Cellular Contributions to Glymphatic and Lymphatic Waste Clearance in the Brain.

Cerebrospinal fluid (CSF) bathes and cushions the brain; however, it also serves a major role in the clearance of metabolic wastes and in the distribution of glucose, lipids, and amino acids. Unlike every other organ in the body, the brain parenchyma lacks a traditional lymphatic system to drain fluids and central nervous system (CNS) antigens. It was historically assumed that all brain wastes were removed by endogenous processing, such as phagocytosis and autophagy, while excess fluids drained directly into the blood. However, the twin discoveries of the glial-lymphatic (glymphatic) system and meningeal lymphatics have transformed our understanding of brain waste clearance. The glymphatic system describes the movement of fluids through the subarachnoid space (SAS), the influx along periarterial spaces into the brain parenchyma, and the ultimate efflux back into the SAS along perivenous spaces where it comes into direct contact with the meningeal lymphatics. The dura mater of the meninges contains a bona fide lymphatic network that can drain CSF that has entered the dura. Together, these pathways provide insights into the clearance of molecules and fluids from the brain, and show that the CNS is physically connected to the adaptive immune system. Here, we outline the glymphatic and lymphatic systems, and describe the cellular components that are important to their function.

Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability.

The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.

An inducible genetic tool to track and manipulate specific microglial states reveals their plasticity and roles in remyelination.

Recent single-cell RNA sequencing studies have revealed distinct microglial states in development and disease. These include proliferative-region-associated microglia (PAMs) in developing white matter and disease-associated microglia (DAMs) prevalent in various neurodegenerative conditions. PAMs and DAMs share a similar core gene signature. However, the extent of the dynamism and plasticity of these microglial states, as well as their functional significance, remains elusive, partly due to the lack of specific tools. Here, we generated an inducible Cre driver line, Clec7a-CreERT2, that targets PAMs and DAMs in the brain parenchyma. Utilizing this tool, we profiled labeled cells during development and in several disease models, uncovering convergence and context-dependent differences in PAM and DAM gene expression. Through long-term tracking, we demonstrated microglial state plasticity. Lastly, we specifically depleted DAMs in demyelination, revealing their roles in disease recovery. Together, we provide a versatile genetic tool to characterize microglial states in CNS development and disease.

Identification of direct connections between the dura and the brain.

The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance1,2. How the arachnoid barrier balances separation and communication is poorly understood. Here, using transcriptomic data, we developed transgenic mice to examine specific anatomical structures that function as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, magnetic resonance imaging tracers transit along bridging veins in a similar manner to access the subarachnoid space. Notably, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also enable cellular trafficking, representing a route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that link the central nervous system with the dura and its immunological diversity and waste clearance systems.

Skull bone marrow channels as immune gateways to the central nervous system.

Decades of research have characterized diverse immune cells surveilling the CNS. More recently, the discovery of osseous channels (so-called 'skull channels') connecting the meninges with the skull and vertebral bone marrow has revealed a new layer of complexity in our understanding of neuroimmune interactions. Here we discuss our current understanding of skull and vertebral bone marrow anatomy, its contribution of leukocytes to the meninges, and its surveillance of the CNS. We explore the role of this hematopoietic output on CNS health, focusing on the supply of immune cells during health and disease.

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.

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.

Hypothiocyanous Acid Disrupts the Barrier Function of Brain Endothelial Cells.

Inflammation is a common feature of neurological diseases. During neuroinflammation, neutrophils are recruited to the brain vasculature, where myeloperoxidase can produce hypochlorous acid and the less well-studied oxidant hypothiocyanous acid (HOSCN). In this study, we exposed primary brain endothelial cells (BECs) to HOSCN and observed a rapid loss of transendothelial electrical resistance (TEER) at sublethal concentrations. Decreased barrier function was associated with a loss of tight junctions at cellular contacts and a concomitant loss of dynamic microtubules. Both tight junction and cytoskeletal disruptions were visible within 30 min of exposure, whereas significant loss of TEER took more than 1 h. The removal of the HOSCN after 30 min prevented subsequent barrier dysfunction. These results indicate that BECs are sensitive to HOSCN, resulting in the eventual loss of barrier function. We hypothesise that this mechanism may be relevant in neutrophil transmigration, with HOSCN facilitating blood-brain barrier opening at the sites of egress. Furthermore, this mechanism may be a way through which neutrophils, residing in the vasculature, can influence neuroinflammation in diseases.

Cerebrospinal fluid regulates skull bone marrow niches via direct access through dural channels.

It remains unclear how immune cells from skull bone marrow niches are recruited to the meninges. Here we report that cerebrospinal fluid (CSF) accesses skull bone marrow via dura-skull channels, and CSF proteins signal onto diverse cell types within the niches. After spinal cord injury, CSF-borne cues promote myelopoiesis and egress of myeloid cells into meninges. This reveals a mechanism of CNS-to-bone-marrow communication via CSF that regulates CNS immune responses.

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.

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.

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.

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.

Macrophage migration inhibitory factor (MIF) enhances hypochlorous acid production in phagocytic neutrophils.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli. METHODS: Production of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay. RESULTS: Exposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA. CONCLUSION: Our results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence.

Cardiac glycosides target barrier inflammation of the vasculature, meninges and choroid plexus.

Neuroinflammation is a key component of virtually all neurodegenerative diseases, preceding neuronal loss and associating directly with cognitive impairment. Neuroinflammatory signals can originate and be amplified at barrier tissues such as brain vasculature, surrounding meninges and the choroid plexus. We designed a high content screening system to target inflammation in human brain-derived cells of the blood-brain barrier (pericytes and endothelial cells) to identify inflammatory modifiers. Screening an FDA-approved drug library we identify digoxin and lanatoside C, members of the cardiac glycoside family, as inflammatory-modulating drugs that work in blood-brain barrier cells. An ex vivo assay of leptomeningeal and choroid plexus explants confirm that these drugs maintain their function in 3D cultures of brain border tissues. These results suggest that cardiac glycosides may be useful in targeting inflammation at border regions of the brain and offer new options for drug discovery approaches for neuroinflammatory driven degeneration.

Identification of a dysfunctional microglial population in human Alzheimer's disease cortex using novel single-cell histology image analysis.

In Alzheimer's disease (AD), microglia are affected by disease processes, but may also drive pathogenesis. AD pathology-associated microglial populations have been identified with single-cell RNA-Seq, but have not been validated in human brain tissue with anatomical context. Here, we quantified myeloid cell markers to identify changes in AD pathology-associated microglial populations. We performed fluorescent immunohistochemistry on normal (n = 8) and AD (n = 8) middle temporal gyri, co-labelling the pan-myeloid cell marker, Iba1, with one of 11 markers of interest (MOIs): CD45, HLA-DR, CD14, CD74, CD33, CD206, CD32, CD163, P2RY12, TMEM119, L-Ferritin. Novel image analyses quantified the single-cell abundance of Iba1 and each MOI. Each cell was gated into one Iba1-MOI population (Iba1low MOIhigh, Iba1high MOIhigh, or Iba1high MOIlow) and the abundance of each population was compared between AD and control. Triple-labelling of L-Ferritin and Iba1 with a subset of MOIs was performed to investigate L-Ferritin-MOI co-expression on Iba1low cells. Iba1low MOIhigh myeloid cell populations delineated by MOIs CD45, HLA-DR, CD14, CD74, CD33, CD32, and L-Ferritin were increased in AD. Further investigation of the Iba1low MOIhigh populations revealed that their abundances correlated with tau, but not amyloid beta, load in AD. The Iba1low microglial population highly expressed L-Ferritin, reflecting microglial dysfunction. The L-Ferritinhigh CD74high HLA-DRhigh phenotype of the Iba1low population mirrors that of a human AD pathology-associated microglial subpopulation previously identified using single-cell RNA-Seq. Our high-throughput immunohistochemical data with anatomical context support the microglial dysfunction hypothesis of AD.

Redox signalling and regulation of the blood-brain barrier.

Neurological disorders are associated with increased oxidative stress. Reactive oxidants damage tissue and promote cell death, but it is apparent that oxidants can have more subtle effects on cell function through the modulation of redox-sensitive signalling pathways. Cells of the blood-brain barrier regulate the brain microenvironment but become dysfunctional during neurological disease. The blood-brain barrier is maintained by many cell types, and is modulated by redox-sensitive pathways, ranging from the cytoskeletal elements responsible for establishing a barrier, to growth factor and cytokine signalling pathways that influence neurovascular cells. During neurological disease, blood-brain barrier cells are exposed to exogenously generated oxidants from immune cells, as well as increasing endogenously oxidant production. These oxidants impair the function of the blood-brain barrier, leading to increased leakage and reduced blood flow. Reducing the impact of oxidants on the function of blood-brain barrier cells may provide new strategies for delaying the progression of neurological 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.