Hippocampal aggregation signatures of pathogenic UBQLN2 in amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic variants in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial amyotrophic lateral sclerosis and/or frontotemporal dementia cases not caused by UBQLN2 pathogenic variants, particularly C9orf72-linked cases. This makes the mechanistic role of mutant ubiquilin 2 protein and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 44 genotypically diverse amyotrophic lateral sclerosis cases with or without frontotemporal dementia, including eight cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, p.T487I (two cases), and p.P497L (three cases)). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43, dipeptide repeat aggregates, and p62, in the hippocampus of controls (n = 6), or amyotrophic lateral sclerosis with or without frontotemporal dementia in sporadic (n = 20), unknown familial (n = 3), SOD1-linked (n = 1), FUS-linked (n = 1), C9orf72-linked (n = 5), and UBQLN2-linked (n = 8) cases. We differentiate between i) ubiquilin 2 aggregation together with phosphorylated TDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 pathogenic variants that cause amyotrophic lateral sclerosis/and frontotemporal dementia. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in amyotrophic lateral sclerosis with or without frontotemporal dementia, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

Ultrastructural localization of Porphyromonas gingivalis gingipains in the substantia nigra of Parkinson's disease brains.

Gingipains are protease virulence factors produced by Porphyromonas gingivalis, a Gram-negative bacterium best known for its role in chronic periodontitis. Gingipains were recently identified in the middle temporal gyrus of postmortem Alzheimer's disease (AD) brains, where gingipain load correlated with AD diagnosis and tau and ubiquitin pathology. Since AD and Parkinson's disease (PD) share some overlapping pathologic features, including nigral pathology and Lewy bodies, the current study explored whether gingipains are present in the substantia nigra pars compacta of PD brains. In immunohistochemical techniques and multi-channel fluorescence studies, gingipain antigens were abundant in dopaminergic neurons in the substantia nigra of both PD and neurologically normal control brains. 3-dimensional reconstructions of Lewy body containing neurons revealed that gingipains associated with the periphery of alpha-synuclein aggregates but were occasionally observed inside aggregates. In vitro proteomic analysis demonstrated that recombinant alpha-synuclein is cleaved by lysine-gingipain, generating multiple alpha-synuclein fragments including the non-amyloid component fragments. Immunogold electron microscopy with co-labeling of gingipains and alpha-synuclein confirmed the occasional colocalization of gingipains with phosphorylated (pSER129) alpha-synuclein. In dopaminergic neurons, gingipains localized to the perinuclear cytoplasm, neuromelanin, mitochondria, and nucleus. These data suggest that gingipains localize in dopaminergic neurons in the substantia nigra and interact with alpha-synuclein.

Aggregate-prone brain regions in Parkinson's disease are rich in unique N-terminus α-synuclein conformers with high proteolysis susceptibility.

In Parkinson's disease (PD), and other α-synucleinopathies, α-synuclein (α-Syn) aggregates form a myriad of conformational and truncational variants. Most antibodies used to detect and quantify α-Syn in the human brain target epitopes within the C-terminus (residues 96-140) of the 140 amino acid protein and may fail to capture the diversity of α-Syn variants present in PD. We sought to investigate the heterogeneity of α-Syn conformations and aggregation states in the PD human brain by labelling with multiple antibodies that detect epitopes along the entire length of α-Syn. We used multiplex immunohistochemistry to simultaneously immunolabel tissue sections with antibodies mapping the three structural domains of α-Syn. Discrete epitope-specific immunoreactivities were visualised and quantified in the olfactory bulb, medulla, substantia nigra, hippocampus, entorhinal cortex, middle temporal gyrus, and middle frontal gyrus of ten PD cases, and the middle temporal gyrus of 23 PD, and 24 neurologically normal cases. Distinct Lewy neurite and Lewy body aggregate morphologies were detected across all interrogated regions/cases. Lewy neurites were the most prominent in the olfactory bulb and hippocampus, while the substantia nigra, medulla and cortical regions showed a mixture of Lewy neurites and Lewy bodies. Importantly, unique N-terminus immunoreactivity revealed previously uncharacterised populations of (1) perinuclear, (2) glial (microglial and astrocytic), and (3) neuronal lysosomal α-Syn aggregates. These epitope-specific N-terminus immunoreactive aggregate populations were susceptible to proteolysis via time-dependent proteinase K digestion, suggesting a less stable oligomeric aggregation state. Our identification of unique N-terminus immunoreactive α-Syn aggregates adds to the emerging paradigm that α-Syn pathology is more abundant and complex in human brains with PD than previously realised. Our findings highlight that labelling multiple regions of the α-Syn protein is necessary to investigate the full spectrum of α-Syn pathology and prompt further investigation into the functional role of these N-terminus polymorphs.

Pericytes take up and degrade α-synuclein but succumb to apoptosis under cellular stress.

Parkinson's disease (PD) is characterised by the progressive loss of midbrain dopaminergic neurons and the presence of aggregated α-synuclein (α-syn). Pericytes and microglia, two non-neuronal cells contain α-syn in the human brain, however, their role in disease processes is poorly understood. Pericytes, found surrounding the capillaries in the brain are important for maintaining the blood-brain barrier, controlling blood flow and mediating inflammation. In this study, primary human brain pericytes and microglia were exposed to two different α-synuclein aggregates. Inflammatory responses were assessed using immunocytochemistry, cytometric bead arrays and proteome profiler cytokine array kits. Fixed flow cytometry was used to investigate the uptake and subsequent degradation of α-syn in pericytes. We found that the two α-syn aggregates are devoid of inflammatory and cytotoxic actions on human brain derived pericytes and microglia. Although α-syn did not induce an inflammatory response, pericytes efficiently take up and degrade α-syn through the lysosomal pathway but not the ubiquitin-proteasome system. Furthermore, when pericytes were exposed the ubiquitin proteasome inhibitor-MG132 and α-syn aggregates, there was profound cytotoxicity through the production of reactive oxygen species resulting in apoptosis. These results suggest that the observed accumulation of α-syn in pericytes in human PD brains likely plays a role in PD pathogenesis, perhaps by causing cerebrovascular instability, under conditions of cellular stress.

Tumour infiltrating lymphocyte density differs by meningioma type and is associated with prognosis in atypical meningioma.

Tumour infiltrating lymphocyte (TIL) density is prognostically significant in various tumours, but few studies have investigated its significance in meningioma. This study aimed to investigate how TIL density differs by meningioma histology and whether it is a predictor of meningioma recurrence. We studied CD3, CD8, CD4, FOXP3 and PD-1 positive (+) TIL density in a continuous cohort of 476 meningiomas resected at Auckland Hospital between 2002 and 2011 using tissue microarrays and computer assisted image analysis. TILs were identified in all meningiomas except one (median CD3+ TIL density across entire cohort 53.0 cells/mm2). Most TILs were CD8+ (median 33.6 cells/mm2) with smaller numbers of CD4+ TILs (median 2.9 cells/mm2). PD-1+ (median 0.32 cells/mm2) and FOXP3+ (median 0.0 cells/mm2) TILs were scarce. Reduced CD3+ (p=0.0066), CD8+ (p=0.0029) and PD-1+ (p=0.0375) TIL density was seen in WHO grade II/III meningioma compared with WHO grade I. Pairwise comparison confirmed statistically significant differences in TIL density existed between meningioma types (CD3, CD8, CD4, p<0.0001; FOXP3, p=0.0096; PD-1, p=0.0090) with chordoid meningioma having the lowest overall CD3+ TIL density (median 12.5 cells/mm2). Despite its low TIL density, chordoid meningioma had a higher FOXP3:CD8 ratio than several meningioma types. Atypical meningioma had a higher FOXP3:CD8 ratio than transitional meningioma (p=0.0045). No association between TIL density and recurrence was seen across the entire cohort or by WHO grade. However, CD3+ and CD8+ TIL density was associated with recurrence in atypical meningioma on multivariable analysis (CD3, p=0.0012; CD8, p=0.0071). A higher CD3+ and CD8+ TIL density was associated with improved recurrence free survival. Our findings suggest CD3+ and CD8+ TIL density is prognostically significant in atypical meningioma. Further investigation of this observation and its biological basis is warranted. The differences in TIL density by meningioma histology may be of relevance in studies of therapeutic immune checkpoint inhibition.

Single-cell image analysis reveals a protective role for microglia in glioblastoma.

BACKGROUND: Microglia and tumor-associated macrophages (TAMs) constitute up to half of the total tumor mass of glioblastomas. Despite these myeloid populations being ontogenetically distinct, they have been largely conflated. Recent single-cell transcriptomic studies have identified genes that distinguish microglia from TAMs. Here we investigated whether the translated proteins of genes enriched in microglial or TAM populations can be used to differentiate these myeloid cells in immunohistochemically stained human glioblastoma tissue. METHODS: Tissue sections from resected low-grade, meningioma, and glioblastoma (grade IV) tumors and epilepsy tissues were immunofluorescently triple-labeled for Iba1 (pan-myeloid marker), CD14 or CD163 (preferential TAM markers), and either P2RY12 or TMEM119 (microglial-specific markers). Using a single-cell-based image analysis pipeline, we quantified the abundance of each marker within single myeloid cells, allowing the identification and analysis of myeloid populations. RESULTS: P2RY12 and TMEM119 successfully discriminated microglia from TAMs in glioblastoma. In contrast, CD14 and CD163 expression were not restricted to invading TAMs and were upregulated by tumor microglia. Notably, a higher ratio of microglia to TAMs significantly correlated with increased patient survival. CONCLUSIONS: We demonstrate the validity of previously defined microglial-specific genes P2RY12 and TMEM119 as robust discriminators of microglia and TAMs at the protein level in human tissue. Moreover, our data suggest that a higher proportion of microglia may be beneficial for patient survival in glioblastoma. Accordingly, this tissue-based method for myeloid population differentiation could serve as a useful prognostic tool.

Isolation of adult mouse microglia using their in vitro adherent properties.

Microglia are the primary innate immune effectors of the central nervous system. Although numerous protocols have been developed to isolate fetal mouse microglia, the isolation of adult mouse microglia has proven more difficult. Here, we present a simple, widely accessible protocol to isolate pure microglia cultures from 4- to 14-month-old mouse brains using their adherent properties in vitro. These isolated microglia recapitulate the adherent properties of adult human microglia and present a more suitable model for studying age-related diseases. For complete details on the use and execution of this protocol in adult human microglia, please refer to Rustenhoven et al. (2016).

fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

The Interplay Between Beta-Amyloid 1-42 (Aß1-42)-Induced Hippocampal Inflammatory Response, p-tau, Vascular Pathology, and Their Synergistic Contributions to Neuronal Death and Behavioral Deficits.

Alzheimer's disease (AD), the most common chronic neurodegenerative disorder, has complex neuropathology. The principal neuropathological hallmarks of the disease are the deposition of extracellular ß-amyloid (Aß) plaques and neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau (p-tau) protein. These changes occur with neuroinflammation, a compromised blood-brain barrier (BBB) integrity, and neuronal synaptic dysfunction, all of which ultimately lead to neuronal cell loss and cognitive deficits in AD. Aß1-42 was stereotaxically administered bilaterally into the CA1 region of the hippocampi of 18-month-old male C57BL/6 mice. This study aimed to characterize, utilizing immunohistochemistry and behavioral testing, the spatial and temporal effects of Aß1-42 on a broad set of parameters characteristic of AD: p-tau, neuroinflammation, vascular pathology, pyramidal cell survival, and behavior. Three days after Aß1-42 injection and before significant neuronal cell loss was detected, acute neuroinflammatory and vascular responses were observed. These responses included the up-regulation of glial fibrillary acidic protein (GFAP), cell adhesion molecule-1 (PECAM-1, also known as CD31), fibrinogen labeling, and an increased number of activated astrocytes and microglia in the CA1 region of the hippocampus. From day 7, there was significant pyramidal cell loss in the CA1 region of the hippocampus, and by 30 days, significant localized up-regulation of p-tau, GFAP, Iba-1, CD31, and alpha-smooth muscle actin (α-SMA) in the Aß1-42-injected mice compared with controls. These molecular changes in Aß1-42-injected mice were accompanied by cognitive deterioration, as demonstrated by long-term spatial memory impairment. This study is reporting a comprehensive examination of a complex set of parameters associated with intrahippocampal administration of Aß1-42 in mice, their spatiotemporal interactions and combined contribution to the disease progression. We show that a single Aß injection can reproduce aspects of the inflammatory, vascular, and p-tau induced pathology occurring in the AD human brain that lead to cognitive deficits.

Isolation and culture of functional adult human neurons from neurosurgical brain specimens.

The ability to characterize and study primary neurons isolated directly from the adult human brain would greatly advance neuroscience research. However, significant challenges such as accessibility of human brain tissue and the lack of a robust neuronal cell culture protocol have hampered its progress. Here, we describe a simple and reproducible method for the isolation and culture of functional adult human neurons from neurosurgical brain specimens. In vitro, adult human neurons form a dense network and express a plethora of mature neuronal and synaptic markers. Most importantly, for the first time, we demonstrate the re-establishment of mature neurophysiological properties in vitro, such as repetitive fast-spiking action potentials, and spontaneous and evoked synaptic activity. Together, our dissociated and slice culture systems enable studies of adult human neurophysiology and gene expression under normal and pathological conditions and provide a high-throughput platform for drug testing on brain cells directly isolated from the adult human brain.

The epidemiology of patients undergoing meningioma resection in Auckland, New Zealand, 2002 to 2011.

The incidence of meningioma is known to vary by gender and ethnicity. This study aimed to describe the epidemiological characteristics of a 10-year cohort of patients undergoing meningioma resection at Auckland City Hospital, Auckland, New Zealand. Of particular interest was whether there was any difference in meningioma incidence and recurrence rates between New Zealand Maori and Pacific Island patients compared with other ethnic groups. The study was a retrospective analysis of 493 patients with pathologically confirmed meningioma over the period 1 January 2002 to 31 December 2011. Based on this neurosurgical cohort, the minimum incidence of meningioma in the Auckland region was 3.39 per 100,000 population per year (95% C.I. 3.02-3.80) for the study period. Meningioma was significantly more common in women than men by a ratio of 4.2:1. New Zealand Maori and Pacific Island patients had a significantly higher incidence of meningioma than other ethnic groups. New Zealand Maori had a meningioma incidence 2.74 times that of Europeans (95% C.I. 2.01-3.73, p < 0.001). Pacific Island patients had 2.03 times higher incidence of meningioma than Europeans (95% C.I. 1.42 - 2.89, p < 0.001). The overall meningioma recurrence rate was 21.6% with a mean follow-up of 77 months. Recurrence rates for meningioma among Pacific Island patients were significantly higher than for other ethnic groups (hazard ratio 1.73, p = 0.008). Multivariate analysis of clinical variables confirmed the significance of traditional prognostic factors such as WHO tumour grade and Simpson grade of surgical excision in predicting meningioma recurrence.

Human in vitro systems for examining synaptic function and plasticity in the brain.

The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue. With recent advances in the optimization of protocols for adult human brain preparations, there is a significant opportunity for neuroscientists to validate their findings in human-based systems. This review addresses the methodological aspects, advantages, and disadvantages of human neuron in vitro systems, focusing on the unique properties of human neurons and synapses in neocortical microcircuits. These in vitro models provide the incomparable advantage of being a direct representation of the neurons that have formed part of the human brain until the point of recording, which cannot be replicated by animal models nor human stem-cell systems. Important distinct cellular mechanisms are observed in human neurons that may underlie the higher order cognitive abilities of the human brain. The use of human brain tissue in neuroscience research also raises important ethical, diversity, and control tissue limitations that need to be considered. Undoubtedly however, these human neuron systems provide critical information to increase the potential of translation of treatments from the laboratory to the clinic in a way animal models are failing to provide.

Assessing fibrinogen extravasation into Alzheimer's disease brain using high-content screening of brain tissue microarrays.

BACKGROUND: Tissue microarrays are commonly used to evaluate disease pathology however methods to automate and quantify pathological changes are limited. NEW METHOD: This article demonstrates the utility of the VSlide scanner (MetaSystems) for automated image acquisition from immunolabelled tissue microarray slides, and subsequent automated image analysis with MetaXpress (Molecular Devices) software to obtain objective, efficient and reproducible data from immunolabelled tissue microarray sections. RESULTS: Significant increases in fibrinogen immunolabelling were observed in 29 Alzheimer's disease cases compared to 28 control cases analysed from a single tissue microarray slide. Western blot analysis also demonstrated significant increases in fibrinogen immunolabelling in 6 Alzheimer's cases compared to 6 control cases. The observed changes were also validated with gold standard blinded manual H-scoring. COMPARISON WITH EXISTING METHOD: VSlide Metafer software offers a 'tissue microarray acquisition' plugin for easy mapping of tissue cores with their original position on the tissue microarray map. High resolution VSlide images are compatible with MetaXpress image analysis software. This article details the coupling of these two technologies to accurately and reproducibly analyse immunolabelled tissue microarrays within minutes, compared to the gold standard method of manual counting using H-scores which is significantly slower and prone to inter-observer variation. CONCLUSIONS: Here, we couple brain tissue microarray technology with high-content screening and automated image analysis as a powerful way to address bottle necks in data generation and improve throughput, as well as sensitivity to study biological/pathological changes in brain disease.

Hypothermic neuroprotection is associated with recovery of spectral edge frequency after asphyxia in preterm fetal sheep.

BACKGROUND AND PURPOSE: Electroencephalographic recovery is predictive of outcome after perinatal hypoxia-ischemia, but it is unknown whether early changes in electroencephalographic can predict the response to therapeutic hypothermia in the preterm brain. METHODS: 0.7 gestation fetal sheep received umbilical cord occlusion or sham occlusion for 25 minutes, followed by sham hypothermia or whole-body cooling started either 30 minutes or 5 hours after occlusion and continued for 72 hours. RESULTS: Early but not delayed hypothermia reduced neuronal loss and microglial induction in the striatum, with faster recovery of spectral edge frequency, reduced seizure burden, and less suppression of electroencephalographic amplitude (P<0.05). CONCLUSIONS: Recovery of higher electroencephalographic frequencies may be a biomarker of effective hypothermic neuroprotection in the preterm-equivalent brain.

High content analysis of histone acetylation in human cells and tissues.

There is increasing demand for automated image analysis of cell nuclei to be fast, objective and informative. Here, we have developed a high content analysis method for quantifying histone acetylation within any given population of cells. To demonstrate the utility of this method we quantified the effect of valproic acid (VPA) on histone H3 acetylation levels in SK-N-SH cells, a human neuroblastomal cell line. VPA, commonly used for treatment of bipolar disorder and epilepsy, has also been shown to act as a histone deacetylase inhibitor (HDACi), and to maintain the N-terminals of susceptible histones in an acetylated and transcriptionally active state. The Discovery-1™ (Molecular Devices) platform was used for automated image acquisition of immunolabelled cells. Multiple parameters of labelled nuclei were analysed in 1.82 s per image using the built-in count nuclei assay from MetaMorph™ (Molecular Devices) image analysis software. Data were presented in two forms: summary graphs or heterogeneity profiles using frequency distributions within GraphPad Prism (SmartDrawNet). Results showed that VPA increased histone H3 acetylation in a concentration- and time-dependent manner in SK-N-SH cells. The same analysis was shown to accurately quantify histone acetylation changes in human tissue sections also. Trichostatin A, a known HDACi was used to validate VPA action. Western blotting was used to validate the specificity of the antibodies. Overall these data demonstrate that this novel method for quantifying average treatment effects and the heterogeneity within any given population of cells, is fast, reproducible and can be applied to many different cellular contexts (immunocyto- and immunohisto-chemistry).

M3 muscarinic receptors promote cell survival through activation of the extracellular regulated kinase (ERK1/2) pathway.

Activation of certain subtypes of the muscarinic acetylcholine receptor can enhance cell survival. In SK-N-SH human neuroblastoma cells, muscarinic acetylcholine receptor activation induces phosphorylation of CREB and induction of EGR1, transcription factors associated with cell growth and survival. We identified the M3 muscarinic acetylcholine receptor subtype as being primarily responsible for these transcription factor responses after stimulation with carbachol, using subtype-preferring receptor antagonists and muscarinic snake toxins. In a cell survival/death model in SK-N-SH cells deprived of serum growth factors, carbachol increased cell viability, an effect blocked by the non-specific muscarinic antagonist atropine and the M3-preferring antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), suggesting that the M3 receptor is also driving the survival response in these cells. This cytoprotection is largely dependent on activation of the p44/42 extracellular regulated kinase (ERK1/2) pathway. Understanding such survival signalling pathways is important for both potential interventions in neurodegenerative disease and for targeting neuroblastoma and malignancies of the central nervous system.

High-throughput quantification of Alzheimer's disease pathological markers in the post-mortem human brain.

Quantitative analysis of amyloid plaques and neurofibrillary tangles is central to many Alzheimer's disease studies. A novel approach for quantitative immunohistochemistry of plaques and tangles has arisen from the need to account for the heterogeneous expression pattern of these markers in the human brain. This approach aims to overcome the human bias inherent to many sampling strategies, to account for the effects of tissue shrinkage resulting from antigen-retrieval procedures, and to accelerate the analysis of large sample sets by using a high-throughput quantification system. The procedure entailed three coordinated steps: acquisition of montaged images of entire tissue sections, randomised sampling across the cortex, and automated quantification of the selected samples with morphometric image analysis software. Two-dimensional estimates of plaque and tangle densities were obtained from the superior temporal gyrus and middle temporal gyrus of Alzheimer's disease and normal human brains. Results showed a robust correlation between the numbers of plaques and tangles quantified by automated image analysis and those acquired by manual counting. Correction for antigen-retrieval tissue shrinkage ensured that density measurements were not over-estimated. The value and applicability of this assay was demonstrated by the statistically significant differences observed between the averaged densities of plaques and tangles within different investigational groups. We report an accurate and objective approach to the quantification of plaques and tangles in human brain tissue. Implementation of a randomised sampling strategy coupled with a reproducible automated quantification system will facilitate more rigorous comparison of quantitative data derived from different immunohistochemical studies.

Extracellular signal-regulated kinase involvement in human astrocyte migration.

Glial scar formation occurs after virtually any injury to the brain. The migration of astrocytes into regions of brain injury underlies the formation of the glial scar. The exact role of the glial scar has yet to be elucidated, although it is likely to impair brain recovery. Understanding astrocyte migration is fundamental to understanding the formation of the glial scar. We have used human astrocytes (NT2A cells), derived from human NT2/D1 precursor cells to study astrocyte migration using an in vitro scratch wound model. Time-lapse microscopy and bromodeoxyuridine labeling revealed that the astrocytes migrated rather than proliferated across the scratch. Time course immunocytochemical studies showed that scratching human astrocytes induced the activation (phosphorylation) of ERK 1/2 at 10 min after scratch. The MEK 1/2 inhibitor U0126 inhibited both the ERK 1/2 phosphorylation and the migration of the astrocytes across the wound after scratch. Thus, the migration of human astrocytes after injury is partly initiated by activation of the MEK-ERK signalling pathway.

High throughput quantification of cells with complex morphology in mixed cultures.

Automated image-based and biochemical assays have greatly increased throughput for quantifying cell numbers in in vitro studies. However, it has been more difficult to automate the counting of specific cell types with complex morphologies in mixed cell cultures. We have developed a fully automated, fast, accurate and objective method for the quantification of primary human GFAP-positive astrocytes and CD45-positive microglia from images of mixed cell populations. This method, called the complex cell count (CCC) assay, utilizes a combination of image processing and analysis operations from MetaMorph (Version 6.2.6, Molecular Devices). The CCC assay consists of four main aspects: image processing with a unique combination of morphology filters; digital thresholding; integrated morphometry analysis; and a configuration of object standards. The time needed to analyze each image is 1.82s. Significant correlations have been consistently achieved between the data obtained from CCC analysis and manual cell counts. This assay can quickly and accurately quantify the number of human astrocytes and microglia in mixed cell culture and can be applied to quantifying a range of other cells/objects with complex morphology in neuroscience research.

Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension.

The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone cells reach the olfactory bulb (OB) in rodents. However, the RMS in the adult human brain has been elusive. We demonstrate the presence of a human RMS, which is unexpectedly organized around a lateral ventricular extension reaching the OB, and illustrate the neuroblasts in it. The RMS ensheathing the lateral olfactory ventricular extension, as seen by magnetic resonance imaging, cell-specific markers, and electron microscopy, contains progenitor cells with migratory characteristics and cells that incorporate 5-bromo-2'-deoxyuridine and become mature neurons in the OB.