Involvement of the tumour necrosis factor receptor system in glioblastoma cell death induced by palbociclib-heptamethine cyanine dye conjugate.

Glioblastoma is the most common and aggressive primary brain tumour in adults. The development of anti-brain cancer agents are challenged by the blood-brain barrier and the resistance conferred by the local tumour microenvironment. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence compounds that have recently emerged as promising agents for drug delivery. We conjugated palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, to an HMCD, MHI-148, and conducted drug activity analysis on primary patient-derived glioblastoma cell lines. In addition to the expected cytostatic activity, our in vitro studies revealed that palbociclib-MHI-148 conjugate resulted in an almost 100-fold increase in cytotoxicity compared to palbociclib alone. This shift of palbociclib from cytostatic to cytotoxic when conjugated to MHI-148 was due to increased DNA damage, as indicated by an increase in γH2AX foci, followed by an increased expression of key extrinsic apoptosis genes, including TP53, TNFR1, TRAIL, FADD and caspase 8. In addition, we observed a time-dependent increase in the cell surface expression of TNFR1, consistent with an observed increase in the secretion TNFα, followed by TNFR1 endocytosis at 48 h. The treatment of patient GBM cells with the palbociclib-MHI-148 conjugate prevented TNFα-induced NFκB translocation, suggesting conjugate-induced TNFR1 signalling favoured the TNFR1-mediated apoptotic response rather than the pro-inflammatory response pathway. Notably, pharmacological inhibition of endocytosis of TNFR1, and siRNA-knockdown of TNFR1 reversed the palbociclib-MHI-148-induced cell death. These results show a novel susceptibility of glioblastoma cells to TNFR1-dependent apoptosis, dependent on inhibition of canonical NFκB signalling using our previously reported palbociclib-HMCD conjugate. Video Abstract.

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.

Quantification of tumorsphere migration with a physics-based machine learning method.

Current analysis techniques available for migration assays only provide quantitative measurements for overall migration. However, the potential of regional migration analyses can open further insight into migration patterns and more avenues of experimentation with the same assays. Previously, we developed an analysis pipeline utilizing the finite element (FE) method to show its potential in analyzing glioblastoma (GBM) tumorsphere migration, especially in characterizing regional changes in the migration pattern. This study aims to streamline and further automate the analysis system by integrating the machine-learning-based U-Net segmentation with the FE method. Our U-Net-based segmentation achieved a 98% accuracy in segmenting our tumorspheres. From the segmentations, FE models made up of 3D hexahedral elements were generated, and the migration patterns of the tumorspheres were analyzed under treatments B and C (under non-disclosure agreements). Our results show that our overall migration analysis correlated very strongly (R2 of 0.9611 and 0.9986 for treatments B and C, respectively) with ImageJ's method of migration area analysis, which is the most common method of tumorsphere migration analysis. Additionally, we were able to quantitatively represent the regional migration patterns in our FE models, which the methods purely based on segmentations could not do. Moreover, the new pipeline improved the efficiency and accessibility of the initial pipeline by implementing machine learning-based automated segmentation onto a mainly open-sourced FE analysis platform. In conclusion, our algorithm enables the development of a high-content and high-throughput in vitro screening platform to elucidate anti-migratory molecules that may reduce the invasiveness of these malignant tumors.

Elucidating the cellular uptake mechanisms of heptamethine cyanine dye analogues for their use as an anticancer drug-carrier molecule for the treatment of glioblastoma.

The development of chemotherapies for glioblastoma is hindered by their limited bioavailability and toxicity on normal brain function. To overcome these limitations, we investigated the structure-dependent activity of heptamethine cyanine dyes (HMCD), a group of tumour-specific and BBB permeable near-infrared fluorescent dyes, in both commercial (U87MG) and patient-derived GBM cell lines. HMCD analogues with strongly ionisable sulphonic acid groups were not taken up by patient-derived GBM cells, but were taken up by the U87MG cell line. HMCD uptake relies on a combination of transporter uptake through organic anion-transporting polypeptides (OATPs) and endocytosis into GBM cells. The uptake of HMCDs was not affected by p-glycoprotein efflux in GBM cells. Finally, we demonstrate structure-dependent cytotoxic activity at high concentrations (EC50 : 1-100 µM), likely due to mitochondrial damage-induced apoptosis. An in vivo orthotopic glioblastoma model highlights tumour-specific accumulation of our lead HMCD, MHI-148, for up to 7 days following a single intraperitoneal injection. These studies suggest that strongly ionisable groups like sulphonic acids hamper the cellular uptake of HMCDs in patient-derived GBM cell lines, highlighting cell line-specific differences in HMCD uptake. We envisage these findings will help in the design and structural modifications of HMCDs for drug-delivery applications for glioblastoma.

Single-cell image analysis reveals over-expression of organic anion transporting polypeptides (OATPs) in human glioblastoma tissue.

Background: Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Whilst the role of the efflux transporters are well established in GBM, the expression and function of uptake transporters, such as the organic anion transporting polypeptide (OATP) family, are not well understood. OATPs possess broad substrate specificity that includes anti-cancer agents; therefore, we sought to investigate the expression of four OATP isoforms in human GBM cell types using patient tumor tissue. Methods: We used fluorescent immunohistochemical labeling of paraffin-embedded surgically resected tissues and single-cell image analysis methods to explore the expression of the OATP isoforms in different tumor cell types through co-labeling with cell-type specific markers, such as IBA1 (pan-myeloid), GFAP (tumor cell), PDGFRß (stromal cell), and UEA-1-lectin (endothelial). Results: We found significant over-expression of all the OATP isoforms (OATP1A2, 2B1, 1C1 and 4A1) in GBM tumor sections when compared to non-neoplastic brain. A single-cell image analysis revealed that OATPs were significantly upregulated throughout the tumor parenchyma, with significantly higher expression found on lectin-positive blood vessels and IBA1-positive myeloid cells in GBM compared to non-tumor brain tissue. Qualitative analysis of the four OATP isoforms demonstrated greater expression of OATP4A1 in peri-necrotic regions of GBM tissue, which correlated with hypoxia-related markers within the Ivy GAP RNAseq dataset. Conclusion: Here, we demonstrate, for the first time, the protein expression of four OATPs in human GBM tissue, including upregulation within the tumor microenvironment by myeloid cells and tumor vasculature, and isoform-specific upregulation within hypoxic niches.

Distinct characteristics of microglia from neurogenic and non-neurogenic regions of the human brain in patients with Mesial Temporal Lobe Epilepsy.

The study of microglia isolated from adult human brain tissue provides unique insight into the physiology of these brain immune cells and their role in adult human brain disorders. Reports of microglia in post-mortem adult human brain tissue show regional differences in microglial populations, however, these differences have not been fully explored in living microglia. In this study biopsy tissue was obtained from epileptic patients undergoing surgery and consisted of both cortical areas and neurogenic ventricular and hippocampal (Hp) areas. Microglia were concurrently isolated from both regions and compared by immunochemistry. Our initial observation was that a greater number of microglia resulted from isolation and culture of ventricular/Hp tissue than cortical tissue. This was found to be due to a greater proliferative capacity of microglia from ventricular/Hp regions compared to the cortex. Additionally, ventricular/Hp microglia had a greater proliferative response to the microglial mitogen Macrophage Colony-Stimulating Factor (M-CSF). This enhanced response was found to be associated with higher M-CSF receptor expression and higher expression of proteins involved in M-CSF signalling DAP12 and C/EBPß. Microglia from the ventricular/Hp region also displayed higher expression of the receptor for Insulin-like Growth Factor-1, a molecule with some functional similarity to M-CSF. Compared to microglia isolated from the cortex, ventricular/Hp microglia showed increased HLA-DP, DQ, DR antigen presentation protein expression and a rounded morphology. These findings show that microglia from adult human brain neurogenic regions are more proliferative than cortical microglia and have a distinct protein expression profile. The data present a case for differential microglial phenotype and function in different regions of the adult human brain and suggest that microglia in adult neurogenic regions are "primed" to an activated state by their unique tissue environment.

The amyotrophic lateral sclerosis-linked protein TDP-43 regulates interleukin-6 cytokine production by human brain pericytes.

Amyotrophic lateral sclerosis (ALS) is a fatal movement disorder involving degeneration of motor neurons through dysfunction of the RNA-binding protein TDP-43. Pericytes, the perivascular cells of the blood-brain, blood-spinal cord, and blood-CSF barriers also degenerate in ALS. Indeed, pericytes are among the earliest cell types to show gene expression changes in pre-symptomatic animal models of ALS. This suggests that pericyte degeneration precedes neurodegeneration and may involve pericyte cell-autonomous TDP-43 dysfunction. Here we determined the effect of TDP-43 dysfunction in human brain pericytes on interleukin 6 (IL-6), a critical secreted inflammatory mediator reported to be regulated by TDP 43. Primary human brain pericytes were cultured from biopsy tissue from epilepsy surgeries and TDP-43 was silenced using siRNA. TDP-43 silencing of pericytes stimulated with pro-inflammatory cytokines, interleukin-1ß or tumour necrosis factor alpha, robustly suppressed the induction of IL-6 transcript and protein. IL-6 regulation by TDP-43 did not involve the assembly of TDP-43 nuclear splicing bodies, and did not occur via altered splicing of IL6. Instead, transcriptome-wide analysis by RNA-Sequencing identified a poison exon in the IL6 destabilising factor HNRNPD (AUF1) as a splicing target of TDP-43. Our data support a model whereby TDP-43 silencing favours destabilisation of IL6 mRNA, via enhanced AU-rich element-mediated decay by HNRNP/AUF1. This suggests that cell-autonomous deficits in TDP-43 function in human brain pericytes would suppress their production of IL-6. Given the importance of the blood-brain and blood-spinal cord barriers in maintaining motor neuron health, TDP-43 in human brain pericytes may represent a cellular target for ALS therapeutics.

Characterization of volumetric growth of intracranial meningiomas in Maori and Pasifika populations in New Zealand.

BACKGROUND: Maori and Pasifika populations in New Zealand have a higher incidence and prevalence of intracranial meningioma (IM). We sought to evaluate the volumetric growth rate of meningiomas under surveillance in these populations. METHODS: From July 2002 to October 2020, 336 patients with a total of 408 IM underwent conservative management with serial radiological surveillance at Auckland City Hospital and met the criteria for the study. Inclusion criteria included: age >16 at diagnosis, ≥2 appropriate scans one or more years apart. Exclusion criteria included previous cranial irradiation, a diagnosis of Neurofibromatosis and prior treatment of meningioma. Demographic and clinical data were obtained from the electronic medical records. Imaging data were recorded from the first and last scans. We utilized open-source image processing software (3D Slicer) for semi-automated segmentation and volume calculation. Consistent with previous literature, we calculated the relative growth rate (RGR, %/year) and annual volume change (AVC, cm3 /year) over time. RESULTS: Four hundred and eight meningiomas were volumetrically characterized for a mean duration of 6.2 years. The Maori and Pasifika populations (n = 134/393) demonstrated a higher RGR (31.41 versus 14.33%/year) (P = 0.026) and AVC (2.05 versus 0.95 cm3 ) (P = 0.025) compared to the control population. They also presented at a younger age and had a higher rate of tumour multiplicity. Males represented only 17.6% of the cohort but exhibited a higher growth rate (AVC = 2.52 cm3 /year) than females (AVC = 0.99 cm3 /year) (P = 0034). CONCLUSIONS: Maori and Pasifika populations in New Zealand have a higher incidence and volumetric growth rate of IM compared to a control population. This warrants further clinical, histopathological and genomic analysis.

Platelet-derived growth factor signalling in neurovascular function and disease.

Platelet-derived growth factors are critical for cerebrovascular development and homeostasis. Abnormalities in this signalling pathway are implicated in neurological diseases, especially those where neurovascular dysfunction and neuroinflammation plays a prominent role in disease pathologies, such as stroke and Alzheimer's disease; the angiogenic nature of this pathway also draws its significance in brain malignancies such as glioblastoma where tumour angiogenesis is profuse. In this review, we provide an updated overview of the actions of the platelet-derived growth factors on neurovascular function, their role in the regulation of perivascular cell types expressing the cognate receptors, neurological diseases associated with aberrance in signalling, and highlight the clinical relevance and therapeutic potentials of this pathway for central nervous system diseases.

Routine culture and study of adult human brain cells from neurosurgical specimens.

When modeling disease in the laboratory, it is important to use clinically relevant models. Patient-derived human brain cells grown in vitro to study and test potential treatments provide such a model. Here, we present simple, highly reproducible coordinated procedures that can be used to routinely culture most cell types found in the human brain from single neurosurgically excised brain specimens. The cell types that can be cultured include dissociated cultures of neurons, astrocytes, microglia, pericytes and brain endothelial and neural precursor cells, as well as explant cultures of the leptomeninges, cortical slice cultures and brain tumor cells. The initial setup of cultures takes ~2 h, and the cells are ready for further experiments within days to weeks. The resulting cells can be studied as purified or mixed population cultures, slice cultures and explant-derived cultures. This protocol therefore enables the investigation of human brain cells to facilitate translation of neuroscience research to the clinic.

Cytoprotective agent troxipide-cyanine dye conjugate with cytotoxic and antiproliferative activity in patient-derived glioblastoma cell lines.

Cytoprotective agents are mainly used to protect the gastrointestinal tract linings and in the treatment of gastric ulcers. These agents are devoid of appreciable cytotoxic or cytostatic effects, and medicinal chemistry efforts to modify them into anticancer agents are rare. A drug repurposing campaign initiated in our laboratory with the primary focus of discovering brain cancer drugs resulted in drug-dye conjugate 1, a combination of the cytoprotective agent troxipide and heptamethine cyanine dye MHI 148. The drug-dye conjugate 1 was evaluated in three different patient-derived adult glioblastoma cell lines, commercially available U87 glioblastoma, and one paediatric glioblastoma cell line. In all cases, the conjugate 1 showed potent cytotoxic activity with nanomolar potency (EC50: 267 nM). Interestingly, troxipide alone does not show any cytotoxic and cytostatic activity in the above cell lines. We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard drug used for glioblastoma treatment, even though the cell lines we used in this study were resistant to TMZ treatment. Herein we disclose the synthesis and in vitro activity of drug-dye conjugate 1 for treatment of difficult-to-treat brain cancers such as glioblastoma.

The Use of Heptamethine Cyanine Dyes as Drug-Conjugate Systems in the Treatment of Primary and Metastatic Brain Tumors.

Effective cancer therapeutics for brain tumors must be able to cross the blood-brain barrier (BBB) to reach the tumor in adequate quantities and overcome the resistance conferred by the local tumor microenvironment. Clinically approved chemotherapeutic agents have been investigated for brain neoplasms, but despite their effectiveness in peripheral cancers, failed to show therapeutic success in brain tumors. This is largely due to their poor bioavailability and specificity towards brain tumors. A targeted delivery system might improve the efficacy of the candidate compounds by increasing the retention time in the tumor tissue, and minimizing the numerous side effects associated with the non-specific distribution of the chemotherapy agent. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence (NIRF) compounds that have recently emerged as promising agents for drug delivery. Initially explored for their use in imaging and monitoring neoplasms, their tumor-targeting properties have recently been investigated for their use as drug carrier systems. This review will explore the recent developments in the tumour-targeting properties of a specific group of NIRF cyanine dyes and the preclinical evidence for their potential as drug-delivery systems in the treatment of primary and metastatic brain tumors.

Extent of resection affects prognosis for patients with glioblastoma in non-eloquent regions.

Glioblastoma (GBM) is a malignant cerebral neoplasm carrying poor prognosis. The importance of extent of resection (EoR) in GBM patient outcomes has been argued in the literature. Previous studies included tumors in eloquent regions of the brain. This confounds the role of EoR by including patients with intrinsically worse outcomes but will be over-represented in the reduced EoR category. In a homogenous group of patients in whom GTR was considered achievable, we investigated the effect of increasing EoR on survival. A retrospective review of 51 patients was undertaken. Quantitative, volumetric analysis of pre-operative and post-operative magnetic resonance image was compared with corresponding clinical details. The primary outcome measured was post-operative overall survival. Median overall survival was 18.3 months for GTR patients compared to 11.6 months for non-GTR (p = 0.025). Median pre-operative contrast-enhancing tumor volume for GTR patients was 54.7 cm3 and 24.9 cm3 for non-GTR. Post-operative median residual tumor volume was 1.1 cm3 in the non-GTR cohort. In multivariate analyses, GTR (HR [95% CI] = 0.973 [0.954-0.994], p = 0.00559) and increasing EoR (HR [95% CI] = 0.964 [0.944-0.985], p = 0.000665) remained predictors of survival. Centile dichotomization of EoR revealed 74% (HR [95% CI] = 0.351 [0.128-0.958], p = 0.0409) as the lowest threshold conferring statistically significant survival benefit. Where technically feasible, both GTR and EoR remained as independent prognostic factors for survival. GTR remains the gold standard for surgical treatment of GBM in patients, 74% being the minimum EoR required to confer survival benefit.

Quantitative immunohistochemical analysis of myeloid cell marker expression in human cortex captures microglia heterogeneity with anatomical context.

Current immunohistochemical methods of studying microglia in the post-mortem human brain do not capture the heterogeneity of microglial function in response to damage and disease. We therefore investigated the expression of eight myeloid cell proteins associated with changes in function alongside Iba1. To study the myeloid cells we used immunohistochemistry on post-mortem human middle temporal gyrus sections from neurologically normal individuals. First we investigated co-labelling between the classical 'activation' marker, HLA-DR and each of the other markers of interest. Significant co-labelling between HLA-DR with CD206, CD32, CD163, or L-Ferritin was observed, although complete overlap of expression of HLA-DR with aforementioned markers was not observed. A qualitative assessment also demonstrated that perivascular macrophages expressed higher levels of the markers of interest we investigated than microglia, suggesting perivascular macrophages show a more phagocytic and antigen presentation state in the human brain. To determine whether the markers of interest were expressed in different functional states, the immunoreactivity for each marker was qualitatively assessed on microglial morphologies. Degenerating marker, L-Ferritin, was specific for dystrophic microglia. We demonstrate that microglial heterogeneity can be investigated in immunohistochemically stain post-mortem human tissue by integrating the single-cell abundance of proteins and cell morphology to infer function.

PARP inhibitor cyanine dye conjugate with enhanced cytotoxic and antiproliferative activity in patient derived glioblastoma cell lines.

We describe the synthesis and in vitro activity of drug-dye conjugate 1, which is a combination of the PARP inhibitor rucaparib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed strong cytotoxic activity with nanomolar potency (EC50: 128 nM), which was a 780 fold improvement over rucaparib itself. We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard drug for treatment for glioblastoma even though these cell lines were resistant to TMZ treatment. We envisage such conjugates to be worth exploring for their utility in the treatment of various brain cancers.

Heptamethine Cyanine Dye Mediated Drug Delivery: Hype or Hope.

This review covers the application of heptamethine cyanine dye (HMCD) mediated drug delivery. A relatively small number of HMCDs possess tumor targeting abilities, and this has spurred interest from research groups to explore them as drug delivery systems. Their tumor selectivity is primarily attributed to their uptake by certain isoforms of organic anion transporting polypeptides (OATPs) which are overexpressed in cancer tissues, although there are other possible mechanisms for the observed selectivity still under investigation. This specificity is confirmed using various cancer cell lines and is accompanied by moderate cytotoxicity. Their retention in tumor tissue is facilitated by the formation of albumin adducts as revealed by published mechanistic studies. HMCDs are also organelle selective dyes with specificity toward mitochondria and lysosomes, and with absorption and emission in the near-infrared region. This makes them valuable tools for biomedical imaging, especially in the field of fluorescence-guided tumor surgery. Furthermore, conjugating antitumor agents to HMCDs is providing novel drugs that await clinical testing. HMCD development as theranostic agents with dual tumor targeting and treatment capability signals a new approach to overcome drug resistance (mediated through evasion of efflux pumps) and systemic toxicity, the two parameters which have long plagued drug discovery.

The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived glioblastoma cell lines.

We describe the synthesis of drug-dye conjugate 1 between anaplastic lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed potent cytotoxic activity with nanomolar potency (EC50: 50.9 nM). We also demonstrate evidence for antiproliferative activity of 1 with single digit nanomolar potency (IC50: 4.7 nM). Furthermore, the cytotoxic effects conveyed a dramatic, 110-fold improvement over Crizotinib. This improvement was even more pronounced (492-fold) when 1 was combined with Temozolomide, the standard drug for treatment for glioblastoma. This work lays the foundation for future exploration of similar tyrosine kinase inhibitor drug-dye conjugates for the treatment of glioblastoma.

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 Aß1-42, 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 Aß1-42 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.

PU.1 regulates Alzheimer's disease-associated genes in primary human microglia.

BACKGROUND: Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer's disease (AD), possibly through limiting neuroinflammatory responses. METHODS: To investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD. RESULTS: Bioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ. CONCLUSIONS: Collectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose.

Markers for human brain pericytes and smooth muscle cells.

Brain pericytes and vascular smooth muscle cells (vSMCs) are a critical component of the neurovascular unit and are important in regulating cerebral blood flow and blood-brain barrier integrity. Identification of subtypes of mural cells in tissue and in vitro is important to any study of their function, therefore we identified distinct mural cell morphologies in neurologically normal post-mortem human brain. Further, the distribution of mural cell markers platelet-derived growth factor receptor-ß (PDGFRß), α-smooth muscle actin (αSMA), CD13, neural/glial antigen-2 (NG2), CD146 and desmin was examined. We determined that PDGFRß, NG2, CD13, and CD146 were expressed in capillary-associated pericytes. NG2, and CD13 were also present on vSMCs in large vessels, however abundant CD146 and desmin staining was also detected in vSMCs on large vessels, co-labelling with αSMA. To determine whether cultures recapitulated observations from tissue, primary human brain pericytes derived from neurologically normal autopsies were analysed for the presence of pericyte markers by immunocytochemistry, western blotting and qPCR. The proteins observed in brain pericytes in tissue (PDGFRß, αSMA, desmin, CD146, CD13, and NG2) were present in vitro, validating a panel of proteins that can be used to label brain pericytes and vSMCs in tissue and in vitro. Finally, we showed that the proteins CD146 and desmin that are expressed on large vessels in situ, are also selective markers of a smooth muscle cell phenotype in vitro.