolysis of β‐amyloid protein precursor (βAPP) by presenilin (PS)/γ‐secretase, ε‐cleavages at the membrane‐cytoplasmic border precede γ‐cleavages at the middle of the transmembrane domain. Generation ratios of Aβ42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 production have been investigated by many pharmaceutical companies. Therefore, the ε‐cleavages, rather than the γ‐cleavage, might be more effective upstream targets for decreasing the relative generation of Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε‐cleavage‐derived), instead of that of Aβ42. Methods: Cell‐free γ‐secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI‐TOF MS analysis was carried out to detect the N‐termini of AICD species. Aβ and AICD species were measured by ELISA and immunoblotting techniques. Results: Effects on the ε‐cleavage by AD‐associated pathological mutations around the ε‐cleavage sites (i.e., βAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε‐cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aβ42 production. Only AICDε51, though, and not Aβ42 production, dramatically changed with modifications to the cell‐free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes. Conclusion: Our current data show that the generation ratio of AICDε51 and Aβ42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aβ42 generation, measurement of the relative level of Aβ42‐related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε‐cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε‐cleavage as a means for decreasing Aβ42 production.