Microglial AGE-albumin is critical for neuronal death in Parkinson's disease: a possible implication for theranostics.

Advanced glycation end products (AGEs) are known to play an important role in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD), by inducing protein aggregation and cross-link, formation of Lewy body, and neuronal death. In this study, we observed that AGE-albumin, the most abundant AGE product in the human PD brain, is synthesized in activated microglial cells and accumulates in the extracellular space. AGE-albumin synthesis in human-activated microglial cells is distinctly inhibited by ascorbic acid and cytochalasin treatment. Accumulated AGE-albumin upregulates the receptor to AGE, leading to apoptosis of human primary dopamine (DA) neurons. In animal experiments, we observed reduced DA neuronal cell death by treatment with soluble receptor to AGE. Our study provides evidence that activated microglial cells are one of the main contributors in AGE-albumin accumulation, deleterious to DA neurons in human and animal PD brains. Finally, activated microglial AGE-albumin could be used as a diagnostic and therapeutic biomarker with high sensitivity for neurodegenerative disorders, including PD.

Changes of calcium binding proteins, c-Fos and COX in hippocampal formation and cerebellum of Niemann-Pick, type C mouse.

Niemann-Pick disease, type C (NPC) is an intractable disease that is accompanied by ataxia, dystonia, neurodegeneration, and dementia due to an NPC gene defect. Disruption of calcium homeostasis in neurons is important in patients with NPC. Thus, we used immunohistochemistry to assess the expression levels of calcium binding proteins (calbindin D28K, parvalbumin, and calretinin), c-Fos and cyclooxygenase-1,2 (COX-1,2) in the hippocampal formation and cerebellum of 4 and 8 week old NPC+/+, NPC+/-, and NPC-/- mice. General expression of these proteins decreased in the hippocampus and cerebellum of NPC-/- compared to that in both young and adult NPC+/+ or NPC+/- mice. Parvalbumin, COX-1,2 or c-Fos-immunoreactive neurons were widely detected in the CA1, CA3, and DG of the hippocampus, but the immunoreactivities were decreased sharply in all areas of hippocampus of NPC-/- compared to NPC+/+ and NPC+/- mice. Taken together, reduction of these proteins may be one of the strong phenotypes related to the neuronal degeneration in NPC-/- mice.

Interleukin-18 attenuates disruption of brain-blood barrier induced by status epilepticus within the rat piriform cortex in interferon-γ independent pathway.

Status epilepticus increases brain-blood barrier (BBB) permeability leading to vasogenic edema. This BBB disruption is usually confined within relatively limited cerebral regions including the piriform cortex (PC), and leads to epileptogenesis and contributes to progression of epilepsy. Although cytokines are at least partly responsible for changes in BBB permeability, the role of interleukin-18 (IL-18) in vasogenic edema is not yet explored in detail. In the present study, we investigated the role of IL-18 in SE-induced vasogenic edema formation. Following SE, IL-18/interferon-γ (IFN-γ) system was up-regulated in astrocytes and microglia/macrophages. Recombinant rat (rr) IL-18 infusion decreased vasogenic edema formation, while anti-rat IL-18 infusion increased it. In contrast, rrIFN-γ, and anti-rat IFN-γ infusion showed reverse effects on vasogenic edema formation. rrIL-18 or anti-rat IFN-γ IgG infusion elevated dystrophin expression accompanied by the reduction in vasogenic edema. However, rr-IFN-γ or anti-rat IL-18 IgG infusion significantly decreased dystrophin immunoreactivity within the PC following SE. These findings indicate that IL-18-mediated up-regulation of dystrophin expression may play either a direct or indirect role in maintenance of BBB function following SE. Therefore, our findings suggest that IL-18 may have protective effect on SE-induced BBB disruption in IFN-γ independent mechanism.

The roles of P2X7 receptor in regional-specific microglial responses in the rat brain following status epilepticus.

Recently, we have reported that astroglial activations in response to status epilepticus (SE) show regional-specific manners in the rat hippocampus. However, it is unknown that microglial responses to SE would show regional-specific patterns. Therefore, the present study was designed to elucidate the regional-specific microglial activation and relationship between P2X7 receptor functions and SE-induced microglial responses in the rat brain. Following SE, microglia appeared amoeboid or phagocytic in the dentate gyrus and the piriform cortex. In contrast, elongated microglia were observed in the CA1 hippocampal regions and the frontoparietal cortex. In the dentate gyrus, the CA1 hippocampal regions, and the frontoparietal cortex, these microglial activation accelerated by BzATP (a P2X7 receptor agonist)-infusion, but inhibited by OxATP (a P2X7 receptor antagonist). However, SE-induced microglial activation in the piriform cortex was not affected by BzATP or OxATP-infusion. Therefore, our findings indicate that SE-induced microglial activation may show regional-specific manners, and suggest that P2X7 receptor function differently modulates SE-induced microglial responses in distinct brain regions.

Up-regulation of endothelial endothelin-1 expression prior to vasogenic edema formation in the rat piriform cortex following status epilepticus.

Endothelin-1 (ET-1) is one of potential factors to induce vasogenic edema formation, since exogenous ET-1 treatment decreases aquaporin 4 (AQP4) expression and increases chemokines induction. To identify the role of endogenous ET-1 in vasogenic edema formation, we examined the correlation between endogenous ET-1 expression and vasogenic edema formation in the pirifom cortex following status epilepticus (SE). In the present study, SMI-71 (a brain-blood barrier marker) immunoreactivity was significantly reduced in blood vessels at 1 day after SE when vasogenic edema and neuronal damage were observed. ET-1 expression was up-regulated in endothelial cells prior to reduction in SMI-71 immunoreactivity. Furthermore, ET-1 expressing endothelial cells showed the absence of SMI-71 immunoreactivity. Increase in ET-1 expression was followed by reduced AQP4 immunoreactivity prior to vasogenic edema formation. Only a few microglia showed monocyte chemotactic protein-1 (a chemokine induced by ET-1) outside vasogenic edema lesion. Taken together, our findings suggest that endothelial ET-1 expression may contribute to SE-induced vasogenic edema formation via brain-blood barrier disruption at AQP4/MCP-1 independent manners.

Alteration of the CNS pathway to the hippocampus in a mouse model of Niemann-Pick, type C disease.

Niemann-Pick type C disease (NPC) is an autosomal recessive disorder that results in premature death due to progressive neurodegeneration including dementia. To understand neuronal pathways connecting to the hippocampus, retrograde transneuronal labeling method with Bartha strain of pseudorabies virus (PRV) was employed in 40 NPC+/+, NPC+/- and NPC-/- mice. Immunohistochemistry using polyclonal antibody against PRV and streological counting were used. The number of neurons and synapse in CA2&3 regions of the hippocampus decreased dramatically in the NPC-/- mouse compared to the NPC+/+ or +/- mouse. The number of PRV positive cell was significantly decreased in several regions including the entorhinal and piriform cortex in the NPC-/- mouse. More severely, lateral septal dorsal nucleus, dorsal entorhinal cortex and medial geniculate body showed no positive labeling in the NPC-/- mouse. However, the hippocampus, medial septal and supramammilary nuclei showed increased immunoreactivity in the NPC-/- mouse. Our data suggest that the synaptic loss and discontinuity of the CNS hippocampal pathway may contribute to understanding the mechanism of symptoms and functional disabilities such as memory and learning disturbance in NPC patients.

Astroglial loss and edema formation in the rat piriform cortex and hippocampus following pilocarpine-induced status epilepticus.

In the present study we analyzed aquaporin-4 (AQP4) immunoreactivity in the piriform cortex (PC) and the hippocampus of pilocarpine-induced rat epilepsy model to elucidate the roles of AQP4 in brain edema following status epilepticus (SE). In non-SE-induced animals, AQP4 immunoreactivity was diffusely detected in the PC and the hippocampus. AQP4 immunoreactivity was mainly observed in the endfeet of astrocytes. Following SE the AQP4-deleted area was clearly detected in the PC, not in the hippocampus. Decreases in dystrophin and α-syntrophin immunoreactivities were followed by reduction in AQP4 immunoreactivity. These alterations were accompanied by the development of vasogenic edema and the astroglial loss in the PC. In addition, acetazolamide (an AQP4 inhibitor) treatment exacerbated vasogenic edema and astroglial loss both in the PC and in the hippocampus. These findings suggest that SE may induce impairments of astroglial AQP4 functions via disruption of the dystrophin/α-syntrophin complex that worsen vasogenic edema. Subsequently, vasogenic edema results in extensive astroglial loss that may aggravate vasogenic edema.

Levetiracetam inhibits interleukin-1 beta inflammatory responses in the hippocampus and piriform cortex of epileptic rats.

Levetiracetam (LEV, 2S-(oxo-1-pyrrolidinyl)butanamide, Keppra, UCB Pharma) is a new anti-epileptic drug used to treat certain types of seizures in epilepsy patients. However, the pharmacodynamics of LEV is still controversial. Recently, interleukin-1 beta (IL-1 beta) has been reported to involve in epileptic phenomena. Therefore, we investigated the effects of LEV on IL-1 beta system in the hippocampus and piriform cortex of chronic epileptic rats. As compared to controls, typical reactive astrogliosis and microgliosis were observed in the hippocampus and piriform cortex of epileptic animals. In addition, both reactive astrocytes and reactive microglia showed strong IL-1 beta and interleukin-1 receptor subtype 1 (IL-1R1) immunoreactivities. LEV reduced reactive gliosis and expression levels of IL-1 beta system in the hippocampus and the piriform cortex, while valproic acid did not. These findings suggest that the LEV may have, at least in part, anti-inflammatory effect, particularly against IL-1 beta system in neuroglia within epileptic brains.

Blockade of P2X receptor prevents astroglial death in the dentate gyrus following pilocarpine-induced status epilepticus.

OBJECTIVE: The P(2) receptor is involved in diverse signal cascades, including the initiation of the rapid release and processing of proinflammatory cytokines, the induction of cytoskeletal rearrangements and transcription factor activation. Therefore, we investigated whether blocking the P(2) receptor would prevent the astroglial death induced by status epilepticus (SE). METHODS: We performed seizure induction and drug treatments. After tissue processing, we executed immunoreactivities: mouse anti-glial fibrillary acidic protein (GFAP) IgG (diluted 1 : 200; Chemicon, Billerica, MA, USA rabbit anti-P(2)X(7) receptor IgG (diluted 1 : 200; Chemicon). RESULTS: In control animals, P(2)X(7) receptor-immunoreactive (P(2)X(7)(+)) microglia had small cell bodies with thin ramified processes. Seven days after SE, P(2)X(7) receptor immunoreactivity in microglia was significantly elevated in the dentate gyrus, and the microglia appeared amoeboid or phagocytic. At this point, loss of GFAP immunoreactivity in the dentate gyrus was even more pronounced, indicating that the network of astrocytes was disrupted and a large empty zone was observed. Treatment with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid and suramin (2, 20 and 200 mg/kg, i.p., respectively) markedly, but not completely, inhibited microglial activation following SE. The morphology of microglia was similar to that of the astrocytes in that they appeared hyper-ramified. In addition, P(2)X(7) receptor antagonist treatments effectively prevented astroglial degeneration. DISCUSSION: These findings suggest that astroglial death induced by ATP-mediated microglia activation may be an important pathophysiological pathway in epileptogenesis.

The expression of somatostatin receptors in the hippocampus of pilocarpine-induced rat epilepsy model.

During the course of this study, we sought examine whether the expression of somatostatin receptors (SSTRs) is altered in the hippocampus following pilocarpine-induced status epilepticus (SE) in order to understand the role/function of SSTRs in the hippocampus after epileptogenic insults. SSTR1 and SSTR4 immunoreactivities were increased in the hippocampus at 1 week after SE. At 4 weeks after SE, SRIF1-family (SSTR 2A, SSTR2B, and SSTR5) immunoreactivity was increased only in neuropil. Both SSTR2A and 2B immunoreactivities were increased in CA2-3 pyramidal cells. However, SSTR3 and SSTR4 immunoreactivities were reduced in the CA1 pyramidal cells of epileptic rat due to neuronal loss. In addition, SSTR5 immunoreactivity was reduced in CA2 pyramidal cells and various interneurons. Both SSTR2B and SSTR4 immunoreactivities were increased within microglia following SE. Our findings suggest that increases in neuron-glial SSTR expressions may be closely related to the enhanced inhibition of the dentate gyrus and regulation of reactive microgliosis in the hippocampus of a pilocarpine model of temporal lobe epilepsy.

Voltage-gated Na+ channel II immunoreactivity is selectively up-regulated in hippocampal interneurons of seizure sensitive gerbils.

In the present study, we investigated the distribution of voltage-gated Na(+) channels (VGSCs) in the normal and epileptic hippocampus of gerbils (a genetic epilepsy model) in order to confirm the relationship between VGSC and seizure activity in these animals. There was no difference of VGSC I immunoreactivity in the hippocampus between seizure-resistant (SR) and seizure sensitive (SS) gerbils. VGSC II immunoreactivity was rarely detected in the perikarya of principal neurons and interneurons in the SR gerbil hippocampus. However, in the SS gerbil hippocampus, VGSC II immunoreactivity was densely observed in the somata of interneurons located in the stratum radiatum and stratum lacunosum-moleculare. Double immunofluorescent study showed immunoreactivity for calretinin (approximately 80% in VGSC II-positive neurons) or calbindin D-28k (approximately 20% in VGSC II-positive neurons) in VGSC II-immunoreactive neurons. VGSC II-immunoreactive neurons did not show parvalbumin immunoreactivity. These findings suggest that seizure activity in SS gerbils may be related to the selective hyperactivation of interneurons in stratum lacunosum-moleculare via the up-regulation of VGSC II expression, which leads to the disinhibition of CA1 pyramidal cells.

Depletion of vesicular zinc in dorsal horn of spinal cord causes increased neuropathic pain in mice.

Zinc enriched (ZEN) neurons and terminals are abundant in the rodent spinal cord. Zinc ions have been suggested to modulate the excitability of primary afferent fibers believed to be important in nociceptive transmission. To test the hypothesis that vesicular zinc concentration is related to neuropathic pain we applied Chung's rodent pain model on BALB/c mice, and traced zinc transporter 3 (ZnT3) proteins and zinc ions with immunohistochemistry and autometallography (AMG), respectively. Under anesthesia the left fifth lumbar spinal nerve was ligated in male mice in order to produced neuropathic pain. The animals were then sacrificed 5 days later. The ZnT3 immunoreactivity was found to have decreased significantly in dorsal horn of fourth, fifth, and sixth lumbar segments. In parallel with the depressed ZnT3 immunoreactivity the amount of vesicular zinc decreased perceptibly in superficial gray matters of especially layer I-IV of the same segments. The transection-induced reduction of vesicular zinc in ZEN terminals of the dorsal horn was synchronic to reduced pain threshold, as measured by von Frey method. In a separate study, we observed intensive zinc selenite precipitation in somata of the smaller spinal ganglion cell, but 5 days after spinal nerve transection zinc precipitation was also found in the lager ganglion cells. The present results indicate that zinc may be involved in pain mechanism in the spinal ganglion level. These results support the hypothesis that vesicular zinc might have a modulatory role for neuropathic pain. Thus, increased pain sensitivity might be related to reduce vesicular zinc level in the dorsal spinal gray matter.

Adrenalectomy-induced ZnT3 downregulation in mouse hippocampus is followed by vesicular zinc depletion.

The effects of adrenalectomy (ADX) on the vesicular zinc content of zinc-enriched (ZEN) terminals in mouse hippocampus were investigated at light microscopic levels using zinc transporter-3 immunohistochemistry (ZnT3IHC) and zinc selenium autometallography (ZnSeAMG). ZnT3 resides in the synaptic vesicle membranes of ZEN neurons and is believed to move zinc ions into the vesicles. ZnT3IHC staining closely corresponds to the ZnSeAMG staining, but in the present study we present evidence of a delayed decrease of ZEN zinc, as compared to downregulation of the ZnT3 protein following ADX. Twenty-four hours after adrenalectomy the level of ZnT3IHC was visibly reduced while the ZnSeAMG staining intensity seemed unchanged. After 10 and 30 days, however, downregulation of ZnT3 was paralleled by a distinct reduction in ZnSeAMG staining. The total protein concentration of ZnT3 was reduced by about 53%, and the total zinc concentration in the hippocampus of the same mice was reduced by 43-64%, 30 days after the adrenalectomy. The present results support previous results suggesting that ZnT3 is responsible for transport of zinc ions into a pool of synaptic vesicles in ZEN terminals.

Dynamic zinc pools in mouse choroid plexus.

We examined the presence of Zn-transporters (ZnT1, ZnT3, ZnT4, and ZnT6) proteins and zinc ions in rat choroid epithelium with immunohistochemistry and zinc selenide autometallography (ZnSe(AMG)). The four ZnT proteins were all expressed in the choroid epithelial cells. ZnT3 immunostaining was found in vesicle membranes in the apical part of the cells, associated to the microvillus membrane. Correspondingly, the ZnSe(AMG) technique revealed zinc ions in small vesicles, in microvilli, and multivesicular bodies in the epithelial cells. Traceable zinc ions were also found in lysosome-like organelles of fenestrated endothelial cells, but here no corresponding ZnT3 immunostaining was seen. The observations suggests that the choroid plexus is instrumental to regulation of the level of zinc ions in the cerebrospinal fluid.

Immunocytochemical localization of zinc transporter 3 in the ependyma of the mouse spinal cord.

We report, for the first time, the light microscopical and ultrastructural appearance of ZnT3-immunoreactivities in the ependymal cells of the central canal of the mouse spinal cord. Light microscopy revealed the presence of ZnT3-immunoreactive (Ir) ependymal cells in 1 microm thick epon sections stained by the ABC method. The ZnT3-Ir cells were observed at all levels of the spinal cord, but were a little more numerous in lumbosacral segments than in cervicothoracic segments. The ZnT3-Ir cells had large, ovoid nuclei with abundant cytoplasm, and protruded into the lumen of the central canal. Our ultrastructural findings suggest that the ZnT3-Ir ependymal cells possess secretory activity directed towards the central canal. We propose that they may play a role in the trans-ependymal mechanism responsible for zinc homeostasis between cerebrospinal fluid and the central area of the gray matter.