Deterioration of axotomy-induced neurodegeneration by group IIA secretory phospholipase A2.

Phospholipase A2 (PLA2) is proposed to play a role in the repair of the ruptured membrane after axotomy. In neonatal rats, we examined the effect of Group IIA secretory PLA2 (sPLA2-IIA) on axotomy-induced cell death of motoneurons. sPLA2-IIA significantly induced death of axotomized motoneurons. Indoxam, a specific inhibitor for sPLA2-IIA, protected motoneurons from the sPLA2-IIA-induced deterioration. The present study indicated that sPLA2-IIA possessed neurotoxic effect rather than neuroprotective effect against facial nerve.     

Apoptotic neurodegeneration following trauma is markedly enhanced in the immature brain.

Age dependency of apoptotic neurodegeneration was studied in the developing rat brain after percussion head trauma. In 7-day-old rats, mechanical trauma, applied by means of a weight drop device, was shown to trigger widespread cell death in the hemisphere ipsilateral to the trauma site, which first appeared at 6 hours, peaked at 24 hours, and subsided by 5 days after trauma. Ultrastructurally, degenerating neurons displayed features consistent with apoptosis. A decrease of bcl-2 in conjunction with an increase of c-jun mRNA levels, which were evident at 1 hour after trauma and were accompanied by elevation of CPP 32-like proteolytic activity and oligonucleosomes in vulnerable brain regions, confirmed the apoptotic nature of this process. Severity of trauma-triggered apoptosis in the brains of 3- to 30-day-old rats was age dependent, was highest in 3- and 7-day-old animals, and demonstrated a subsequent rapid decline. Adjusting the mechanical force in accordance with age-specific brain weights revealed a similar vulnerability profile. Thus, apoptotic neurodegeneration contributes in an age-dependent fashion to neuropathological outcome after head trauma, with the immature brain being exceedingly vulnerable. These results help explain unfavorable outcomes of very young pediatric head trauma patients and imply that, in this group, an antiapoptotic regimen may constitute a successful neuroprotective approach.     

Cytosolic phospholipase A alpha modulates NMDA neurotoxicity in mouse hippocampal cultures

The arachidonic acid-specific cytosolic phospholipase A(2) alpha (cPLA(2)alpha) has been implicated in the generation of neurological injuries. cPLA(2)alpha-dependent neurological injury has been postulated to be mediated through inflammatory and eicosanoid pathways. We determined if cPLA(2)alpha amplifies the injury of a non-inflammatory, excitotoxic stimulus by modifying a well-described toxicity assay to measure the toxicity of N-methyl-d-aspartate (NMDA) in the CA1 region of organotypic, mouse hippocampal cultures. Hippocampal cultures from wild-type and cPLA(2)alpha knockout mice were exposed to 5, 7.5 or 10 microm NMDA for 1 h. Toxicity was measured 23 h later. Cultures derived from cPLA(2)alpha(-/-) mice and cultures treated with the selective inhibitor AACOCF(3) were significantly protected from NMDA toxicity, as compared with wild-type cultures. To determine if cPLA(2)alpha-dependent toxicity is cyclooxygenase (COX)-2 dependent, COX-2 and PGE(2) levels were measured 7 and 25 h after NMDA treatment. NMDA treatment failed to induce COX-2 protein or increase PGE(2) in the culture media in either genotype at either time. In contrast, phorbol 12-myristate 13-acetate and ionophore treatment caused robust induction of COX-2 and PGE(2) in both genotypes. We conclude that cPLA(2)alpha may have a hitherto unrecognized direct effect on excitatory neurotoxicity, suggesting that cPLA(2)alpha inhibition is a therapeutic candidate for treatment of the early, excitotoxic injury observed in stroke.     

The SHH/Gli pathway is reactivated in reactive glia and drives proliferation in response to neurodegeneration‐induced lesions

In response to neurodegeneration, the adult mammalian brain activates a cellular cascade that results in reactive astrogliosis and microgliosis. The mechanism through which astrocytes become reactive and the physiological consequences of their activation in response to neurodegeneration is complex. While the activation and proliferation of astrocytes has been shown to occur during massive neuronal cell death, the functional relationship between these two events has not been clearly elucidated. Here we show that in response to kainic acid‐ (KA) induced neurodegeneration, the mitogen sonic hedgehog (SHH) is upregulated in reactive astrocytes. SHH activity peaks at 7 days and is accompanied by increased Gli activity and elevated proliferation in several cell types. To determine the functional role of SHH‐Gli signaling following KA lesions, we used a pharmacological approach to show that SHH secreted by astrocytes drives the activation and proliferation of astrocytes and microglia. The consequences of SHH‐Gli signaling in KA‐induced lesions appear to be independent of the severity of neurodegeneration. GLIA 2014;62:1595–1607     

Apolipoprotein E is upregulated in olfactory bulb glia following peripheral receptor lesion in mice.

Apolipoprotein E (apoE), a lipid transporting protein, has been postulated to participate in nerve regeneration. To better clarify apoE function in the olfactory system, we evaluated the amount and distribution of apoE in the olfactory bulb following olfactory nerve lesion in mice. Olfactory nerve was lesioned in 2- to 4-month-old mice by intranasal irrigation with Triton X-100. Olfactory bulbs were collected at 0, 3, 7, 21, 42, and 56 days postlesion, and both apoE concentrations and apoE distribution were determined. ApoE levels, as determined by immunoblot analysis, were twofold greater than normal during nerve degeneration at 3 days. ApoE levels remained elevated by approximately 1.5 times normal levels at 7 through 21 days after injury and returned to baseline by 56 days. Immunocytochemical studies supported these observations. ApoE immunoreactivity was prominent on the olfactory nerve at 3 days after lesion and decreased to baseline levels at later time periods. Double-labeling immunocytochemical studies confirmed that both reactive astroglia and microglia produced detectable amounts of apoE following the lesion. Return of apoE expression to baseline paralleled measures of olfactory nerve maturation as measured by olfactory marker protein. These data suggest that apoE increases concurrent with nerve degeneration. ApoE may facilitate efficient regeneration perhaps by recycling lipids from degenerating fibers for use by growing axons. The association of apoE genotype with dementing illnesses may represent a diminished ability to support a lifetime of nerve regeneration.     

Epidermal growth factor receptor in proliferating reactive glia following transient focal ischemia in the rat brain

Severe transient focal cerebral ischemia causes brain infarction with a strong glial reaction. We have studied whether postischemic reactive glial cells express epidermal growth factor receptor (EGFR) following middle cerebral artery occlusion in the rat. We have also looked for signs of proliferating activity, as EGFR is known to be involved in cell growth and proliferation in certain non-neural cells. EGFR was studied using three different antibodies which were found to stain for a tyrosine-phosphorylated protein (p170) corresponding to the membrane-anchored EGFR. Neurons of the control brain were strongly immunoreactive to EGFR, but a decrease of EGFR-immunoreactivity was seen in the ipsilateral brain side from 24 h postischemia due to neuronal loss. However, the presence of abundant glial cells strongly immunoreactive to EGFR became apparent in this area from 4 days postischemia onward. The use of microglial (lectin or OX-42) and astroglial (GFAP) markers showed that these postischemic EGFR-stained cells were reactive microglia/macrophages and astroglia. The subcellular localization of EGFR in reactive microglia/macrophages was compatible with the network of the Golgi apparatus, as revealed with an antibody against a peripheral membrane-bound protein of the Golgi. The presence of abundant proliferating cells in the ischemic brain was detected from 4 days postischemia with an antibody against proliferating cell nuclear antigen. Proliferating reactive microglia/macrophages were abundant within the infarcted brain side, whereas proliferating astrocytes were found mainly in the immediate periphery of the infarct limiting the necrotic area from the undamaged tissue. These proliferating cells were immunoreactive to EGFR. The results show the presence of EGFR in postischemic reactive glial cells and suggest that EGFR-dependent pathways mediate signal transduction in reactive glia following transient focal cerebral ischemia. GLIA 23:120–129, 1998. © 1998 Wiley-Liss, Inc.     

Lipoprotein-associated phospholipase A2 is associated with risk of dementia

OBJECTIVE: High levels of the inflammatory marker lipoprotein-associated phospholipase A2 (Lp-PLA2) have been proposed to be a predictor of coronary heart disease and stroke. Because both inflammation and vascular disease are associated with dementia, the objective of the present study was to examine the association between Lp-PLA2 and the risk of dementia. METHODS: Within the Rotterdam Study, a population-based prospective cohort study, we performed a case-cohort study. Of the 6,713 participants at risk for dementia, a random sample of 1,742 individuals was drawn. During follow-up (mean, 5.7 years), 302 incident dementia cases were identified. Cox proportional hazard models were used to estimate the association of Lp-PLA2 and dementia. RESULTS: We found that subjects with higher levels of Lp-PLA2 had an increased risk of dementia. Compared with the first quartile of Lp-PLA2, age- and sex-adjusted hazard ratios (HRs; 95% confidence interval [CI]) for dementia for the second, third, and fourth quartiles were 1.19 (0.78-1.81), 1.15 (0.74-1.79), and 1.56 (1.03-2.37), respectively (p value for trend 0.04). Additional adjustment for cardiovascular and inflammatory factors did not change the estimates. INTERPRETATION: This is the first study to our knowledge that shows that Lp-PLA2 is associated with the risk of dementia independent of cardiovascular and inflammatory factors and provides evidence for a potential role of Lp-PLA2 in identifying subjects at risk for dementia.     

Deterioration of axotomy-induced neurodegeneration by group IIA secretory phospholipase A2

Phospholipase A₂ (PLA₂) is proposed to play a role in the repair of the ruptured membrane after axotomy. In neonatal rats, we examined the effect of Group IIA secretory PLA₂ (sPLA₂-IIA) on axotomy-induced cell death of motoneurons. sPLA₂-IIA significantly induced death of axotomized motoneurons. Indoxam, a specific inhibitor for sPLA₂-IIA, protected motoneurons from the sPLA₂-IIA-induced deterioration. The present study indicated that sPLA₂-IIA possessed neurotoxic effect rather than neuroprotective effect against facial nerve.     

Spatiotemporal progression of neurodegeneration and glia activation in the wobbler neuropathy of the mouse

The wobbler mouse (phenotype WR; genotype wr/wr) has been investigated as a model for neurodegenerative diseases like SMA and ALS. A new diagnostic marker based on a polymorphism in the closely linked chaperonine gene Cct4 enabled us to diagnose the allelic status at the wr locus within the original background strain C57BL/6. Using this marker, we investigated the spatiotemporal progression of neuropathology in WR mice from postnatal day (d.p.n.) 10 to 60. Neurodegeneration starts at 13 d.p.n. in the thalamus (N. ventralis), in deep cerebellar nuclei, brain stem (N. vestibularis) and spinal cord interneurons. The motor nuclei of spinal nerves and motoneurons degenerate from 15 d.p.n. onward. Reactive astrocytes are observed around 17 d.p.n. in the white and grey matter of the spinal cord. Microgliosis occurs only from 23 d.p.n. onward. Our data demonstrate that in the WR disease, neurodegeneration in thalamus, cerebellum, and brain stem precedes motoneuron degeneration, astrogliosis and microgliosis.     

Expression and location of mRNAs encoding multiple forms of secretory phospholipase A2 in the rat retina

Low-molecular-weight secretory phospholipases A(2) (sPLA(2)s) are a subgroup of PLA(2)s, which are secreted, bind to receptors, and may act as intercellular signaling modulators. At least 10 different groups have been characterized in mammals, and there is expanding evidence of the significance of sPLA(2)s in neuronal signaling and survival [Kolko et al. (1996) J. Biol. Chem. 271: 32722-32728]. To date, no retinal sPLA(2)s have been cloned or characterized. We evaluated the existence and abundance of sPLA(2) subtypes in rat retina and explored their possible involvement in light-induced retinal damage. We designed primers to identify the sPLA(2)s in rat retina, based on known sequences of sPLA(2)-specific mRNAs in other tissues. RNA was isolated from rat retina, and cDNA was produced and used for PCR cloning to identify the novel subtypes of sPLA(2). Our study revealed the presence of mRNAs encoding sPLA(2)-IB, -X, -V, -IIE, -IIA, and -IIF in the retina, and quantification by real-time PCR revealed different abundances of the sPLA(2)s. We showed a time-dependent gene induction of sPLA(2)-X, -IB, and -V in light-induced retinal damage. We further explored the location of sPLA(2)-IB by in situ hybridization and immunohistochemistry. This study is the first to reveal the presence, abundance, and induction of mRNAs encoding sPLA(2)s in rat retina. We suggest that these enzymes are themselves intercellular signaling modulators of retinal cell function and perhaps also of retinal degeneration.     

Severe periodontitis is associated with the serum levels of hypersensitive C reactive protein and lipoprotein-associated phospholipase A2 in the patients of acute ischemic stroke

BackgroundPeriodontitis is associated with the pathogenesis of atherosclerotic plaque, and hypersensitive C reactive protein (hs-CRP) and lipoprotein-associated phospholipase A2 (Lp-PLA2) are the serum biomarkers of the stability of atherosclerotic plaque. Whether periodontitis is associated with the serum level of hs-CRP and Lp-PLA2 of acute ischemic stroke remains unclear.Material and MethodsWe recruited 103 cases with acute ischemic stroke within 7 days after stroke onset. Pocket depth and clinical attachment loss were assessed by oral examination to define the severe periodontitis. Demographic information including gender, age and body weight index, income level, education level, past medical history include smoking history, drinking history, ischemic stroke history, coronary heart disease, hypertension, diabetes and hyperlipidemia were collected, and serum biomarkers including white blood cell (WBC), fibrinogen, total cholesterol (TC), triglyceride (TG), lower density lipoprotein (LDL-C), high density lipoprotein (HDL-C), hs-CRP, HemoglobinA1c (HbAlc), Homocysteine (HCY) and Lp-PLA2 were tested.Results65 (63.1%) cases were diagnosed as severe periodontitis. Severe periodontitis group showed more male, age, drinking history, higher levels of hs-CRP and Lp-PLA2. Multivariate logistic regression showed that severe periodontitis was were significantly associated with hs-CRP (OR = 2.367, 95%CI: 1.182–4.738; P = .015) and Lp-PLA2 (OR = 2.577, 95% CI: 1.010–6.574; P = .048).ConclusionsSevere periodontitis is independently associated with the serum Level of hs-CRP and Lp-PLA2 in patients with acute ischemic stroke. Whether the improvement of periodontitis could decrease the occurrence and re-occurrence of ischemic stroke by stablizating atherosclerotic plaque need be further studied in future.     

Microglial response is poorly correlated with neurodegeneration following chronic, low-dose MPTP administration in monkeys

Many investigators have reported extensive microglial activation in the mouse substantia nigra and striatum following acute, high-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum in monkeys that had received a partial dopaminergic lesion using a low-dose, chronic MPTP administration paradigm. To characterize the microglial response, we utilized HLA-DR (LN3) to immunolabel the class II major histocompatibility complex (MHC II). In MPTP-treated monkeys, there was an intense microglial response in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically heterogeneous, with commingled ramified, activated, and multicellular morphologies throughout the extent of these basal ganglia structures. Surprisingly, there was little evidence of microglial reactivity in the striatum despite evidence of neurodegeneration-by silver labeling and by loss of TH immunolabeling. Moreover, this pattern of microglial reactivity was the same in all animals that had received MPTP and seemed to be independent of the degree of neurotoxin-induced neurodegeneration. Thus, we conclude that microglial reactivity, per se, is not consistently associated with neurodegeneration, but depends on regional differences.     

Cytosolic phospholipase A2 (cPLA2) distribution in murine brain and functional studies indicate that cPLA2 does not participate in muscarinic receptor-mediated signaling in neurons

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of membrane phospholipids and has been suggested as an effector in the receptor-mediated release of arachidonic acid in signal transduction. The potential role of cPLA2 as an effector in muscarinic acetylcholine receptor signaling was investigated through ectopic expression of either the m1 or m5 receptor in combination with cPLA2 in COS-1, CHO and U-373 MG cell lines. U-373 MG and COS-1 cells express undetectable or very low levels of cPLA2. CHO cell extracts are characterized by a significant endogenous PLA2 activity that was increased over 20-fold following transient expression with cPLA2 cDNA. However, in none of the cells lines did the co-expression of muscarinic receptor and cPLA2 result in a significant increase in muscarinic receptor-mediated arachidonic acid release over cells expressing muscarinic receptor alone. The distribution of cPLA2 mRNA and cPLA2 immunoreactivity in murine brain were determined in order to investigate a potential role for cPLA2 in neurotransmission. cPLA2 mRNA was expressed in white matter, including cells contained within linear arrays characteristic of interfascicular oligodendrocytes. cPLA2 immunoreactivity in white matter was evident throughout the processes of fibrous astrocytes. cPLA2 expression in gray matter was confined to astrocytes at the pial surface of the brain. cPLA2 mRNA was detected in pia mater, both at the brain surface and inner core of the choroid plexus. cPLA2 may not be directly linked to neurotransmission since enzyme expression, mRNA, and cPLA2 immunoreactivity were undetectable in neurons of murine brain. Support or regulation of neurotransmission may be provided through the activity of cPLA2 in glial cells.     

Inhibition of sodium glucose cotransporters following status epilepticus induced by intrahippocampal pilocarpine affects neurodegeneration process in hippocampus

Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, starting from secondary functional disorders due to several insults, including self-sustaining continuous seizures identified as status epilepticus (SE). Although hypoglycemia has been associated with SE, the effect of inhibition of the Na⁺/glucose cotransporters (SGLTs) on hippocampus during SE is still unknown. Here we evaluated the functional role of SGLT in the pattern of limbic seizures and neurodegeneration process after pilocarpine (PILO)-induced SE. Vehicle (VEH, 1 μL) or phlorizin, a specific SGLT inhibitor (PZN, 1 μL, 50 μg/μL), was administered in the hippocampus of rats 30 min before PILO (VEH + PILO or PZN + PILO, respectively). The limbic seizures were classified using the Racine's scale, and the amount of wet dog shakes (WDS) was quantified before and during SE. Neurodegeneration process was evaluated by Fluoro-Jade C (FJ-C), and FJ-C-positive neurons (FJ-C +) were counted 24 h and 15 days after SE. The PZN-treated rats showed higher (p < 0.05) number of WDS when compared with VEH + PILO. There was no difference in seizure severity between PZN + PILO and VEH + PILO groups. However, the pattern of limbic seizures significantly changed in PZN + PILO. Indeed, the class 5 seizures repeated themselves more times (p < 0.05) than the other classes in the PZN group at 50 min after SE induction. The PZN + PILO animals had a higher (p < 0.05) number of FJ-C + cells in the dentate gyrus (DG), hilus, and CA3 and CA1 of hippocampus, when compared with VEH + PILO. The PZN + PILO animals had a decreased number (p < 0.05) of FJ-C + cells in CA1 compared with VEH + PILO 15 days after SE induction. Taken together, our data suggest that SGLT inhibition with PZN increased the severity of limbic seizures during SE and increased neurodegeneration in hippocampus 24 h after SE, suggesting that SGLT1 and SGLT2 could participate in the modulation of earlier stages of epileptogenic processes.