Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling

To reduce damage from toxic insults such as glutamate excitotoxicity and oxidative stresses, neurons may deploy an array of neuroprotective mechanisms. Recent reports show that progranulin (PGRN) gene null or missense mutations leading to inactive protein, are linked to frontotemporal lobar degeneration (FTLD), suggesting that survival of certain neuronal populations needs full expression of functional PGRN. Here we show that extracellular PGRN stimulates phosphorylation/activation of the neuronal MEK/extracellular regulated kinase (ERK)/p90 ribosomal S6 kinase (p90RSK) and phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathways and rescues cortical neurons from cell death induced by glutamate or oxidative stress. Pharmacological inhibition of MEK/ERK/p90RSK signaling blocks the PGRN-induced phosphorylation and neuroprotection against glutamate toxicity while inhibition of either MEK/ERK/p90RSK or PI3K/Akt blocks PGRN protection against neurotoxin MPP⁺. Inhibition of both pathways had synergistic effects on PGRN-dependent neuroprotection against MPP⁺ toxicity suggesting both pathways contribute to the neuroprotective activities of PGRN. Extracellular PGRN is remarkably stable in neuronal cultures indicating neuroprotective activities are associated with full-length protein. Together, our data show that extracellular PGRN acts as a neuroprotective factor and support the hypothesis that in FTLD reduction of functional brain PGRN results in reduced survival signaling and decreased neuronal protection against excitotoxicity and oxidative stress leading to accelerated neuronal cell death. That extracellular PGRN has neuroprotective functions against toxic insults suggests that in vitro preparations of this protein may be used therapeutically.     

Nrf2 is closely related to allergic airway inflammatory responses induced by low-dose diesel exhaust particles in mice

We have recently reported that disruption of nuclear erythroid 2 P45-related factor 2 (Nrf2) enhances susceptibility to airway inflammatory responses induced by low-dose diesel exhaust particles (DEP) in mice. C57BL/6 Nrf2 knockout (Nrf2^−/−) mice and wild-type (Nrf2^+/+) mice were further exposed to low-dose DEP for 7 h/day, 5 days/week, for a maximum of 8 weeks. After exposure to DEP for 5 weeks, allergic airway inflammation was generated in the mice by intraperitoneal sensitization with OVA followed by intranasal challenge. Nrf2^−/− mice exposed to relatively low-dose DEP showed significantly increased percentage changes relative to the OVA alone group in terms of airway hyperresponsiveness (AHR) and inflammatory cells, levels of IL-5 and thymus and activation regulated chemokine (TARC) in bronchoalveolar lavage (BAL) fluid than did Nrf2^+/+ mice. Lung tissues of Nrf2^−/− mice after DEP exposure showed inflammatory cell infiltrates, and increased PAS staining-positive mucus cell hyperplasia. In contrast, the percentage changes relative to the OVA group in the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in whole blood was higher in Nrf2^+/+ mice than in Nrf2^−/− mice. By using Nrf2^−/− mice, it was shown for the first time that relatively low-dose DEP exposure induces oxidant stress, and that host anti-oxidant responses play a key role in the development of DEP-induced exacerbation of allergic airway inflammation.     

A study in male and female 5-HT transporter knockout rats: An animal model for anxiety and depression disorders

Human studies have shown that a reduction of 5-HT transporter (SERT) increases the vulnerability for anxiety and depression. Moreover, women are more vulnerable to develop depression and anxiety disorders than men. For that reason we hypothesized that homozygous 5-HT transporter knockout rat (SERT^−/−) models, especially female, are valuable and reliable animal models for humans with an increased vulnerability for anxiety- and depression-related disorders. As rats are extensively used in neuroscience research, we used the unique 5-HT transporter knockout rat, that was recently generated using N-ethyl-N-nitrosurea (ENU) -driven mutagenesis, to test this hypothesis. Behavioral testing revealed that male and female SERT^−/− rats spent less time in the center of the open field and spent less time on the open arm of the elevated plus maze compared with wild-type 5-HT transporter knockout rats (SERT^+/+). In the novelty suppressed feeding test, only male SERT^−/− rats showed a higher latency before starting to eat in a bright novel arena compared with SERT^+/+ controls. Both male and female SERT^−/− rats showed a higher escape latency from their home cage than SERT^+/+ littermates. Moreover, SERT^−/− rats were less mobile in the forced swim test, and sucrose consumption was reduced in SERT^−/− rats relative to SERT^+/+ rats. Both effects were sex-independent. Neurochemically, basal extracellular 5-HT levels were elevated to a similar extent in male and female SERT^−/− rats, which was not influenced by the selective 5-HT reuptake inhibitor citalopram. 5-HT immunostaining revealed no difference between SERT^+/+ and SERT^−/− rats in the dorsal raphe nuclei, in both males and females. These findings demonstrate that SERT^−/− rats show anxiety and depression-related behavior, independent of sex. Genetic inactivation of the SERT has apparently such a great impact on behavior, that hardly any differences are found between male and female rats. This knockout rat model may provide a valuable model to study anxiety- and depression-related disorders in male and female rats.     

LP-211 is a brain penetrant selective agonist for the serotonin 5-HT7 receptor

We have determined the pharmacological profile of the new serotonin 5-HT₇ receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211). Radioligand binding assays were performed on a panel of 5-HT receptor subtypes. The compound was also evaluated in vivo by examining its effect on body temperature regulation in mice lacking the 5-HT₇ receptor (5-HT₇^−/−) and their 5-HT₇^+/+ sibling controls. Disposition studies were performed in mice of both genotypes. It was found that LP-211 was brain penetrant and underwent metabolic degradation to 1-(2-diphenyl)piperazine (RA-7). In vitro binding assays revealed that RA-7 possessed higher 5-HT₇ receptor affinity than LP-211 and a better selectivity profile over a panel of 5-HT receptor subtypes. In vivo it was demonstrated that LP-211, and to a lesser degree RA-7, induced hypothermia in 5-HT₇^+/+ but not in 5-HT₇^−/− mice. Our results suggest that LP-211 can be used as a 5-HT₇ receptor agonist in vivo.     

Molecular and functional interactions between tumor necrosis factor-alpha receptors and the glutamatergic system in the mouse hippocampus: Implications for seizure susceptibility

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine acting on two distinct receptor subtypes, namely p55 and p75 receptors. TNF-alpha p55 and p75 receptor knockout mice were previously shown to display a decreased or enhanced susceptibility to seizures, respectively, suggesting intrinsic modifications in neuronal excitability. We investigated whether alterations in glutamate system function occur in these naive knockout mice with perturbed cytokine signaling that could explain their different propensity to develop seizures. Using Western blot analysis of hippocampal homogenates, we found that p55^−/− mice have decreased levels of membrane GluR3 and NR1 glutamate receptor subunits while GluR1, GluR2, GluR6/7 and NR2A/B were unchanged as compared to wild-type mice. In p75^−/− mice, GluR2, GluR3, GluR6/7 and NR2A/B glutamate receptor subunits were increased in the hippocampus while GluR1 and NR1 did not change. Extracellular single-cell recordings of the electrical activity of hippocampal neurons were carried out in anesthetized mice by standard electrophysiological techniques. Microiontophoretic application of glutamate increased the basal firing rate of hippocampal neurons in p75^−/− mice versus wild-type mice, and this effect was blocked by 2-amino-5-phosphopentanoic acid and 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione denoting the involvement of N-methyl-d-aspartic acid and AMPA receptors. In p55^−/− mice, hippocampal neurons responses to glutamate were similar to wild-type mice. Spontaneous glutamate release measured by in vivo hippocampal microdialysis was significantly decreased only in p55^−/− mice. No changes were observed in KCl-induced glutamate release in both receptor knockout mice strains versus wild-type mice. These findings highlight specific molecular and functional interactions between p55 and p75 receptor-mediated signaling and the glutamate system. These interactions may be relevant for controlling neuronal excitability in physiological and pathological conditions.     

Midkine-deficient mice delayed degeneration and regeneration after skeletal muscle injury

Midkine (MK), a heparin-binding growth factor, was previously found to be expressed in the rat myotube-forming stage. We investigated MK gene-deficient (Mdk^−/−) mice in terms of skeletal muscle degeneration and regeneration after injury by bupivacaine injection into the tibialis anterior muscle. Injured muscles showed intense inflammatory cell infiltration. Myotubes, myofibers with centrally located nuclei in their cytoplasm, were significantly smaller in Mdk^−/− mice than in wild type (Mdk^+/+) mice 7 days after injury (p = 0.02). The distribution of myotube sizes showed quantitative differences between the two groups at 5 and 7 days, but not at 14 days. Many small myotubes were found in the regenerative area of Mdk^−/− mice compared with that of Mdk^+/+mice 5 and 7 days after injury. The expression of Iba1, a macrophage marker, was significantly lower in Mdk^−/− mice 3 days after injury (p = 0.01). The number of desmin-positive cells like myoblasts in Mdk^−/− mice was significantly fewer than that in Mdk^+/+ mice 3 days after injury. Our results suggested that deletion of MK results in a delay in regeneration, preceded by decelerated migration of macrophages to the damaged area, and that MK has a role in cell differentiation and maturation after skeletal muscle injury.     

Enlarged extracellular space of aquaporin-4–deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers

Aquaporin-4 (AQP4) water channels expressed on glia have been implicated in maintaining the volume of extracellular space (ECS). A previous diffusion study employing small cation tetramethylammonium and a real-time iontophoretic (RTI) method demonstrated an increase of about 25% in the ECS volume fraction (α) in the neocortex of AQP4^−/− mice compared to AQP4^+/+ mice but no change in the hindrance imposed to diffusing molecules (tortuosity λ). In contrast, other diffusion studies employing large molecules (dextran polymers) and a fluorescence recovery after photobleaching (FRAP) method measured a decrease of about 10%–20% in λ in the neocortex of AQP4^−/− mice. These conflicting findings on λ would imply that large molecules diffuse more readily in the enlarged ECS of AQP4^−/− mice than in wild type but small molecules do not. To test this hypothesis, we used integrative optical imaging (IOI) to measure tortuosity with a small Alexa Fluor 488 (molecular weight [MW] 547, λ_AF) and two large dextran polymers (MW 3000, λ_dex3 and MW 75,000, λ_dex75) in the in vitro neocortex of AQP4^+/+ and AQP4^−/− mice. We found that λ_AF=1.59, λ_dex3=1.76 and λ_dex75=2.30 obtained in AQP4^−/− mice were not significantly different from λ_AF=1.61, λ_dex3=1.76, and λ_dex75=2.33 in AQP4^+/+ mice. These IOI results demonstrate that λ measured with small and large molecules each remain unchanged in the enlarged ECS of AQP4^−/− mice compared to values in AQP4^+/+ mice. Further analysis suggests that the FRAP method yields diffusion parameters not directly comparable with those obtained by IOI or RTI methods. Our findings have implications for the role of glial AQP4 in maintaining the ECS structure.     

Alzheimer neuropathology without frontotemporal lobar degeneration hallmarks (TAR DNA‐binding protein 43 inclusions) in missense progranulin mutation Cys139Arg

Null mutations in progranulin gene (GRN) reduce the progranulin production resulting in haploinsufficiency and are tightly associated with tau‐negative frontotemporal lobar degeneration with TAR DNA‐binding protein 43‐positive inclusions (FTLD‐TDP). Missense mutations of GRN were also identified, but their effects are not completely clear, in particular unanswered is the question of what neuropathology they elicit, also considering that their occurrence has been reported in patients with typical clinical features of Alzheimer disease. They describe two fraternal twins carrying the missense GRN Cys139Arg mutation affected by late‐onset dementia and we report the neuropathological study of one of them. Both patients were examined by neuroimaging, neuropsychological assessment and genetic analysis of GRN and other genes associated with dementia. The brain of one was obtained at autopsy and examined neuropathologically. One sister presented clinical and MRI features leading to the diagnosis of Alzheimer disease. The other underwent autopsy and the brain showed neuropathological hallmarks of Alzheimer disease with abundant Aβ‐amyloid deposition and Braak stage V of neurofibrillary pathology, in the absence of the hallmark lesions of FTLD‐TDP. Their findings may contribute to better clarify the role of progranulin in neurodegenerative diseases indicating that some GRN mutations, in particular missense ones, may act as strong risk factor for Alzheimer disease rather than induce FTLD‐TDP.     

Loss of tau elicits axonal degeneration in a mouse model of Alzheimer's disease

A central issue in the pathogenesis of tauopathy is the question of how tau protein dysfunction leads to neurodegeneration. We have previously demonstrated that the absence of tau protein is associated with destabilization of microtubules and impaired neurite outgrowth (13 and 64). We now hypothesize that the absence of functional tau protein may render the central nervous system more vulnerable to secondary insults such as the overexpression of mutated beta amyloid precursor protein (APP) and traumatic brain injury. We therefore crossed tau knockout mice (Dawson et al., 2001) to mice overexpressing a mutated human APP (APP_670,671, A_sw) (Hsiao et al., 1996) and created a mouse model (A_sw/mTau^−/−) that provides evidence that the loss of tau function causes degeneration of neuronal processes. The overexpression of APP_670,671 in tau knockout mice, elicits the extensive formation of axonal spheroids. While spheroids are only found associated with Aβ plaques in mice expressing APP_670,671 on an endogenous mouse tau background (Irizarry et al., 1997), A_sw/mTau^−/− mice have spheroids not only surrounding Aβ plaques but also in white matter tracks and in the neuropil. Plaque associated and neuropil dystrophic neurites and spheroids are prominent features of Alzheimer's disease (52, 74 and 72), and our current data suggests that loss of tau function may lead to neurodegeneration.     

Role of α7- and β4-Containing Nicotinic Acetylcholine Receptors in the Affective and Somatic Aspects of Nicotine Withdrawal: Studies in Knockout Mice

To assess which nicotinic acetylcholine receptors (nAChRs) are involved in the aversive aspects of nicotine withdrawal, brain reward function and the somatic signs of nicotine withdrawal were assessed in mice that lack α7 and β4 nAChR subunits. Brain reward function was assessed with the intracranial self-stimulation (ICSS) procedure, in which elevations in ICSS thresholds reflect an anhedonic mood state. At 3–6 h of spontaneous nicotine/saline withdrawal, thresholds were elevated in nicotine-withdrawing α7^+/+ and β4^+/+, but not α7^−/− or β4^−/−, mice compared with saline-withdrawing mice, indicating a delay in the onset of withdrawal in the knockout mice. From 8 to 100 h of withdrawal, thresholds in α7^+/+ and α7^−/− mice were equally elevated, whereas thresholds in β4^+/+ and β4^−/− mice returned to baseline levels. Somatic signs were attenuated in nicotine-withdrawing β4^−/−, but not α7^−/−, mice. Administration of a low dose of the nAChR antagonist mecamylamine induced threshold elevations in α7^−/−, but not α7^+/+, mice, whereas the highest dose tested only elevated thresholds in α7^+/+ mice. Mecamylamine-induced threshold elevations were similar in β4^−/− and β4^+/+ mice. In conclusion, null mutation of the α7 and β4 nAChR subunits resulted in a delayed onset of the anhedonic aspects of the spontaneous nicotine withdrawal syndrome. Previous findings of attenuated somatic signs of nicotine withdrawal in β4^−/−, but not α7^−/−, mice were confirmed in the present study, indicating an important role for β4-containing nAChRs in the somatic signs of nicotine withdrawal. The mecamylamine-precipitated withdrawal data suggest that compensatory adaptations may occur in constitutive α7^−/− mice or that mecamylamine may interact with other receptors besides nAChRs in these mice. In summary, the present results indicate an important role for α7 and β4-containing nAChRs in the anhedonic or somatic signs of nicotine withdrawal.     

Novel progranulin variants do not disrupt progranulin secretion and cleavage

A subset of frontotemporal dementia cases are neuropathologically defined by tau-negative, TAR DNA-binding protein-43, and ubiquitin-positive inclusions in the brain and are associated with mutations in the progranulin gene (GRN). Deep sequencing of families exhibiting late-onset dementia revealed several novel variants in GRN. Because of the small size of these families and limited availability of samples, it was not possible to determine whether the variants segregated with the disease. Furthermore, none of these families had autopsy confirmation of diagnosis. We sought to determine if these novel GRN variants alter progranulin (PGRN) protein stability, PGRN secretion, and PGRN cleavage in cultured cells. All the novel GRN variants behave like PGRN wild-type protein, suggesting that these variants represent rare polymorphisms. However, it remains possible that these variants affect other aspects of PGRN function or represent risk factors for dementia when combined with other modifying genes.     

Seizure resistance without parkinsonism in aged mice after tau reduction

Tau is an emerging target for Alzheimer's disease (AD) and other conditions with epileptiform activity. Genetic tau reduction (in Tau^+/− and Tau^−/− mice) prevents deficits in AD models and has an excitoprotective effect, increasing resistance to seizures, without causing apparent neuronal dysfunction. However, most studies of tau reduction have been conducted in <1-year-old mice, and the effects of tau reduction in aged mice are less clear. Specifically, whether the excitoprotective effects of tau reduction persist with aging is unknown and whether tau reduction causes neuronal dysfunction, including parkinsonism, with aging is controversial. Here, we performed a comprehensive analysis of 2-year-old Tau^+/+, Tau^+/−, and Tau^−/− mice. In aged mice, tau reduction still conferred resistance to pentylenetetrazole-induced seizures. Moreover, tau reduction did not cause parkinsonian abnormalities in dopamine levels or motor function and did not cause iron accumulation or impaired cognition, although Tau^−/− mice had mild hyperactivity and decreased brain weight. Importantly, the excitoprotective effect in aged Tau^+/− mice was not accompanied by detectable abnormalities, indicating that partially reducing tau or blocking its function may be a safe and effective therapeutic approach for AD and other conditions with increased excitability.     

B-1 cells promote immunosurveillance against murine melanoma in host absence of CCR5: New perspective in autologous vaccination therapy

Autologous vaccination with tumor-primed dendritic cells increases immune response against tumor, which seems to be improved in host absence of CCR5. Because B-1 lymphocytes modulate the activity of different immune cells, we decided to study their influence in the resistance against murine B16F10 melanoma in a CCR5 deprived environment. Adoptive transfer of peritoneal B-1 CCR5^+/+ lymphocytes to CCR5^−/− animals inhibited the establishment of lung metastasis and melanoma cell growth, in comparison to saline-treated CCR5^−/− mice. In loco cell analysis demonstrated that the adoptive transfer of B-1 CCR5^+/+ lymphocytes to CCR5 deficient host was associated with a more intense influx of T CD8⁺ to tumor site, indicating that the presence of CCR5^+/+ B-1 cells in the tumor environment induces the migration of T CD8 CCR5^−/− cells to the implantation site. To corroborate this idea, CCR5^−/− mice were injected with non B-1 peritoneal cells from wild type (WT) mice before B16F10 inoculation. In this regimen, CCR5^−/− mice were not protected from tumor growth reinforcing the idea that, in host absence of CCR5, B-1 cells are essential to confer tumor resistance. This work indicates that, in the host absence of CCR5, naive B-1 cells may activate CD8T lymphocytes thereby promoting tumor resistance. Our results strongly suggest that autologous vaccination with B-1 lymphocytes in combination with CCR5 antagonists can be an alternative approach to tumor therapy.     

Aggravated chronic brain injury after focal cerebral ischemia in aquaporin-4-deficient mice

The water channel aquaporin-4 (AQP4) is important in brain water homeostasis, and is also involved in astrocyte growth and glial scar formation. It has been reported that AQP4 deficiency attenuates acute ischemic brain injury as a result of reducing cytotoxic edema. Here, we determined whether AQP4 deficiency influences chronic brain injury after focal cerebral ischemia induced by 30 min of middle cerebral artery occlusion (MCAO). AQP4^−/− mice exhibited a lower survival rate and less body weight gain than wild-type mice, but their neurological deficits were similar to wild-type mice during 35 days after MCAO. At 35 days after MCAO, AQP4^−/− mice showed more severe brain atrophy and cavity formation in the ischemic hemisphere as well as more neuronal loss in the hippocampus. Furthermore, astrocyte proliferation and glial scar formation were impaired in AQP4^−/− mice. Therefore, AQP4 deficiency complicated by astrocyte dysfunction aggravates chronic brain injury after focal cerebral ischemia, suggesting that AQP4 may be important in the chronic phase of the post-ischemic recovery process.     

Mice lacking rhes show altered morphine analgesia,tolerance, and dependence

Rhes, the Ras Homolog Enriched in Striatum, is an intermediate-size GTP binding protein. Although its full functions are not yet known, it has been shown to affect signaling and behaviors mediated by G protein-coupled receptors. Here we have tested whether Rhes affects behaviors mediated by opioid receptors. Wild type and rhes-deficient mice were administered morphine and tested for analgesia in formalin and tail flick tests. Rhes^−/− mice showed significantly enhanced analgesia in both tests relative to rhes^+/+ mice. Furthermore, rhes^−/− mice did not display tolerance to repeated morphine administration and displayed significantly less withdrawal than rhes^+/+ mice. These findings indicate that Rhes is involved in behaviors mediated by mu opioid receptors and in the adaptive response to repeated morphine administration.     

In vivo anti- and pro-tumour activities of the TLR2 ligand FSL-1

TLR ligands as Th1 inducers have been investigated as potential anti-tumour agents. However, few attempts have been made to investigate the anti-tumour activity of TLR ligands as Th2 inducers. This study, therefore, was carried out to determine whether the TLR2 ligand FSL-1 as a Th2 inducers affects the growth of a QRsP tumour, a fibrosarcoma derived from the C57BL/6 (TLR2^+/+) mouse in vivo. Tumour volumes in TLR2^+/+ mice immunized with both FSL-1 and tumour-associated antigens were significantly smaller than those in control mice. Immunization with both FSL-1 and tumour-associated antigens increased the survival rate of TLR2^+/+ mice. However, surprisingly, immunization with FSL-1 alone significantly enhanced the growth of tumour. Both anti- and pro-tumour activities of FSL-1 were not observed in TLR2^−/− mice. Immunization of both FSL-1 and tumour-associated antigens induced tumour-associated antigen-specific cytolytic T cells, antibody-dependent cell-mediated cytotoxicity of natural killer cells by production of the tumour-specific antibodies, tumour lysis by complement activation and reduction of the number of regulatory T cells in the draining lymph node. Immunization with FSL-1 alone increased the number of regulatory T cells in the draining lymph node, and in vivo administration of anti-CD25 antibody into mice abrogated the pro-tumour activity of FSL-1, suggesting that regulatory T cells are involved in the pro-tumour activity.This study demonstrated that FSL-1 exhibited TLR2-mediated anti- and pro-tumour activities when immunized with and without tumour-associated antigens, respectively.     

Role of Interleukin-6 in Immune Complex Induced Models of Vascular Injury

Previous studies have suggested that Interleukin-6 (IL-6) acts as a marker of vasculitis. To determine the role of IL-6 in vasculitis we utilized two models of immune complex induced vascular injury (dermal Arthus and acute pulmonary alveolitis) in IL-6 deficient (IL-6^−/−) and IL-6 sufficient (IL-6^+/+) mice. Plasma and bronchoalveolar lavage (BAL) levels of IL-6 were elevated in the injured IL-6^+/+ mice with acute alveolitis and in the plasma of IL-6^+/+ mice with dermal Arthus vasculitis. While, IL-6 levels in IL-6^−/− mice were near or below the levels of detection. Histological examination of the intensity of vascular injury response demonstrated no significant differences between IL-6^−/− and IL6^+/+ mice. More specifically, lung permeability (total protein in the BAL) in the lung injury model in IL-6^−/− mice was the same as injured IL-6^+/+ mice. As a corollary, assessment of vascular permeability in both models was the same in the IL-6^−/− as the IL-6^+/+ mice. Quantification of leukocyte influx into the injured tissues in both models also revealed no differences between the IL-6^−/− and IL-6^+/+ mice. These data demonstrate that while IL-6 is upregulated in acute vascular injury it does not appear to be critical in the development of the vascular inflammatory response.This work is supported in part by: Pfizer Global Research & Development