Shuei Sugama

A pivotal role of matrix metalloproteinase-3 activity in dopaminergic neuronal degeneration via microglial activation

Recent studies have demonstrated that activated microglia play an important role in dopamine (DA) neuronal degeneration in Parkinson disease (PD) by generating NADPH‐oxidase (NADPHO)‐derived superoxide. However, the molecular mechanisms that underlie microglial activation in DA cell death are still disputed. We report here that matrix metalloproteinase‐3 (MMP‐3) was newly induced and activated in stressed DA cells, and the active form of MMP‐3 (actMMP‐3) was released into the medium. The released actMMP‐3, as well as catalytically active recombinant MMP‐3 (cMMP‐3) led to microglial activation and superoxide generation in microglia and enhanced DA cell death. cMMP‐3 caused DA cell death in mesencephalic neuron‐glia mixed culture of wild‐type (WT) mice, but this was attenuated in the culture of NADPHO subunit null mice (gp91phox‐/‐), suggesting that NADPHO mediated the cMMP‐3‐induced microglial production of superoxide and DA cell death. Furthermore, in the N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐injected animal model of PD, nigrostriatal DA neuronal degeneration, microglial activation, and superoxide generation were largely attenuated in MMP‐3‐/‐mice. These results indicate that actMMP‐3 released from stressed DA neurons is responsible for microglial activation and generation of NADPHO‐derived superoxide and eventually enhances nigrostriatal DA neuronal degeneration. Our results could lead to a novel therapeutic approach to PD. Kim, Y. S., Choi, D. H., Block, M. L., Lorenzl, S., Yang, L., Kim, Y. J., Sugama, S., Cho, B. P., Ywang, O., Browne, S. E., Kim, S. Y., Hong, J.‐S., Beal, M. F., Jon, T. H. A pivotal role of matrix metalloproteinase‐3 activity in dopaminergic neuronal degeneration via microglial activation. FASEB J. 21, 179–187 (2007)

Minocycline enhances MPTP toxicity to dopaminergic neurons.

Minocycline has been shown previously to have beneficial effects against ischemia in rats as well as neuroprotective properties against excitotoxic damage in vitro, nigral cell loss via 6‐hydroxydopamine, and to prolong the life‐span of transgenic mouse models of Huntingtons disease (HD) and amyotrophic lateral sclerosis (ALS). We investigated whether minocycline would protect against toxic effects of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), a toxin that selectively destroys nigrostriatal dopaminergic (DA) neurons and produces a clinical state similar to Parkinsons disease (PD) in rodents and primates. We found that although minocycline inhibited microglial activation, it significantly exacerbated MPTP‐induced damage to DA neurons. We present evidence suggesting that this effect may be due to inhibition of DA and 1‐methyl‐4‐phenylpridium (MPP+) uptake into striatal vesicles.

Pathological dynamics of activated microglia following medial forebrain bundle transection

To elucidate the role and pathological dynamics of activated microglia, this study assessed the phagocytic, immunophenotypic, morphological, and migratory properties of activated microglia in the medial forebrain bundle (MFB) axotomized rat brain. Activated microglia were identified using two different monoclonal antibodies: ED1 for phagocytic activity and OX6 for major histocompatibility complex (MHC) class II. Phagocytic microglia, characterized by ED1‐immunoreactivity or ED1‐ and OX6‐immunoreactivity, appeared in the MFB and substantia nigra (SN) as early as 1–3 days post‐lesion (dpl), when there was no apparent loss of SN dopamine (DA) neurons. Thereafter, a great number of activated microglia selectively adhered to degenerating axons, dendrites and DA neuronal somas of the SN. This was followed by significant loss of these fibers and nigral DA neurons. Activation of microglia into phagocytic stage was most pronounced between 14∼28 dpl and gradually subsided, but phagocytic microglia persisted until 70 dpl, the last time point examined. ED1 expression preceded MHC II expression in phagocytic microglia. All phagocytic microglia sticking to DA neurons showed activated but ramified form with enlarged somas and thickened processes. They were recruited to the SNc from cranial, dorsal and ventral aspects along various structures and finally stuck to DA neurons of the SNc. Characteristic rod‐shaped microglia in the white matter were thought to migrate a long distance. The present study strongly suggests that neurons undergoing delayed neurodegeneration may be phagocytosed by numerous phagocytic, ramified microglia at various sites where specific surface signals are exposed or diffusible molecules are released.

Neurons of the superior nucleus of the medial habenula and ependymal cells express IL-18 in rat CNS.

The habenular-interpeduncular pathway is involved in the modulation of several functions including neuroendocrine and stress responses. Interleukin-18 (IL-18) is a pro-inflammatory cytokine predominantly studied as a modulator of immune functions and also produced in the adrenal cortex following activation of the hypothalamic-pituitary-adrenal axis. In the central nervous system, IL-18 was demonstrated to induce sleep and to influence long-term potentiation and was proposed to mediate local inflammatory reactions. The present study investigated the localization of IL-18 and its expression following either acute or chronic restraint stress in the brain of adult male Wistar rats. Using immunocytochemistry and in situ hybridization we report the unprecedented localization of IL-18 in the neurons of the superior part of the medial habenula (MHbS), their projections to the interpenducular nucleus and its expression in the ependymal cells surrounding the third and the lateral ventricles. In addition, acute (2 h) or chronic (6 h/day for 3 weeks) restraint stress induced a strong elevation of IL-18 immunostaining in the MHbS but not in ependymal cells. The present data suggest that IL-18 may participate in the modulation of stress responses in the MHbS. They also suggest that ependymal cells may be the source of IL-18 previously reported in the cerebrospinal fluid (CSF). The role of IL-18 in the ependyma and the CSF remains to be elucidated.

Temporal and sequential analysis of microglia in the substantia nigra following medial forebrain bundle axotomy in rat

Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis after transection of the medial forebrain bundle. We have assessed the temporal and sequential activities of microglia in these events by examining the complement-3 (OX-42), major histocompatibility complex class II antigen presentation (OX-6) and phagocytic activity (ED1), and correlating these indicators with dopaminergic neuronal loss. Microglia in the ipsilateral substantia nigra pars reticulata evinced activation morphology at 12 h postaxotomy. Phagocytic microglia apposed dying dopaminergic neurons in the pars compacta starting at 3 days postlesion; their number increased through 14 days and slowly decreased. Nuclear chromatin condensation and significant loss of tyrosine hydroxylase-positive dopaminergic neurons occurred around 7 days postlesion. In contrast to microglial expression of interleukin-1beta and inducible nitric oxide synthase at the axotomy site, nigral microglia were interleukin-1beta and inducible nitric oxide synthase-negative. Consistently, RNase protection assays showed that interleukin-1beta and inducible nitric oxide synthase transcripts in nigra were equivocal. The present data support the idea that phagocytosis of axotomized neurons by activated microglia is not limited to dead neurons but includes dying neurons probably without cytotoxic effects of inflammatory substances, such as interleukin-1beta or nitric oxide.

Tissue-Specific Expression of Rat IL-18 Gene and Response to Adrenocorticotropic Hormone Treatment

IL-18 is a pleiotropic cytokine also proposed to have a role in modulating immune function during stress. Initially found in immune cells, IL-18 mRNA is detectable in several tissues including the cells of the zona reticularis and the zona fasciculata of the adrenal cortex, where its levels are elevated by acute stress or adrenocorticotropic hormone treatment. In the present study, we compared the expression of IL-18 in the adrenal cortex with that of spleen and duodenum, two other IL-18-positive tissues. In situ hybridization showed that, in contrast to the adrenal cortex, in spleen and duodenum IL-18 is primarily localized to immune cells. In duodenum, IL-18 mRNA was also detectable in epithelial cells. Northern blot demonstrated that while the adrenal gland synthesized IL-18 mRNA of 1.1 kb, spleen and duodenum produced a 0.9-kb transcript. RT-PCR, sequencing, Western blot, primer extension, and rapid amplification of cDNA end analysis demonstrated that the three tissues synthesize IL-18 mRNAs containing the same coding region and produce the same IL-18 peptide, but differ in the length of their 5′-untranslated region, indicating tissue-specific usage of the promoter region. Finally, in contrast to the adrenal gland, adrenocorticotropic hormone treatment did not increase the levels of IL-18 mRNA in spleen and duodenum. These results demonstrate tissue-specific expression and promoter usage of IL-18 gene and suggest that the adrenal cortex and not immune cells may be the source of IL-18 during stress.

Modulation of IL-18 Production in the Adrenal Cortex following Acute ACTH or Chronic Corticosterone Treatment

Interleukin (IL)-18 is a proinflammatory cytokine and a stimulator of cell-mediated immune responses. We have previously reported that acute stress stimulates the production of IL-18 mRNA in the glucocorticoid (GC)-producing cells of the adrenal cortex. In order to investigate the mechanisms governing the expression of IL-18 in the adrenal cortex, the effects of acute ACTH or chronic corticosterone treatment on the levels of IL-18 mRNA and protein were examined by in situ hybridization and Northern and Western blot assays. Adult male Sprague-Dawley rats received a subcutaneous injection of ACTH or subcutaneous implantation of slow-release corticosterone pellets followed by an injection of saline or ACTH. After 4 h, ACTH induced a 4-fold increase in IL-18 mRNA levels and elevated the content of pro-IL-18 peptide. Six days of chronic corticosterone treatment did not alter the basal levels of IL-18 mRNA and reduced those of pro-IL-18. Finally, ACTH treatment of animals under the corticosterone regimen induced a 2-fold increase in IL-18 mRNA and elevated the levels of the pro-IL-18 protein. The levels of the precursor, p45, and the active subunit p10 peptides of the IL-18-processing enzyme, IL-1β-converting enzyme (ICE), showed no substantial differences in all the conditions tested. IL-1β was not detected under these experimental conditions. These data demonstrate that the production of IL-18 in the adrenal cortex is stimulated by ACTH treatment and is not inhibited by the direct action of corticosterone. In contrast to the anti-inflammatory action of GCs, IL-18 may have an immunostimulatory role during acute stress.

Microglial Phagocytosis of Dopamine Neurons at Early Phases of Apoptosis

Transection of the medial forebrain bundle caused apoptosis of dopamine neurons in the rat substantia nigra. Immunohistochemical localization of activated microglia and tyrosine hydroxylase in the axotomized substantia nigra showed that activation of microglia was rapid and OX-6 (MHC-II marker)-positive and ED1 (lysosomal phagocytic marker)-positive microglia were apposed to structurally intact tyrosine hydroxylase-positive dopamine neurons, indicating microglial phagocytosis of degenerating dopamine neurons. The occurrence of microglial phagocytosis at early stages of apoptosis may indicate the evolution of apoptosis into an irreversible state. Alternatively, interventions that suppress early activation of microglia might lead to novel mechanisms for neuron protection.

APP knockout attenuates microglial activation and enhances neuron survival in substantia nigra compacta after axotomy.

Focal microglial activation and progressive dopaminergic neurodegeneration in substantia nigra compacta (SNc) have characterized Parkinsons disease (PD). We have hypothesized that the microglial response may be provoked by molecular signals from chronically stressed SNc neurons. To test whether amyloid precursor protein (APP) could serve as such a signal, we evaluated microglial activation in SN after unilateral transection of the medial forebrain bundle (MFB) in mice either wild‐type (WT) or null (KO) for APP. WT and KO mice displayed comparable microglial response at the MFB transection site. In WT mice microglial activation was first apparent in the ipsilateral SN at 3 days postlesion (dpl), marked by morphological change and increased isolectin immunoreactivity. The microglial response intensified at 7 dpl and persisted in the medial nigra through 14 dpl. In contrast, in KO mice activated microglia appeared predominantly at 7 dpl, with little activation at 3 dpl and none at 14 dpl. Neuron number in affected WT SNc at 14 dpl was significantly reduced compared with loss in affected KO SNc. The delayed and limited local microglial activation and increased neuron survival in response to distal axotomy of SNc neurons in APP KO mice are consistent with the important role APP in neuronal stress responses in vivo. GLIA 38:174–178, 2002.

Amyloid precursor protein gene disruption attenuates degeneration of substantia nigra compacta neurons following axotomy.

Our past work has shown that the C-terminal fragment of amyloid precursor protein (APP) translocated to the nucleus in neurons destined for delayed excitotoxic degeneration. To test whether nuclear APP fragments also play a role in the progressive loss of dopaminergic (DA) substantia nigra compacta (SNc) neurons, we performed unilateral medial forebrain bundle (MFB) transection on APP wild type (WT) and on mice with disruption of the APP gene (KO). In WT mice immunoreactivity for APP C-terminal, beta-amyloid and Alz90 epitopes appeared in the nuclei of axotomized DA neurons at 3 days post-lesion (dpl), persisted at 7 dpl and was absent in 14 dpl mice. APP N-terminal immunoreactivity was restricted to the cytosol at all time points, precluding the possibility of full length APP in the nucleus. Nuclear localization of APP epitopes was absent in neurons of the contralateral SNc or in neurons of the ipsilateral ventral tegmental area and SN reticulata. The presence of APP C-terminal and Alz90 domains was confirmed by Western blotting performed on the nuclear fraction of the SN ipsilateral to the axotomy. Quantitative morphometric analysis revealed that WT mice demonstrated earlier and more profound loss of tyrosine hydroxylase+SNc neurons than did KO mice. These data showed that a novel nuclear C-terminal fragment appeared coincident with SNc neuron degeneration, and that APP deficiency correlated with significant neuroprotection in vivo.