PSA-NCAM Regulatory Gene Expression Changes in the Alzheimer's Disease Entorhinal Cortex Revealed with Multiplexed in situ Hybridization.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia and is characterized by a substantial reduction of neuroplasticity. Our previous work demonstrated that neurons involved in memory function may lose plasticity because of decreased protein levels of polysialylated neural cell adhesion molecule (PSA-NCAM) in the entorhinal cortex (EC) of the human AD brain, but the cause of this decrease is unclear. OBJECTIVE: To investigate genes involved in PSA-NCAM regulation which may underlie its decrease in the AD EC. METHODS: We subjected neurologically normal and AD human EC sections to multiplexed fluorescent in situ hybridization and immunohistochemistry to investigate genes involved in PSA-NCAM regulation. Gene expression changes were sought to be validated in both human tissue and a mouse model of AD. RESULTS: In the AD EC, a cell population expressing a high level of CALB2 mRNA and a cell population expressing a high level of PST mRNA were both decreased. CALB2 mRNA and protein were not decreased globally, indicating that the decrease in CALB2 was specific to a sub-population of cells. A significant decrease in PST mRNA expression was observed with single-plex in situ hybridization in middle temporal gyrus tissue microarray cores from AD patients, which negatively correlated with tau pathology, hinting at global loss in PST expression across the AD brain. No significant differences in PSA-NCAM or PST protein expression were observed in the MAPT P301S mouse brain at 9 months of age. CONCLUSION: We conclude that PSA-NCAM dysregulation may cause subsequent loss of structural plasticity in AD, and this may result from a loss of PST mRNA expression. Due PSTs involvement in structural plasticity, intervention for AD may be possible by targeting this disrupted plasticity pathway.

Progressive Spread of Beta-amyloid Pathology in an Olfactory-driven Amyloid Precursor Protein Mouse Model.

Years before Alzheimer's disease (AD) is diagnosed, patients experience an impaired sense of smell, and ß-amyloid plaques accumulate within the olfactory mucosa and olfactory bulb (OB). The olfactory vector hypothesis proposes that external agents cause ß-amyloid to aggregate and spread from the OB to connected downstream brain regions. To reproduce the slow accumulation of ß-amyloid that occurs in human AD, we investigated the progressive accumulation of ß-amyloid across the brain using a conditional mouse model that overexpresses a humanized mutant form of the amyloid precursor protein (hAPP) in olfactory sensory neurons. Using design-based stereology, we show the progressive accumulation of ß-amyloid plaques within the OB and cortical olfactory regions with age. We also observe reduced OB volumes in these mice when hAPP expression begins prior-to but not post-weaning which we tracked using manganese-enhanced MRI. We therefore conclude that the reduced OB volume does not represent progressive degeneration but rather disrupted OB development. Overall, our data demonstrate that hAPP expression in the olfactory epithelium can lead to the accumulation and spread of ß-amyloid through the olfactory system into the hippocampus, consistent with an olfactory system role in the early stages of ß-amyloid-related AD progression.

Human pericytes degrade diverse α-synuclein aggregates.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of α-synuclein (α-syn) aggregates. Central to disease progression is the gradual spread of pathological α-syn. α-syn aggregation is closely linked to progressive neuron loss. As such, clearance of α-syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence is increasing that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy. Our previous work has shown that pericytes-vascular mural cells that regulate the blood-brain barrier-contain α-syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar α-syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes cleave fibrillar α-syn aggregates (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110), with cleaved α-syn remaining present for up to 21 days. The number of α-syn aggregates/cell and average aggregate size depends on the type of strain, but differences disappear within 5 five hours of treatment. Our results highlight the role brain vasculature may play in reducing α-syn aggregate burden in PD.

Persistent cortical and white matter inflammation after therapeutic hypothermia for ischemia in near-term fetal sheep.

BACKGROUND: Therapeutic hypothermia significantly improves outcomes after moderate-severe hypoxic-ischemic encephalopathy (HIE), but it is partially effective. Although hypothermia is consistently associated with reduced microgliosis, it is still unclear whether it normalizes microglial morphology and phenotype. METHODS: Near-term fetal sheep (n = 24) were randomized to sham control, ischemia-normothermia, or ischemia-hypothermia. Brain sections were immunohistochemically labeled to assess neurons, microglia and their interactions with neurons, astrocytes, myelination, and gitter cells (microglia with cytoplasmic lipid granules) 7 days after cerebral ischemia. Lesions were defined as areas with complete loss of cells. RNAscope® was used to assess microglial phenotype markers CD86 and CD206. RESULTS: Ischemia-normothermia was associated with severe loss of neurons and myelin (p < 0.05), with extensive lesions, astrogliosis and microgliosis with a high proportion of gitter cells (p < 0.05). Microglial wrapping of neurons was present in both the ischemia groups. Hypothermia improved neuronal survival, suppressed lesions, gitter cells and gliosis (p < 0.05), and attenuated the reduction of myelin area fraction. The "M1" marker CD86 and "M2" marker CD206 were upregulated after ischemia. Hypothermia partially suppressed CD86 in the cortex only (p < 0.05), but did not affect CD206. CONCLUSIONS: Hypothermia prevented lesions after cerebral ischemia, but only partially suppressed microglial wrapping and M1 marker expression. These data support the hypothesis that persistent upregulation of injurious microglial activity may contribute to partial neuroprotection after hypothermia, and that immunomodulation after rewarming may be an important therapeutic target.

Neutrophil-vascular interactions drive myeloperoxidase accumulation in the brain in Alzheimer's disease.

INTRODUCTION: Neutrophil accumulation is a well-established feature of Alzheimer's disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain. MATERIALS AND METHODS: We used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD. RESULTS: There was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature. CONCLUSION: Our report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD.

Characterisation of PDGF-BB:PDGFRß signalling pathways in human brain pericytes: evidence of disruption in Alzheimer's disease.

Platelet-derived growth factor-BB (PDGF-BB):PDGF receptor-ß (PDGFRß) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer's disease (AD) is well documented. We found that PDGF-BB:PDGFRß signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB. We hypothesised that reduced PDGF-BB:PDGFRß signalling in pericytes may impact on the BBB. We therefore tested the effects of PDGF-BB on primary human brain pericytes in vitro to define pathways related to BBB function. Using pharmacological inhibitors, we dissected distinct aspects of the PDGF-BB response that are controlled by extracellular signal-regulated kinase (ERK) and Akt pathways. PDGF-BB promotes the proliferation of pericytes and protection from apoptosis through ERK signalling. In contrast, PDGF-BB:PDGFRß signalling through Akt augments pericyte-derived inflammatory secretions. It may therefore be possible to supplement PDGF-BB signalling to stabilise the cerebrovasculature in AD.

Lamina-specific immunohistochemical signatures in the olfactory bulb of healthy, Alzheimer's and Parkinson's disease patients.

Traditional neuroanatomy immunohistology studies involve low-content analyses of a few antibodies of interest, typically applied and compared across sequential tissue sections. The efficiency, consistency, and ultimate insights of these studies can be substantially improved using high-plex immunofluorescence labelling on a single tissue section to allow direct comparison of many markers. Here we present an expanded and efficient multiplexed fluorescence-based immunohistochemistry (MP-IHC) approach that improves throughput with sequential labelling of up to 10 antibodies per cycle, with no limitation on the number of cycles, and maintains versatility and accessibility by using readily available commercial reagents and standard epifluorescence microscopy imaging. We demonstrate this approach by cumulatively screening up to 100 markers on formalin-fixed paraffin-embedded sections of human olfactory bulb sourced from neurologically normal (no significant pathology), Alzheimer's (AD), and Parkinson's disease (PD) patients. This brain region is involved early in the symptomology and pathophysiology of AD and PD. We also developed a spatial pixel bin analysis approach for unsupervised analysis of the high-content anatomical information from large tissue sections. Here, we present a comprehensive immunohistological characterisation of human olfactory bulb anatomy and a summary of differentially expressed biomarkers in AD and PD using the MP-IHC labelling and spatial protein analysis pipeline.

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.

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.

RNA Quality in Post-mortem Human Brain Tissue Is Affected by Alzheimer's Disease.

Gene expression studies of human post-mortem brain tissue are useful for understanding the pathogenesis of neurodegenerative disease. These studies rely on the assumption that RNA quality is consistent between disease and neurologically normal cases; however, previous studies have suggested that RNA quality may be affected by neurodegenerative disease. Here, we compared RNA quality in human post-mortem brain tissue between neurologically normal cases (n = 14) and neurodegenerative disease cases (Alzheimer's disease n = 10; Parkinson's disease n = 11; and Huntington's disease n = 9) in regions affected by pathology and regions that are relatively devoid of pathology. We identified a statistically significant decrease in RNA integrity number (RIN) in Alzheimer's disease tissue relative to neurologically normal tissue (mixed effects model, p = 0.04). There were no statistically significant differences between neurologically normal cases and Parkinson's disease or Huntington's disease cases. Next, we investigated whether total RNA quality affected mRNA quantification, by correlating RIN with qPCR threshold cycle (CT). CT values for all six genes investigated were strongly correlated with RIN (p < 0.05, Pearson correlation); this effect was only partially mitigated by normalization to RPL30. Our results indicate that RNA quality is decreased in Alzheimer's disease tissue. We recommend that RIN should be considered when this tissue is used in gene expression analyses.

Huntingtin Aggregates in the Olfactory Bulb in Huntington's Disease.

Olfactory deficits are an early and prevalent non-motor symptom of Huntington's disease (HD). In other neurodegenerative diseases where olfactory deficits occur, such as Alzheimer's disease and Parkinson's disease, pathological protein aggregates (tau, ß-amyloid, α-synuclein) accumulate in the anterior olfactory nucleus (AON) of the olfactory bulb (OFB). Therefore, in this study we determined whether aggregates are also present in HD OFBs; 13 HD and five normal human OFBs were stained for mutant huntingtin (mHtt), tau, ß-amyloid, TDP-43, and α-synuclein. Our results show that mHtt aggregates detected with 1F8 antibody are present within all HD OFBs, and mHtt aggregate load in the OFB does not correlate with Vonsattel grading scores. The majority of the aggregates were located in the AON and in similar abundance in each anatomical segment of the AON. No mHtt aggregates were found in controls; 31% of HD cases also contained tau neurofibrillary tangles within the AON. This work demonstrates HD pathology in the OFB and indicates that disease-specific protein aggregation in the AON is a common feature of neurodegenerative diseases that show olfactory deficits.

Polysialic acid masks neural cell adhesion molecule antigenicity.

The neural cell adhesion molecule (NCAM) is a transmembrane protein involved in major cellular processes. The addition of polysialic acid (PSA), a post-translational modification (PTM) almost exclusively carried by NCAM, alters NCAM properties and functions and is therefore tightly regulated. Changes in NCAM and PSA-NCAM take place during development and ageing and occur in various diseases. The presence of PTMs can reduce the accessibility of antibodies to their epitopes and lead to false negative results. Thus, it is vital to identify antibodies that can specifically detect their target regardless of the presence of PTMs. In the present study, four commercially available NCAM antibodies were characterized by western blot and immunocytochemistry. Antibody specificity was determined by decreasing NCAM expression with small interfering RNA and subsequently determining whether the antibodies still produced a signal. In addition, PSA was digested with endoneuraminidase N to assess whether removing PSA improves NCAM detection with these antibodies. Our study revealed that the presence of PSA on NCAM reduced antibody accessibility to the epitope and consequently masked NCAM antigenicity for both techniques investigated. Moreover, three of the four antibodies tested were specific for the detection of NCAM by western blot and by immunocytochemistry. Altogether, this study demonstrates the importance of choosing the correct antibody to study NCAM depending on the technique of interest and underlines the importance of taking PTMs into account when using antibody-based techniques for the study of NCAM.