Molecular signatures underlying neurofibrillary tangle susceptibility in Alzheimer's disease.

Tau aggregation in neurofibrillary tangles (NFTs) is closely associated with neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the molecular signatures that distinguish between aggregation-prone and aggregation-resistant cell states are unknown. We developed methods for the high-throughput isolation and transcriptome profiling of single somas with NFTs from the human AD brain, quantified the susceptibility of 20 neocortical subtypes for NFT formation and death, and identified both shared and cell-type-specific signatures. NFT-bearing neurons shared a marked upregulation of synaptic transmission-related genes, including a core set of 63 genes enriched for synaptic vesicle cycling. Oxidative phosphorylation and mitochondrial dysfunction were highly cell-type dependent. Apoptosis was only modestly enriched, and the susceptibilities of NFT-bearing and NFT-free neurons for death were highly similar. Our analysis suggests that NFTs represent cell-type-specific responses to stress and synaptic dysfunction. We provide a resource for biomarker discovery and the investigation of tau-dependent and tau-independent mechanisms of neurodegeneration.

All-trans-retinoic acid reduces BACE1 expression under inflammatory conditions via modulation of nuclear factor κB (NFκB) signaling.

Insulin resistance and neuroinflammation have emerged as two likely key contributors in the pathogenesis of Alzheimer disease (AD), especially in those sporadic AD cases compromised by diabetes or cardiovascular disease. Amyloid-ß (Aß) deposition and its associated inflammatory response are hallmarks in sporadic AD brains. Elevated expression and activity of ß-secretase 1 (BACE1), the rate-limiting enzyme responsible for the ß-cleavage of amyloid precursor proteins to Aß peptides, are also observed in sporadic AD brains. Previous studies have suggested that there is therapeutic potential for retinoic acid in treating neurodegeneration based on decreased Aß. Here we discovered that BACE1 expression is elevated in the brains of both Tg2576 transgenic mice and mice on high fat diets. These conditions are associated with a neuroinflammatory response. We found that administration of all-trans-retinoic acid (atRA) down-regulated the expression of BACE1 in the brains of Tg2576 mice and in mice fed a high fat diet. Moreover, in LPS-treated mice and cultured neurons, BACE1 expression was repressed by the addition of atRA, correlating with the anti-inflammatory efficacy of atRA. Mutations of the NFκB binding site in BACE1 promoter abolished the suppressive effect of atRA. Furthermore, atRA disrupted LPS-induced nuclear translocation of NFκB and its binding to BACE1 promoter as well as promoting the recruitment of the corepressor NCoR. Our findings indicate that atRA represses BACE1 gene expression under inflammatory conditions via the modulation of NFκB signaling.

Partial eNOS deficiency causes spontaneous thrombotic cerebral infarction, amyloid angiopathy and cognitive impairment.

BACKGROUND: Cerebral infarction due to thrombosis leads to the most common type of stroke and a likely cause of age-related cognitive decline and dementia. Endothelial nitric oxide synthase (eNOS) generates NO, which plays a crucial role in maintaining vascular function and exerting an antithrombotic action. Reduced eNOS expression and eNOS polymorphisms have been associated with stroke and Alzheimer's disease (AD), the most common type of dementia associated with neurovascular dysfunction. However, direct proof of such association is lacking. Since there are no reports of complete eNOS deficiency in humans, we used heterozygous eNOS(+/-) mice to mimic partial deficiency of eNOS, and determine its impact on cerebrovascular pathology and perfusion of cerebral vessels. RESULTS: Combining cerebral angiography with immunohistochemistry, we found thrombotic cerebral infarctions in eNOS(+/-) mice as early as 3-6 months of age but not in eNOS(+/+) mice at any age. Remarkably, vascular occlusions in eNOS(+/-) mice were found almost exclusively in three areas: temporoparietal and retrosplenial granular cortexes, and hippocampus this distribution precisely matching the hypoperfused areas identified in preclinical AD patients. Moreover, progressive cerebral amyloid angiopaphy (CAA), blood brain barrier (BBB) breakdown, and cognitive impairment were also detected in aged eNOS(+/-) mice. CONCLUSIONS: These data provide for the first time the evidence that partial eNOS deficiency results in spontaneous thrombotic cerebral infarctions that increase with age, leading to progressive CAA and cognitive impairments. We thus conclude that eNOS(+/-) mouse may represent an ideal model of ischemic stroke to address early and progressive damage in spontaneously-evolving chronic cerebral ischemia and thus, study vascular mechanisms contributing to vascular dementia and AD.

Genetic variability to diet-induced hippocampal dysfunction in BXD recombinant inbred (RI) mouse strains.

Evidence has emerged suggesting that diet-induced obesity can have a negative effect on cognitive function. Here, we exploited a mouse genetic reference population to look for the linkage between these two processes on a genome-wide scale. The focus of this report is to determine whether the various BXD RI strains exhibited different behavioral performance and hippocampal function under high fat dietary (HFD) condition. We quantified genetic variation in body weight gain and consequent influences on behavioral tests in a cohort of 14 BXD strains of mice (8-12 mice/strain, n = 153), for which we have matched data on gene expression and neuroanatomical changes in the hippocampus. It showed that BXD66 was the most susceptible, whereas BXD77 was the least susceptible strain to dietary influences. The performance of spatial reference memory tasks was strongly correlated with body weight gain (P < 0.05). The obesity-prone strains displayed more pronounced spatial memory defects compared to the obesity-resistant strains. These abnormalities were associated with neuroinflammation, synaptic dysfunction, and neuronal loss in the hippocampus. The biological relevance of DSCAM gene polymorphism was assessed using the trait correlation analysis tool in Genenetwork. Furthermore, a significant strain-dependent gene expression difference of DSCAM was detected in the hippocampus of obese BXD strains by real-time quantitative PCR. In conclusion, a variety of across-strain hippocampal alterations and genetic predispositions to diet-induced obesity were found in a set of BXD strains. The obesity-prone and obesity-resistant lines we have identified should be highly useful to study the molecular genetics of diet-induced cognitive decline.

Hsp90 chaperone inhibitor 17-AAG attenuates Aß-induced synaptic toxicity and memory impairment.

The excessive accumulation of soluble amyloid peptides (Aß) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), particularly in synaptic dysfunction. The role of the two major chaperone proteins, Hsp70 and Hsp90, in clearing misfolded protein aggregates has been established. Despite their abundant presence in synapses, the role of these chaperones in synapses remains elusive. Here, we report that Hsp90 inhibition by 17-AAG elicited not only a heat shock-like response but also upregulated presynaptic and postsynaptic proteins, such as synapsin I, synaptophysin, and PSD95 in neurons. 17-AAG treatment enhanced high-frequency stimulation-evoked LTP and protected neurons from synaptic damage induced by soluble Aß. In AD transgenic mice, the daily administration of 17-AAG over 7 d resulted in a marked increase in PSD95 expression in hippocampi. 17-AAG treatments in wild-type C57BL/6 mice challenged by soluble Aß significantly improved contextual fear memory. Further, we demonstrate that 17-AAG activated synaptic protein expression via transcriptional mechanisms through the heat shock transcription factor HSF1. Together, our findings identify a novel function of Hsp90 inhibition in regulating synaptic plasticity, in addition to the known neuroprotective effects of the chaperones against Aß and tau toxicity, thus further supporting the potential of Hsp90 inhibitors in treating neurodegenerative diseases.

Regulated expression of lentivirus-mediated GDNF in human bone marrow-derived mesenchymal stem cells and its neuroprotection on dopaminergic cells in vitro.

Gene regulation remains one of the major challenges for gene therapy in clinical trials. In the present study, we first generated a binary tetracycline-on (Tet-On) system based on two lentivirus vectors, one expressing both human glial cell line-derived neurotrophic factor (hGDNF) and humanized recombinant green fluorescent protein (hrGFP) genes under second-generation tetracycline response element (TRE), and the other expressing the advanced reverse tetracycline-controlled transactivator--rtTA2S-M2 under a human minimal cytomegalovirus immediate early (CMV-IE) promoter. This system allows simultaneous expression of hGDNF and hrGFP genes in the presence of doxycycline (Dox). Human bone marrow-derived mesenchymal stem cells (hMSCs) were transduced with the binary Tet-On lentivirus vectors and characterized in vitro in the presence (On) or absence (Off) of Dox. The expression of hGDNF and hrGFP transgenes in transduced hMSCs was tightly regulated as determined by flow cytometry (FCM), GDNF enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR). There was a dose-dependent regulation for hrGFP transgene expression. The levels of hGDNF protein in culture medium were correlated with the mean fluorescence intensity (MFI) units of hrGFP. The levels of transgene background expression were very low in the absence of Dox. The treatment of the conditioned medium from cultures of transduced hMSCs in the presence of Dox protected SH-SY5Y cells against 6-hydroxydopamine (6-OHDA) toxicity as determined by cell viability using 3, [4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) assay. The treatment of the conditioned medium was also found to improve the survival of dopaminergic (DA) neurons of ventral mesencephalic (VM) tissue in serum-free culture conditions as assessed by cell body area, the number of neurites and dendrite branching points, and proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) cells. Our inducible lentivirus-mediated hGDNF gene delivery system may provide useful tools for basic research on gene therapy for chronic neurological disorders such as Parkinson's disease (PD).

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo.

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.

Inactivation of the central nucleus of the amygdala reduces the effect of punishment on cocaine self-administration in rats.

Continued cocaine use despite the negative consequences is a hallmark of cocaine addiction. One such consequence is punishment, which is often used by society to curb cocaine use. Unfortunately, we know little about the mechanism involved in regulation by punishment of cocaine use. The fact that cocaine addicts continue to use cocaine despite potentially severe punishment suggests that the mechanism may be impaired. Such impairment is expected to critically contribute to compulsive cocaine use. This study was aimed at testing the hypothesis that the central nucleus of the amygdala (CeN) plays a critical role in such regulation. To this end, rats were trained to press a lever to self-administer cocaine under a chained schedule: a response on one lever (cocaine-seeking lever) led to access to the other lever (cocaine-taking lever), on which a response was reinforced by cocaine and cues. Thereafter, responses on the seeking lever were punished by footshock with a probability of 0.5. Cocaine self-administration (SA) was significantly suppressed by punishment in an intensity-dependent manner. Interestingly, rats trained with daily 6-h (extended access) but not 2-h (limited access) sessions showed resistance to the lower intensity of punishment. Inactivation of the CeN induced a robust anti-punishment effect in both groups. These data provided evidence that the CeN is a critical neural substrate involved in regulation by punishment of cocaine SA. Rats with a history of extended cocaine SA appeared to be less sensitive to punishment. The decreased sensitivity could result from the neuroplastic changes induced by extended cocaine SA in the CeN.

Regulation of cocaine-induced reinstatement by group II metabotropic glutamate receptors in the ventral tegmental area.

RATIONALE: A high rate of relapse is a daunting challenge facing clinical treatment of cocaine addiction. Recent studies have shown that drugs of abuse enhance glutamate neurotransmission in dopamine neurons in the ventral tegmental area (VTA) and such enhancement may contribute to the risk of relapse. OBJECTIVES: Given the important role of group II metabotropic glutamate receptors (mGluR2/3s) in regulating glutamate release from the glutamatergic terminals, this study aimed to test whether activation of mGluR2/3s in the VTA can inhibit cocaine-induced reinstatement of cocaine-seeking behavior, a model of relapse to drug-seeking behavior. METHODS: Rats were trained to self-administer intravenous cocaine (0.25 mg/infusion) under a modified fixed-ratio 5 schedule. After rats reached the training criteria, they went through extinction training to extinguish cocaine-seeking behavior. Then the dose-response effects of a selective mGluR2/3 agonist LY 379268 microinjected into the VTA on cocaine-induced reinstatement of cocaine-seeking behavior were assessed. RESULTS: LY 379268 (0.032-0.1 µg/side) dose-dependently decreased cocaine-induced reinstatement. The effect could not be fully attributed to diffusion of the drug to the neighboring substantia nigra or to motor impairment. Interestingly, LY 379268 has a less potent effect on cocaine-induced reinstatement than on sucrose-induced reinstatement of sucrose-seeking behavior. CONCLUSIONS: Our data support the idea that glutamate release in the VTA is critically involved in cocaine-induced reinstatement and indicate that loss of mGluR2/3-mediated regulation of glutamate release in the VTA may critically contribute to the risk of relapse.

Activation of D2-like receptors in rat ventral tegmental area inhibits cocaine-reinstated drug-seeking behavior.

Relapse is a hallmark of cocaine addiction. Cocaine-induced neuroplastic changes in the mesocorticolimbic circuits critically contribute to this phenomenon. Pre-clinical evidence indicates that relapse to cocaine-seeking behavior depends on activation of dopamine neurons in the ventral tegmental area. Thus, blocking such activation may inhibit relapse. Because the activity of dopamine neurons is regulated by D2-like autoreceptors expressed on somatodendritic sites, this study, using the reinstatement model, aimed to determine whether activation of D2-like receptors in the ventral tegmental area can inhibit cocaine-induced reinstatement of extinguished cocaine-seeking behavior. Rats were trained to self-administer i.v. cocaine (0.25 mg/infusion) under a modified fixed-ratio 5 schedule. After such behavior was well learned, rats went through extinction training to extinguish cocaine-seeking behavior. The effect of quinpirole, a selective D2-like receptor agonist microinjected into the ventral tegmental area, on cocaine-induced reinstatement was then assessed. Quinpirole (0-3.2 µg/side) dose-dependently decreased cocaine-induced reinstatement and such effects were reversed by the selective D2-like receptor antagonist eticlopride when co-microinjected with quinpirole into the ventral tegmental area. The effect appeared to be specific to the ventral tegmental area because quinpirole microinjected into the substantia nigra had no effect. Because D2-like receptors are expressed on rat ventral tegmental area dopamine neurons projecting to the pre-frontal cortex and nucleus accumbens, our data suggest that these dopamine circuits may play a critical role in cocaine-induced reinstatement. The role of potential changes in D2-like receptors and related signaling molecules of dopamine neurons in the vulnerability to relapse was discussed.

Regulation of cocaine-reinstated drug-seeking behavior by kappa-opioid receptors in the ventral tegmental area of rats.

RATIONALE: Relapse is one of the main challenges facing the current treatment of cocaine addiction. Understanding its neurobiological mechanism is a critical step toward developing effective anti-relapse therapies. OBJECTIVES: Emerging evidence indicates that glutamate-mediated activation of dopamine (DA) neurons in the ventral tegmental area (VTA) may be critically involved in cocaine-induced relapse to drug-seeking behavior. Activity of VTA DA neurons is modulated by multiple neurotransmitter systems including opioids, serotonin, dopamine, and acetylcholine. Recent studies demonstrated that activation of kappa-opioid receptors (kappaORs) in the rat VTA directly inhibits the activity of a subpopulation of DA neurons projecting to the prefrontal cortex (PFC) and amygdala. Because we previously showed that blockade of DA receptors in the dorsal PFC inhibits cocaine-induced reinstatement of extinguished cocaine-seeking behavior suggesting a critical role of the VTA-PFC DA circuit in this process, we tested the hypothesis that activation of kappaORs in the VTA will block cocaine-induced reinstatement in rats. METHODS: Rats were trained to self-administer intravenous cocaine (0.125 mg/infusion) under a modified fixed-ratio five schedule. After extinction of the learned behavior, the effects of activation of VTA kappaORs on cocaine-induced reinstatement were studied. RESULTS: The kappaOR agonist U50 488 (0-5.6 microg/side) microinjected into the VTA dose-dependently decreased cocaine-induced reinstatement. The effects could not be explained by either a disruption of operant behavior or diffusion of the drug to the areas surrounding the VTA. Moreover, the effect was reversed by norbinaltorphimine. CONCLUSIONS: The VTA DA neurons expressing functional kappaORs are critically involved in cocaine-induced reinstatement in rats.

Brain self-protection: the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia.

Convincing evidence has shown that brain ischemia causes the proliferation of neural stem cells/neural progenitor cells (NSCs/NPCs) in both the subventricular zone (SVZ) and the subgranular zone (SGZ) of adult brain. The role of brain ischemia-induced NSC/NPC proliferation, however, has remained unclear. Here we have determined whether brain ischemia-induced amplification of the NSCs/NPCs in adult brain is required for brain self-protection. The approach of intracerebroventricular (ICV) infusion of cytosine arabinoside (Ara-C), an inhibitor for cell proliferation, for the first 7days after brain ischemia was used to block ischemia-induced NSC/NPC proliferation. We observed that ICV infusion of Ara-C caused a complete blockade of NSC/NPC proliferation in the SVZ and a dramatic reduction of NSC/NPC proliferation in the SGZ. Additionally, as a result of the inhibition of ischemia-induced NSC/NPC pool amplification, the number of neurons in the hippocampal CA1 and CA3 was significantly reduced, the infarction size was significantly enlarged, and neurological deficits were significantly worsened after focal brain ischemia. We also found that an NSC/NPC-conditioned medium showed neuroprotective effects in vitro and that adult NSC/NPC-released brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are required for NSC/NPC-conditioned medium-induced neuroprotection. These data suggest that NSC/NPC-generated trophic factors are neuroprotective and that brain ischemia-triggered NSC/NPC proliferation is crucial for brain protection. This study provides insights into the contribution of endogenous NSCs/NPCs to brain self-protection in adult brain after ischemia injury.

The role of stem cell factor and granulocyte-colony stimulating factor in brain repair during chronic stroke.

Chronic stroke is a highly important but under-investigated scientific problem in neurologic research. We have reported earlier that stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) treatment during chronic stroke improves functional outcomes. Here we have determined the contribution of bone marrow-derived cells in angiogenesis and neurogenesis, which are enhanced by SCF+G-CSF treatment during chronic stroke. Using bone marrow tracking, flow cytometry, 2-photon live brain imaging, and immunohistochemistry, we observed that the levels of circulating bone marrow stem cells (BMSCs) (CD34+/c-kit+) were significantly increased by SCF+G-CSF treatment. In addition, live brain imaging revealed that numerous bone marrow-derived cells migrate into the brain parenchyma in the treated mice. We also found that bone marrow-derived cells, bone marrow-derived endothelial cells, vascular density, and bone marrow-derived neurons were significantly augmented by SCF+G-CSF. It is interesting that, in addition to the increase in bone marrow-derived endothelial cells, the number of bone marrow-derived pericytes was reduced after SCF+G-CSF treatment during chronic stroke. These data suggest that SCF+G-CSF treatment can enhance repair of brain damage during chronic stroke by mobilizing BMSCs, and promoting the contribution of bone marrow-derived cells to angiogenesis and neurogenesis.

[Effects of hydrocortisone on oxygen free radicals released by polymorphonuclear neutrophils in lipopolysaccharide-induced acute lung injury in mice].

BACKGROUND: Corticosteroid treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) has been one of the most controversial clinical issues in critical care. Although the administration of high-dose corticosteroids does not benefit patients with early septic shock and ARDS, recent clinical trials have indicated that treatment with relatively low-dose corticosteroids (2 to 3 mg/kg/day of methylprednisolone or equivalent) may improve outcome when used for late ARDS or persistent septic shock. The underlying mechanism was not fully clarified. Whether the administration of corticosteroids can arrest neutrophil-driven organ injury once started remains to be elucidated. OBJECTIVE: To observe the effects of hydrocortisone (HC, 6 mg/kg) on oxygen free radicals (OFR) released by PMN and pulmonary pathological changes in rat ALI model induced by lipopolysaccharide (LPS), to investigate the possible mechanism through which corticosteroids exert protective effect on ALI. METHODS: A rat model of ALI was induced by peritoneal injection of 2 x 10(12) Escherichia coli/kg. Fifty-six rats were randomly divided into three groups: normal control group, LPS group and HC group (6 mg/kg). Samples were collected 2 h, 4 h and 6 h after giving LPS to LPS and HC group (6 h after giving normal saline in normal control group) to measure the level of OFR released by PMN using chemiluminescence method based on lumino, and to compae of pulmonary pathological changes among the three groups. RESULTS: Pathological examination with light microscope in LPS group showed thickened pulmonary interstitia, inflammatory cell infiltration, edema and hemorrhage, which were in accordance with the features of ALI. There were significant differences in the release of OFR by PMN among the three groups (P < 0.01). The level of OFR released by PMN in LPS group was significantly higher than that of the control group, and continued to increase during the observation period (2 - 6 h after LPS). The release of OFR by PMN in HC group was significantly suppressed as compared with LPS group, which was peaked at 4 h after LPS injection (to 98.2%); there were also significant differences in the grades of ALI pathologic changes among the three groups (P < 0.01). The grades of ALI pathologic changes in LPS group were significantly increased when compared with the normal control group (P < 0.05) while significantly decreased in HC group as compared with LPS group (P < 0.05). CONCLUSION: It was demonstrated in the LPS induced ALI model that OFR might play an important role in onset of ALI. Intervening with HC (6 mg/kg) treatment could ameliorate the lung injury and exert significant and sustained suppression on the release of OFR by PMN, showing that HC has a protective effect on LPS induced ALI and its theraputic effect occurs possibly through suppression on the release of OFR by PMN.