Minho Moon

Neuroprotective Effect of Ghrelin in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Mouse Model of Parkinson’s Disease by Blocking Microglial Activation

Ghrelin is an endogenous ligand for growth hormone (GH) secretagogue receptor 1a (GHS-R1a) and is produced and released mainly from the stomach. It was recently demonstrated that ghrelin can function as a neuroprotective factor by inhibiting apoptotic pathways. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity in rodents; previous studies suggest that activated microglia actively participate in the pathogenesis of Parkinson’s disease (PD) neurodegeneration. However, the role of microglia in the neuroprotective properties of ghrelin is still unknown. Here we show that, in the mouse MPTP PD model generated by an acute regimen of MPTP administration, systemic administration of ghrelin significantly attenuates the loss of substantia nigra pars compacta (SNpc) neurons and the striatal dopaminergic fibers through the activation of GHS-R1a. We also found that ghrelin reduced nitrotyrosine levels and improved the impairment of rota-rod performance. Ghrelin prevents MPTP-induced microglial activation in the SNpc and striatum, the expression of pro-inflammatory molecules tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), and the activation of inducible nitric oxide synthase. The inhibitory effect of ghrelin on the activation of microglia appears to be indirect by suppressing matrix metalloproteinase-3 (MMP-3) expression in stressed dopaminergic neurons because GHS-R1a is not expressed in SNpc microglial cells. Finally, in vitro administration of ghrelin prevented 1-methyl-4-phenylpyridinium-induced dopaminergic cell loss, MMP-3 expression, microglial activation, and the subsequent release of TNF-α, IL-1β, and nitrite in mesencephalic cultures. Our data indicate that ghrelin may act as a survival factor for dopaminergic neurons by functioning as a microglia-deactivating factor and suggest that ghrelin may be a valuable therapeutic agent for neurodegenerative diseases such as PD.

Aβ1–42-RAGE Interaction Disrupts Tight Junctions of the Blood–Brain Barrier Via Ca2+-Calcineurin Signaling

The blood–brain barrier (BBB), which is formed by adherens and tight junctions (TJs) of endothelial cells, maintains homeostasis of the brain. Disrupted intracellular Ca2+ homeostasis and breakdown of the BBB have been implicated in the pathogenesis of Alzheimers disease (AD). The receptor for advanced glycation end products (RAGE) is known to interact with amyloid β-peptide (Aβ) and mediate Aβ transport across the BBB, contributing to the deposition of Aβ in the brain. However, molecular mechanisms underlying Aβ-RAGE interaction-induced alterations in the BBB have not been identified. We found that Aβ1–42 induces enhanced permeability, disruption of zonula occludin-1 (ZO-1) expression in the plasma membrane, and increased intracellular calcium and matrix metalloproteinase (MMP) secretion in cultured endothelial cells. Neutralizing antibodies against RAGE and inhibitors of calcineurin and MMPs prevented Aβ1–42-induced changes in ZO-1, suggesting that Aβ-RAGE interactions alter TJ proteins through the Ca2+-calcineurin pathway. Consistent with these in vitro findings, we found disrupted microvessels near Aβ plaque-deposited areas, elevated RAGE expression, and enhanced MMP secretion in microvessels of the brains of 5XFAD mice, an animal model for AD. We have identified a potential molecular pathway underlying Aβ-RAGE interaction-induced breakage of BBB integrity. This pathway might play an important role in the pathogenesis of AD.

Ghrelin Ameliorates Cognitive Dysfunction and Neurodegeneration in Intrahippocampal Amyloid-β1-42 Oligomer-Injected Mice

Alzheimers disease (AD) is an age-related neurodegenerative disorder characterized by cognitive deficits, neuroinflammation, and loss of neurons. Recently, it has been shown that ghrelin, a 28 amino acid peptide hormone produced from the stomach and hypothalamus, has been reported as a potential therapeutic agent for several neurological disorders, including Parkinsons disease (PD), stroke, epilepsy, multiple sclerosis, and spinal cord injury. Here we determined the effects of ghrelin on memory impairments and neuropathological changes in an AD mouse model induced by intrahippocampal injection of amyloid-β oligomers (AβO). We report that ghrelin: 1) rescues memory deficits in mice injected with AβO in the hippocampus; 2) decreases AβO-induced microgliosis in hippocampus; 3) attenuates hippocampal neuronal loss mediated by AβO; 4) prevents AβO-associated synaptic degeneration including cholinergic fiber loss. Taken together, our findings demonstrate that ghrelin can ameliorate AβO-induced cognitive impairment associated with neuroinflammation and neuronal loss. These results suggest that ghrelin may be a promising therapeutic agent for the treatment of AD.

Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model

Immune responses in the brain are thought to play a role in disorders of the central nervous system, but an understanding of the process underlying how immune cells get into the brain and their fate there remains unclear. In this study, we used a 2-photon microscopy to reveal that neutrophils infiltrate brain and migrate toward amyloid plaques in a mouse model of Alzheimers disease. These findings suggest a new molecular process underlying the pathophysiology of Alzheimers disease.

Cistanches Herba enhances learning and memory by inducing nerve growth factor

Nerve growth factor (NGF) has potent biological activities such as preventing neuronal death, promoting neurite outgrowth, supporting synapse formation, and enhancing memory function. NGF and NGF-like molecules can potentially be used to treat neurodegenerative disorders, such as dementia. This study investigated the effects of Cistanches Herba, a widely used medicinal herb, on NGF regulation and its neuronal actions, including neurite outgrowth, synapse formation, and learning and memory enhancement. Cistanches Herba extract (CHE; 250 μg/ml) increased NGF induction in C6 cells and led to neurite extension in PC12 cells. It also stimulated NGF secretion in the cortex and hippocampus of the mouse brain at 5 and 20mg/kg/day (3 days, p.o.). Furthermore, CHE increased neuronal cell differentiation, neurite length, and synapse formation in the mouse hippocampus. CHE significantly enhanced learning and memory, as demonstrated by passive avoidance test and novel object recognition test. These results suggest that CHE is useful for improving memory function via its action in upregulating NGF.

Disruption of blood-brain barrier in Alzheimer disease pathogenesis

Blood-brain barrier (BBB) regulates transport of various molecules and maintains brain homeostasis. Perturbed intracellular Ca2+ homeostasis and BBB damage have been implicated in the pathogenesis of Alzheimer disease (AD). Although receptor for advanced glycation end products (RAGE) is known to mediate Aβ transcytosis across the BBB, molecular mechanisms underlying Aβ-RAGE interaction-induced BBB alterations are largely unknown. We found enhanced permeability, decreased zonula occludin-1 (ZO-1) expression and increased intracellular calcium and MMP secretion in endothelial cells exposed to Aβ1–42. Aβ-induced changes in ZO-1 were attenuated by neutralizing antibodies against RAGE and inhibitors of calcineurin (CaN) and MMPs, suggesting that Aβ-RAGE interactions disrupt tight junction proteins via the Ca2+-CaN pathway. We also found disrupted microvessels near Aβ plaque-deposited areas, elevated RAGE expression and enhanced MMP secretion in microvessels of the brains of 5XFAD mice, an animal model of AD. These results identify a potential molecular pathway underlying Aβ-RAGE interaction-induced breakage of BBB integrity.

Impaired hippocampal neurogenesis and its enhancement with ghrelin in 5XFAD mice.

Alzheimers disease (AD) is an age-related neurological disorder characterized by the deposition of amyloid-β (Aβ), cognitive deficits, and neuronal loss. The decline in neurogenic capacity could participate in neuronal vulnerability and contribute to memory impairment in AD. In our longitudinal study with AD model mice (5XFAD mice), we found that the number of doublecortin (neurogenesis marker)-positive cells in 5XFAD mice was significantly decreased compared to wild-type littermate mice. Using Aβ immunostaining with 4G8 antibody, we observed that impairment in neurogenesis might be associated with the deposits of amyloid plaques. To investigate the effect of the neurogenic hormone ghrelin on defective neurogenesis in the AD brain, 5XFAD mice were administered peripherally with ghrelin. We found that treatment with ghrelin increased the number of doublecortin, HH3, and calretinin-stained cells in the hippocampus of 5XFAD mice. In 5XFAD mice treated with ghrelin, the 4G8-positive area was not significantly different from the saline-treated 5XFAD mice. Together, these findings suggest that hippocampal neurogenesis is impaired in 5XFAD mice and that treatment with ghrelin successfully rescued the abnormality of neurogenesis in 5XFAD mice without affecting Aβ pathology.

Intracellular Amyloid-β Accumulation in Calcium-Binding Protein-Deficient Neurons Leads to Amyloid-β Plaque Formation in Animal Model of Alzheimer's Disease

One of the major hallmarks of Alzheimers disease (AD) is the extracellular deposition of amyloid-β (Aβ) as senile plaques in specific brain regions. Clearly, an understanding of the cellular processes underlying Aβ deposition is a crucial issue in the field of AD research. Recent studies have found that accumulation of intraneuronal Aβ (iAβ) is associated with synaptic deficits, neuronal death, and cognitive dysfunction in AD patients. In this study, we found that Aβ deposits had several shapes and sizes, and that iAβ occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice, an animal model of AD. We also observed pyroglutamate-modified Aβ (N3pE-Aβ), which has been suggested to be a seeding molecule for senile plaques, inside the Aβ plaques only after iAβ accumulation, which argues against its seeding role. In addition, we found that iAβ accumulates in calcium-binding protein (CBP)-free neurons, induces neuronal death, and then develops into senile plaques in 2-4-month-old 5XFAD mice. These findings suggest that N3pE-Aβ-independent accumulation of Aβ in CBP-free neurons might be an early process that triggers neuronal damage and senile plaque formation in AD patients. Our results provide new insights into several long-standing gaps in AD research, namely how Aβ plaques are formed, what happens to iAβ and how Aβ causes selective neuronal loss in AD patients.

RNA-Seq Analysis of Frontal Cortex and Cerebellum from 5XFAD Mice at Early Stage of Disease Pathology

The pathogenesis of Alzheimers disease (AD), especially the early events of AD pathology, remains unknown because of the complexity of AD and limitation of analysis methods. Transcriptome analysis has provided comprehensive insights to investigate the complex cellular activity in brain, but the transcriptome profiles from AD patients with microarray have generated discordant results. Here, for the first time, we performed transcriptome analysis of frontal cortex and cerebellum in 7-week-old 5XFAD transgenic mice (before extracellular amyloid plaque deposits) using high-throughput RNA-Seq analysis. Specific functional annotations were identified with differentially expressed genes (DEGs) of frontal cortex (a typically vulnerable region of AD pathology) and cerebellum (a typically non-vulnerable region of AD pathology). Cardiovascular disease-related genes were significantly found in down-regulated DEGs of frontal cortex, and mitochondrial dysfunction-related genes were evident in down-regulated DEGs of cerebellum. Additionally, we found RNA variants at the nucleotide level in transgenic mice compared with non-transgenic mice. Our results indicate that both frontal cortex and cerebellum in 5XFAD transgenic mice show specific pathological processes in the early pathophysiology of AD.

FK506 Reduces Amyloid Plaque Burden and Induces MMP-9 in AβPP/PS1 Double Transgenic Mice

Deposition of amyloid-β peptide (Aβ) and neurofibrillary tangles are pathological hallmarks of Alzheimers disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Recently, calcineurin (CaN) has been reported as a potential modulator of memory function, synaptic plasticity, and neural degeneration in brains of AD animal models. In the present study, we examined the relationship between Aβ accumulations and CaN activity in brains of the AβPP/PS1 double transgenic mice. Treatment with FK506, a CaN inhibitor, significantly reduces Aβ burden and restores synaptic proteins (synaptophysin and postsynaptic density protein-95; PSD-95) while inducing matrix metallopeptidase-9 (MMP-9) expression in GFAP-positive astrocytes in the brain. These results suggest a role of FK506 and control of CaN activity in neuroprotection associated with Aβ deposition in AD.