Adult Human Brain Neural Progenitor Cells (NPCs) and Fibroblast-Like Cells Have Similar Properties In Vitro but Only NPCs Differentiate into Neurons

The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5–6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments

Convulsant Doses of a Dopamine D1 Receptor Agonist Result in Erk-Dependent Increases in Zif268 and Arc/Arg3.1 Expression in Mouse Dentate Gyrus

Activation of dopamine D1 receptors (D1Rs) has been shown to induce epileptiform activity. We studied the molecular changes occurring in the hippocampus in response to the administration of the D1-type receptor agonist, SKF 81297. SKF 81297 at 2.5 and 5.0 mg/kg induced behavioural seizures. Electrophysiological recordings in the dentate gyrus revealed the presence of epileptiform discharges peaking at 30–45 min post-injection and declining by 60 min. Seizures were prevented by the D1-type receptor antagonist, SCH 23390, or the cannabinoid CB1 receptor agonist, CP 55,940. The effect of SKF 81297 was accompanied by increased phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK), in the granule cells of the dentate gyrus. This effect was also observed in response to administration of other D1-type receptor agonists, such as SKF83822 and SKF83959. In addition, SKF 81297 increased the phosphorylation of the ribosomal protein S6 and histone H3, two downstream targets of ERK. These effects were prevented by genetic inactivation of D1Rs, or by pharmacological inhibition of ERK. SKF 81297 was also able to enhance the levels of Zif268 and Arc/Arg3.1, two immediate early genes involved in transcriptional regulation and synaptic plasticity. These changes may be involved in forms of activity-dependent plasticity linked to the manifestation of seizures and to the ability of dopamine to affect learning and memory

Porphyromonas gingivalis in Alzheimer's disease brains : evidence for disease causation and treatment with small-molecule inhibitors

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease

Propofol induces MAPK/ERK cascade dependant expression of cFos and Egr-1 in rat hippocampal slices

Background: Propofol is a commonly used intravenous anesthetic agent, which produce rapid induction of and recovery from general anesthesia. Numerous clinical studies reported that propofol can potentially cause amnesia and memory loss in human subjects. The underlying mechanism for this memory loss is unclear but may potentially be related to the induction of memory-associated genes such as c-Fos and Egr-1 by propofol. This study explored the effects of propofol on c-Fos and Egr-1 expression in rat hippocampal slices. Findings: Hippocampal brain slices were exposed to varying concentrations of propofol at multiple time intervals. The transcription of the immediate early genes, c-Fos and Egr-1, was quantified using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). MAPK/ERK inhibitors were used to investigate the mechanism of action. We demonstrate that propofol induced the expression of c-Fos and Egr-1 within 30 and 60 min of exposure time. At 16.8 μM concentration, propofol induced a 110% increase in c-Fos transcription and 90% decrease in the transcription of Egr-1. However, at concentrations above 100 μM, propofol failed to induce expression of c-Fos but did completely inhibit the transcription of Egr-1. Propofol-induced c-Fos and Egr-1 transcription was abolished by inhibitors of RAS, RAF, MEK, ERK and p38-MAPK in the MAPK/ERK cascade. Conclusions: Our study shows that clinically relevant concentrations of propofol induce c-Fos and down regulated Egr-1 expression via an MAPK/ERK mediated pathway. We demonstrated that propofol induces a time and dose dependant transcription of IEGs c-Fos and Egr-1 in rat hippocampal slices. We further demonstrate for the first time that propofol induced IEG expression was mediated via a MAPK/ERK dependant pathway. These novel findings provide a new avenue to investigate transcription-dependant mechanisms and suggest a parallel pathway of action with an unclear role in the activity of general anesthetics

AI-powered transmitted light microscopy for functional analysis of live cells

Transmitted light microscopy can readily visualize the morphology of living cells. Here, we introduce artificial-intelligence-powered transmitted light microscopy (AIM) for subcellular structure identification and labeling-free functional analysis of live cells. AIM provides accurate images of subcellular organelles; allows identification of cellular and functional characteristics (cell type, viability, and maturation stage); and facilitates live cell tracking and multimodality analysis of immune cells in their native form without labeling

A Very Large Number of GABAergic Neurons Are Activated in the Tuberal Hypothalamus during Paradoxical (REM) Sleep Hypersomnia

We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD67 mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD67 in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD+, Fos-ir/MCH+, and GAD+/MCH+ double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD+ neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis

Amphetamine-evoked c- fos mRNA expression in the caudate-putamen: the effects of DA and NMDA receptor antagonists vary as a function of neuronal phenotype and environmental context

Dopamine (DA) and glutamate neurotransmission is thought to be critical for psychostimulant drugs to induce immediate early genes (IEGs) in the caudate-putamen (CPu). We report here, however, that the ability of DA and glutamate NMDA receptor antagonists to attenuate amphetamine-evoked c- fos mRNA expression in the CPu depends on environmental context. When given in the home cage, amphetamine induced c- fos mRNA expression predominately in preprodynorphin and preprotachykinin mRNA-containing neurons (Dyn-SP+ cells) in the CPu. In this condition, all of the D1R, D2R and NMDAR antagonists tested dose-dependently decreased c- fos expression in Dyn-SP+ cells. When given in a novel environment, amphetamine induced c- fos mRNA in both Dyn-SP+ and preproenkephalin mRNA-containing neurons (Enk+ cells). In this condition, D1R and non-selective NMDAR antagonists dose-dependently decreased c- fos expression in Dyn-SP+ cells, but neither D2R nor NR2B-selective NMDAR antagonists had no effect. Furthermore, amphetamine-evoked c- fos expression in Enk+ cells was most sensitive to DAR and NMDAR antagonism; the lowest dose of every antagonist tested significantly decreased c- fos expression only in these cells. Finally, novelty-stress also induced c- fos expression in both Dyn-SP+ and Enk+ cells, and this was relatively resistant to all but D1R antagonists. We suggest that the mechanism(s) by which amphetamine evokes c- fos expression in the CPu varies depending on the stimulus (amphetamine vs. stress), the striatal cell population engaged (Dyn-SP+ vs. Enk+ cells), and environmental context (home vs. novel cage).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66272/1/j.1471-4159.2003.01815.x.pd

Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response

<p>Abstract</p> <p>Background</p> <p>Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data.</p> <p>Results</p> <p>A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p < 0.0001) with microarray results.</p> <p>Conclusions</p> <p>Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against neuroinflammation. Pathologic activity of the complement/coagulation cascade has been shown in patients suffering from a chronic form of ciguatera poisoning and is of particular interest in this model. Anti-inflammatory processes were at work not only in the brain but were also seen in whole blood and liver of these animals, creating a systemic anti-inflammatory environment to protect against the initial cellular damage caused by the toxin.</p

BDNF Facilitates L-LTP Maintenance in the Absence of Protein Synthesis through PKMζ

Late-phase long term potentiation (L-LTP) is thought to be the cellular basis for long-term memory (LTM). While LTM as well as L-LTP is known to depend on transcription and translation, it is unclear why brain-derived neurotrophic factor (BDNF) could sustain L-LTP when protein synthesis is inhibited. The persistently active protein kinase ζ (PKMζ) is the only molecule implicated in perpetuating L-LTP maintenance. Here, in mouse acute brain slices, we show that inhibition of PKMζ reversed BDNF-dependent form of L-LTP. While BDNF did not alter the steady-state level of PKMζ, BDNF together with the L-LTP inducing theta-burst stimulation (TBS) increased PKMζ level even without protein synthesis. Finally, in the absence of de novo protein synthesis, BDNF maintained TBS-induced PKMζ at a sufficient level. These results suggest that BDNF sustains L-LTP through PKMζ in a protein synthesis-independent manner, revealing an unexpected link between BDNF and PKMζ

Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening

Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutaminemediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington’s Disease (HD) model.National Institutes of Health (U.S.) (Grant