Thomas I.H. Park

Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices.

During a period of acute ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 mum) obtained from Wistar rats were submerged in ACSF at 36 degrees C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La3+, Gd3+, 2-APB) or lowering temperature by 3 degrees C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 degrees C) and La3+ or Gd3+ (100 microM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.

TGF-beta1 regulates human brain pericyte inflammatory processes involved in neurovasculature function

BackgroundTransforming growth factor beta 1 (TGFβ1) is strongly induced following brain injury and polarises microglia to an anti-inflammatory phenotype. Augmentation of TGFβ1 responses may therefore be beneficial in preventing inflammation in neurological disorders including stroke and neurodegenerative diseases. However, several other cell types display immunogenic potential and identifying the effect of TGFβ1 on these cells is required to more fully understand its effects on brain inflammation. Pericytes are multifunctional cells which ensheath the brain vasculature and have garnered recent attention with respect to their immunomodulatory potential. Here, we sought to investigate the inflammatory phenotype adopted by TGFβ1-stimulated human brain pericytes.MethodsMicroarray analysis was performed to examine transcriptome-wide changes in TGFβ1-stimulated pericytes, and results were validated by qRT-PCR and cytometric bead arrays. Flow cytometry, immunocytochemistry and LDH/Alamar Blue® viability assays were utilised to examine phagocytic capacity of human brain pericytes, transcription factor modulation and pericyte health.ResultsTGFβ1 treatment of primary human brain pericytes induced the expression of several inflammatory-related genes (NOX4, COX2, IL6 and MMP2) and attenuated others (IL8, CX3CL1, MCP1 and VCAM1). A synergistic induction of IL-6 was seen with IL-1β/TGFβ1 treatment whilst TGFβ1 attenuated the IL-1β-induced expression of CX3CL1, MCP-1 and sVCAM-1. TGFβ1 was found to signal through SMAD2/3 transcription factors but did not modify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) translocation. Furthermore, TGFβ1 attenuated the phagocytic ability of pericytes, possibly through downregulation of the scavenger receptors CD36, CD47 and CD68. Whilst TGFβ did decrease pericyte number, this was due to a reduction in proliferation, not apoptotic death or compromised cell viability.ConclusionsTGFβ1 attenuated pericyte expression of key chemokines and adhesion molecules involved in CNS leukocyte trafficking and the modulation of microglial function, as well as reduced the phagocytic ability of pericytes. However, TGFβ1 also enhanced the expression of classical pro-inflammatory cytokines and enzymes which can disrupt BBB functioning, suggesting that pericytes adopt a phenotype which is neither solely pro- nor anti-inflammatory. Whilst the effects of pericyte modulation by TGFβ1 in vivo are difficult to infer, the reduction in pericyte proliferation together with the elevated IL-6, MMP-2 and NOX4 and reduced phagocytosis suggests a detrimental action of TGFβ1 on neurovasculature.

α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson’s disease patients

Parkinson’s disease (PD) is characterized by the presence of inclusions known as Lewy bodies, which mainly consist of α-synuclein (α-syn) aggregates. There is growing evidence that α-syn self-propagates in non-neuronal cells, thereby contributing to the progression and spread of PD pathology in the brain. Tunneling nanotubes (TNTs) are long, thin, F-actin-based membranous channels that connect cells and have been proposed to act as conduits for α-syn transfer between cells. SH-SY5Y cells and primary human brain pericytes, derived from postmortem PD brains, frequently form TNTs that allow α-syn transfer and long-distance electrical coupling between cells. Pericytes in situ contain α-syn precipitates like those seen in neurons. Exchange through TNTs was rapid, but dependent on the size of the protein. Proteins were able to spread throughout a network of cells connected by TNTs. Transfer through TNTs was not restricted to α-syn; fluorescent control proteins and labeled membrane were also exchanged through TNTs. Most importantly the formation of TNTs and transfer continued during mitosis. Together, our results provide a detailed description of TNTs in SH-SY5Y cells and human brain PD pericytes, demonstrating their role in α-syn transfer and further emphasize the importance that non-neuronal cells, such as pericytes play in disease progression.

Isolation of highly enriched primary human microglia for functional studies.

Microglia, the resident macrophages of the central nervous system play vital roles in brain homeostasis through clearance of pathogenic material. Microglia are also implicated in neurological disorders through uncontrolled activation and inflammatory responses. To date, the vast majority of microglial studies have been performed using rodent models. Human microglia differ from rodent counterparts in several aspects including their response to pharmacological substances and their inflammatory secretions. Such differences highlight the need for studies on primary adult human brain microglia and methods to isolate them are therefore required. Our procedure generates microglial cultures of >95% purity from both biopsy and autopsy human brain tissue using a very simple media-based culture procedure that takes advantage of the adherent properties of these cells. Microglia obtained in this manner can be utilised for research within a week. Isolated microglia demonstrate phagocytic ability and respond to inflammatory stimuli and their purity makes them suitable for numerous other forms of in vitro studies, including secretome and transcriptome analysis. Furthermore, this protocol allows for the simultaneous isolation of neural precursor cells during the microglial isolation procedure. As human brain tissue is such a precious and valuable resource the simultaneous isolation of multiple cell types is highly beneficial.

Insulin and IGF1 modulate turnover of polysialylated neural cell adhesion molecule (PSA-NCAM) in a process involving specific extracellular matrix components.

Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM‐interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post‐translational modification, polysialic acid (PSA). Regulation of cell‐surface PSA‐NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca2+‐dependent activity, we demonstrate in TE671 cells that downregulation of PSA‐NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell‐surface PSA‐NCAM; instead, PSA‐NCAM turnover required internalization of the molecule into the cytosol. PSA‐NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin‐like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA‐NCAM turnover at the cell surface.

An anti-inflammatory role for C/EBPδ in human brain pericytes

Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1β-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.

Cultured pericytes from human brain show phenotypic and functional differences associated with differential CD90 expression

The human brain is a highly vascular organ in which the blood-brain barrier (BBB) tightly regulates molecules entering the brain. Pericytes are an integral cell type of the BBB, regulating vascular integrity, neuroinflammation, angiogenesis and wound repair. Despite their importance, identifying pericytes amongst other perivascular cell types and deciphering their specific role in the neurovasculature remains a challenge. Using primary adult human brain cultures and fluorescent-activated cell sorting, we identified two CD73+CD45− mesenchymal populations that showed either high or low CD90 expression. CD90 is known to be present on neurons in the brain and peripheral blood vessels. We found in the human brain, that CD90 immunostaining localised to the neurovasculature and often associated with pericytes. In vitro, CD90+ cells exhibited higher basal proliferation, lower expression of markers αSMA and CD140b, produced less extracellular matrix (ECM) proteins, and exhibited lesser pro-inflammatory responses when compared to the CD90− population. Thus, CD90 distinguishes two interrelated, yet functionally distinct pericyte populations in the adult human brain that may have discrete roles in neurovascular function, immune response and scar formation.

Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

BackgroundBrain pericytes ensheathe the endothelium and contribute to formation and maintenance of the blood–brain-barrier. Additionally, pericytes are involved in several aspects of the CNS immune response including scarring, adhesion molecule expression, chemokine secretion, and phagocytosis. In vitro cultures are routinely used to investigate these functions of brain pericytes, however, these are highly plastic cells and can display differing phenotypes and functional responses depending on their culture conditions. Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.MethodsPericytes were isolated from adult human brain biopsy tissue and cultured in DMEM/F12 (D-pericytes) or Pericyte Medium (P-pericytes). Immunocytochemistry, qRT-PCR, and EdU incorporation were used to determine how this altered their basal phenotype, including the expression of pericyte markers, proliferation, and cell morphology. To determine whether culture media altered the inflammatory response in human brain pericytes, immunocytochemistry, qRT-PCR, cytometric bead arrays, and flow cytometry were used to investigate transcription factor induction, chemokine secretion, adhesion molecule expression, migration, phagocytosis, and response to inflammatory-related growth factors.ResultsP-pericytes displayed elevated proliferation and a distinct bipolar morphology compared to D-pericytes. Additionally, P-pericytes displayed lower expression of pericyte-associated markers NG2, PDGFRβ, and fibronectin, with notably lower αSMA, CD146, P4H and desmin, and higher Col-IV expression. Nuclear NF-kB translocation in response to IL-1β stimulation was observed in both cultures, however, P-pericytes displayed elevated expression of the transcription factor C/EBPδ, and lower expression of the adhesion molecule ICAM-1. P-pericytes displayed elevated phagocytic and migratory ability. Both cultures responded similarly to stimulation by the growth factors TGFβ1 and PDGF-BB.ConclusionsDespite differences in their phenotype and magnitude of response, both P-pericytes and D-pericytes responded similarly to all examined functions, indicating that the neuroinflammatory phenotype of these cells is robust to culture conditions.

Identifying Neural Progenitor Cells in the Adult Human Brain

The discovery, in 1998, that the adult human brain contains at least two populations of progenitor cells and that progenitor cells are upregulated in response to a range of degenerative brain diseases has raised hopes for their use in replacing dying brain cells. Since these early findings the race has been on to understand the biology of progenitor cells in the human brain and they have now been isolated and studied in many major neurodegenerative diseases. Before these cells can be exploited for cell replacement purposes it is important to understand how to: (1) find them, (2) label them, (3) determine what receptors they express, (4) isolate them, and (5) examine their electrophysiological properties when differentiated. In this chapter we have described the methods we use for studying progenitor cells in the adult human brain and in particular the tissue processing, immunohistochemistry, autoradiography, progenitor cell culture, and electrophysiology on brain cells. The Neurological Foundation of New Zealand Human Brain Bank has been receiving human tissue for approximately 20 years during which time we have developed a number of unique ways to examine and isolate progenitor cells from resected surgical specimens as well as from postmortem brain tissue. There are ethical and technical considerations that are unique to working with human brain tissue and these, as well as the processing of this tissue and the culturing of it for the purpose of studying progenitor cells, are the topic of this chapter.

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

BackgroundMicroglia 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.MethodsTo 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.ResultsBioinformatic 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.ConclusionsCollectively, 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.