Microglia directly associate with pericytes in the central nervous system.

Cerebral blood flow (CBF) is important for the maintenance of brain function and its dysregulation has been implicated in Alzheimer's disease (AD). Microglia associations with capillaries suggest they may play a role in the regulation of CBF or the blood-brain-barrier (BBB). We explored the relationship between microglia and pericytes, a vessel-resident cell type that has a major role in the control of CBF and maintenance of the BBB, discovering a spatially distinct subset of microglia that closely associate with pericytes. We termed these pericyte-associated microglia (PEM). PEM are present throughout the brain and spinal cord in NG2DsRed × CX3 CR1+/GFP mice, and in the human frontal cortex. Using in vivo two-photon microscopy, we found microglia residing adjacent to pericytes at all levels of the capillary tree and found they can maintain their position for at least 28 days. PEM can associate with pericytes lacking astroglial endfeet coverage and capillary vessel width is increased beneath pericytes with or without an associated PEM, but capillary width decreases if a pericyte loses a PEM. Deletion of the microglia fractalkine receptor (CX3 CR1) did not disrupt the association between pericytes and PEM. Finally, we found the proportion of microglia that are PEM declines in the superior frontal gyrus in AD. In summary, we identify microglia that specifically associate with pericytes and find these are reduced in number in AD, which may be a novel mechanism contributing to vascular dysfunction in neurodegenerative diseases.

Vascular perfusion differs in two distinct PDGFRß-positive zones within the ischemic core of male mice 2 weeks following photothrombotic stroke.

Stroke therapy has largely focused on preventing damage and encouraging repair outside the ischemic core, as the core is considered irreparable. Recently, several studies have suggested endogenous responses within the core are important for limiting the spread of damage and enhancing recovery, but the role of blood flow and capillary pericytes in this process is unknown. Using the Rose Bengal photothrombotic model of stroke, we illustrate blood vessels are present in the ischemic core and peri-lesional regions 2 weeks post stroke in male mice. A FITC-albumin gel cast of the vasculature revealed perfusion of these vessels, suggesting cerebral blood flow (CBF) may be partially present, without vascular leakage. The length of these vessels is significantly reduced compared to uninjured regions, but the average width is greater, suggesting they are either larger vessels that survived the initial injury, smaller vessels that have expanded in size (i.e., arteriogenesis), or that neovascularization begins with larger vessels. Concurrently, we observed an increase in platelet-derived growth factor receptor beta (PDGFRß, a marker of pericytes) expression within the ischemic core in two distinct patterns, one which resembles pericyte-derived fibrotic scarring at the edge of the core, and one which is vessel associated and may represent blood vessel recovery. We find little evidence for dividing cells on these intralesional blood vessels 2 weeks post stroke. Our study provides evidence flow is present in PDGFRß-positive vessels in the ischemic core 2 weeks post stroke. We hypothesize intralesional CBF is important for limiting injury and for encouraging endogenous repair following cerebral ischemia.

Automated Analysis of PD1 and PDL1 Expression in Lymph Nodes and the Microenvironment of Transmissible Tumors in Tasmanian Devils.

The wild Tasmanian devil (Sarcophilus harrisii) population has suffered a devastating decline due to two clonal transmissible cancers. The first devil facial tumor 1 (DFT1) was observed in 1996, followed by a second genetically distinct transmissible tumor, the devil facial tumor 2 (DFT2), in 2014. DFT1/2 frequently metastasize, with lymph nodes being common metastatic sites. MHC-I downregulation by DFT1 cells is a primary means of evading allograft immunity aimed at polymorphic MHC-I proteins. DFT2 cells constitutively express MHC-I, and MHC-I is upregulated on DFT1/2 cells by interferon gamma, suggesting other immune evasion mechanisms may contribute to overcoming allograft and anti-tumor immunity. Human clinical trials have demonstrated PD1/PDL1 blockade effectively treats patients showing increased expression of PD1 in tumor draining lymph nodes, and PDL1 on peritumoral immune cells and tumor cells. The effects of DFT1/2 on systemic immunity remain largely uncharacterized. This study applied the open-access software QuPath to develop a semiautomated pipeline for whole slide analysis of stained tissue sections to quantify PD1/PDL1 expression in devil lymph nodes. The QuPath protocol provided strong correlations to manual counting. PD-1 expression was approximately 10-fold higher than PD-L1 expression in lymph nodes and was primarily expressed in germinal centers, whereas PD-L1 expression was more widely distributed throughout the lymph nodes. The density of PD1 positive cells was increased in lymph nodes containing DFT2 metastases, compared to DFT1. This suggests PD1/PDL1 exploitation may contribute to the poorly immunogenic nature of transmissible tumors in some devils and could be targeted in therapeutic or prophylactic treatments.Abbreviations: PD1: programmed cell death protein 1; PDL1: programmed death ligand 1; DFT1: devil facial tumor 1; DFT2: devil facial tumor 2; DFTD: devil facial tumor disease; MCC: Matthew's correlation coefficient; DAB: diaminobenzidine; ROI: region of interest.

Time dependent degeneration of the nigrostriatal tract in mice with 6-OHDA lesioned medial forebrain bundle and the effect of activin A on L-Dopa induced dyskinesia.

BACKGROUND: Accurately assessing promising therapeutic interventions for human diseases depends, in part, on the reproducibility of preclinical disease models. With the development of transgenic mice, the rapid adaptation of a 6-OHDA mouse model of Parkinson's disease that was originally described for the use in rats has come with a lack of a comprehensive characterization of lesion progression. In this study we therefore first characterised the time course of neurodegeneration in the substantia nigra pars compacta and striatum over a 4 week period following 6-OHDA injection into the medial forebrain bundle of mice. We then utilised the model to assess the anti-dyskinetic efficacy of recombinant activin A, a putative neuroprotectant and anti-inflammatory that is endogenously upregulated during the course of Parkinson's disease. RESULTS: We found that degeneration of fibers in the striatum was fully established within 1 week following 6-OHDA administration, but that the loss of neurons continued to progress over time, becoming fully established 3 weeks after the 6-OHDA injection. In assessing the anti-dyskinetic efficacy of activin A using this model we found that treatment with activin A did not significantly reduce the severity, or delay the time-of-onset, of dyskinesia. CONCLUSION: First, the current study concludes that a 3 week duration is required to establish a complete lesion of the nigrostriatal tract following 6-OHDA injection into the medial forebrain bundle of mice. Second, we found that activin A was not anti-dyskinetic in this model.

Questions concerning the role of amyloid-ß in the definition, aetiology and diagnosis of Alzheimer's disease.

The dominant hypothesis of Alzheimer's disease (AD) aetiology, the neuropathological guidelines for diagnosing AD and the majority of high-profile therapeutic efforts, in both research and in clinical practice, have been built around one possible causal factor, amyloid-ß (Aß). However, the causal link between Aß and AD remains unproven. Here, in the context of a detailed assessment of historical and contemporary studies, we raise critical questions regarding the role of Aß in the definition, diagnosis and aetiology of AD. We illustrate that a holistic view of the available data does not support an unequivocal conclusion that Aß has a central or unique role in AD. Instead, the data suggest alternative views of AD aetiology are potentially valid, at this time. We propose that an unbiased way forward for the field, beyond the current Aß-centric approach, without excluding a role for Aß, is required to come to an accurate understanding of AD dementia and, ultimately, an effective treatment.

Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility.

BACKGROUND: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). METHODS: We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. RESULTS: iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators. CONCLUSIONS: iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

Time-dependent changes in gene expression induced by secreted amyloid precursor protein-alpha in the rat hippocampus.

BACKGROUND: Differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer's disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15). RESULTS: Treatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer's disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα. CONCLUSIONS: Our results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.

Microglia: a new frontier for synaptic plasticity, learning and memory, and neurodegenerative disease research.

We focus on emerging roles for microglia in synaptic plasticity, cognition and disease. We outline evidence that ramified microglia, traditionally thought to be functionally "resting" (i.e. quiescent) in the normal brain, in fact are highly dynamic and plastic. Ramified microglia continually and rapidly extend processes, contact synapses in an activity and experience dependent manner, and play a functionally dynamic role in synaptic plasticity, possibly through release of cytokines and growth factors. Ramified microglial also contribute to structural plasticity through the elimination of synapses via phagocytic mechanisms, which is necessary for normal cognition. Microglia have numerous mechanisms to monitor neuronal activity and numerous mechanisms also exist to prevent them transitioning to an activated state, which involves retraction of their surveying processes. Based on the evidence, we suggest that maintaining the ramified state of microglia is essential for normal synaptic and structural plasticity that supports cognition. Further, we propose that change of their ramified morphology and function, as occurs in inflammation associated with numerous neurological disorders such as Alzheimer's and Parkinson's disease, disrupts their intricate and essential synaptic functions. In turn altered microglia function could cause synaptic dysfunction and excess synapse loss early in disease, initiating a range of pathologies that follow. We conclude that the future of learning and memory research depends on an understanding of the role of non-neuronal cells and that this should include using sophisticated molecular, cellular, physiological and behavioural approaches combined with imaging to causally link the role of microglia to brain function and disease including Alzheimer's and Parkinson's disease and other neuropsychiatric disorders.

Quantification of AMPA receptor subunits and RNA editing-related proteins in the J20 mouse model of Alzheimer's disease by capillary western blotting.

Introduction: Accurate modelling of molecular changes in Alzheimer's disease (AD) dementia is crucial for understanding the mechanisms driving neuronal pathology and for developing treatments. Synaptic dysfunction has long been implicated as a mechanism underpinning memory dysfunction in AD and may result in part from changes in adenosine deaminase acting on RNA (ADAR) mediated RNA editing of the GluA2 subunit of AMPA receptors and changes in AMPA receptor function at the post synaptic cleft. However, few studies have investigated changes in proteins which influence RNA editing and notably, AD studies that focus on studying changes in protein expression, rather than changes in mRNA, often use traditional western blotting. Methods: Here, we demonstrate the value of automated capillary western blotting to investigate the protein expression of AMPA receptor subunits (GluA1-4), the ADAR RNA editing proteins (ADAR1-3), and proteins known to regulate RNA editing (PIN1, WWP2, FXR1P, and CREB1), in the J20 AD mouse model. We describe extensive optimisation and validation of the automated capillary western blotting method, demonstrating the use of total protein to normalise protein load, in addition to characterising the optimal protein/antibody concentrations to ensure accurate protein quantification. Following this, we assessed changes in proteins of interest in the hippocampus of 44-week-old J20 AD mice. Results: We observed an increase in the expression of ADAR1 p110 and GluA3 and a decrease in ADAR2 in the hippocampus of 44-week-old J20 mice. These changes signify a shift in the balance of proteins that play a critical role at the synapse. Regression analysis revealed unique J20-specific correlations between changes in AMPA receptor subunits, ADAR enzymes, and proteins that regulate ADAR stability in J20 mice, highlighting potential mechanisms mediating RNA-editing changes found in AD. Discussion: Our findings in J20 mice generally reflect changes seen in the human AD brain. This study underlines the importance of novel techniques, like automated capillary western blotting, to assess protein expression in AD. It also provides further evidence to support the hypothesis that a dysregulation in RNA editing-related proteins may play a role in the initiation and/or progression of AD.

The Q/R editing site of AMPA receptor GluA2 subunit acts as an epigenetic switch regulating dendritic spines, neurodegeneration and cognitive deficits in Alzheimer's disease.

BACKGROUND: RNA editing at the Q/R site of GluA2 occurs with ~99% efficiency in the healthy brain, so that the majority of AMPARs contain GluA2(R) instead of the exonically encoded GluA2(Q). Reduced Q/R site editing infcreases AMPA receptor calcium permeability and leads to dendritic spine loss, neurodegeneration, seizures and learning impairments. Furthermore, GluA2 Q/R site editing is impaired in Alzheimer's disease (AD), raising the possibility that unedited GluA2(Q)-containing AMPARs contribute to synapse loss and neurodegeneration in AD. If true, then inhibiting expression of unedited GluA2(Q), while maintaining expression of GluA2(R), may be a novel strategy of preventing synapse loss and neurodegeneration in AD. METHODS: We engineered mice with the 'edited' arginine codon (CGG) in place of the unedited glutamine codon (CAG) at position 607 of the Gria2 gene. We crossbred this line with the J20 mouse model of AD and conducted anatomical, electrophysiological and behavioural assays to determine the impact of eliminating unedited GluA2(Q) expression on AD-related phenotypes. RESULTS: Eliminating unedited GluA2(Q) expression in AD mice prevented dendritic spine loss and hippocampal CA1 neurodegeneration as well as improved working and reference memory in the radial arm maze. These phenotypes were improved independently of Aß pathology and ongoing seizure susceptibility. Surprisingly, our data also revealed increased spine density in non-AD mice with exonically encoded GluA2(R) as compared to their wild-type littermates, suggesting an unexpected and previously unknown role for unedited GluA2(Q) in regulating dendritic spines. CONCLUSION: The Q/R editing site of the AMPA receptor subunit GluA2 may act as an epigenetic switch that regulates dendritic spines, neurodegeneration and memory deficits in AD.

Automated Quantification of Multiple Cell Types in Fluorescently Labeled Whole Mouse Brain Sections Using QuPath.

The quantification of labeled cells in tissue sections is crucial to the advancement of biological knowledge. Traditionally, this was a tedious process, requiring hours of careful manual counting in small portions of a larger tissue section. To overcome this, many automated methods for cell analysis have been developed. Recent advances in whole slide scanning technologies have provided the means to image cells in entire tissue sections. However, common automated analysis tools do not have the capacity to deal with the large image files produced. Herein, we present a protocol for the quantification of two fluorescently labeled cell populations, namely pericytes and microglia, in whole brain tissue sections. This protocol uses custom-made scripts within the open source software QuPath to provide a framework for the careful optimization and validation of automated cell detection parameters. Images obtained from a whole-slide scanner are first loaded into a QuPath project. Manual counts are performed on small sample regions to optimize cell detection parameters prior to automated quantification of cells across entire brain regions. Even though we have quantified pericytes and microglia, any fluorescently labeled cell with clear labeling in and around the nucleus can be analyzed using these methods. This protocol provides a user-friendly and cost-effective framework for the automated analysis of whole tissue sections.

An Automated Approach to Improve the Quantification of Pericytes and Microglia in Whole Mouse Brain Sections.

Whole slide scanning technology has enabled the generation of high-resolution images from complete tissue sections. However, commonly used analysis software is often unable to handle the large data files produced. Here, we present a method using the open-source software QuPath to detect, classify and quantify fluorescently-labeled cells (microglia and pericytes) in whole coronal brain tissue sections. Whole-brain sections from both male and female NG2DsRed x CX3CR1+/GFP mice were analyzed. Small regions of interest were selected and manual counts were compared with counts generated from an automated approach, across a range of detection parameters. The optimal parameters for detecting cells and classifying them as microglia or pericytes in each brain region were determined and applied to annotations corresponding to the entire somatosensory and motor cortices, hippocampus, thalamus, and hypothalamus in each section. 3.74% of all detected cells were classified as pericytes; however, this proportion was significantly higher in the thalamus (6.20%) than in other regions. In contrast, microglia (4.51% of total cells) were more abundant in the cortex (5.54%). No differences were detected between male and female mice. In conclusion, QuPath offers a user-friendly solution to whole-slide image analysis which could lead to important new discoveries in both health and disease.

A new mouse line with reduced GluA2 Q/R site RNA editing exhibits loss of dendritic spines, hippocampal CA1-neuron loss, learning and memory impairments and NMDA receptor-independent seizure vulnerability.

Calcium (Ca2+)-permeable AMPA receptors may, in certain circumstances, contribute to normal synaptic plasticity or to neurodegeneration. AMPA receptors are Ca2+-permeable if they lack the GluA2 subunit or if GluA2 is unedited at a single nucleic acid, known as the Q/R site. In this study, we examined mice engineered with a point mutation in the intronic editing complementary sequence (ECS) of the GluA2 gene, Gria2. Mice heterozygous for the ECS mutation (named GluA2+/ECS(G)) had a ~ 20% reduction in GluA2 RNA editing at the Q/R site. We conducted an initial phenotypic analysis of these mice, finding altered current-voltage relations (confirming expression of Ca2+-permeable AMPA receptors at the synapse). Anatomically, we observed a loss of hippocampal CA1 neurons, altered dendritic morphology and reductions in CA1 pyramidal cell spine density. Behaviourally, GluA2+/ECS(G) mice exhibited reduced motor coordination, and learning and memory impairments. Notably, the mice also exhibited both NMDA receptor-independent long-term potentiation (LTP) and vulnerability to NMDA receptor-independent seizures. These NMDA receptor-independent seizures were rescued by the Ca2+-permeable AMPA receptor antagonist IEM-1460. In summary, unedited GluA2(Q) may have the potential to drive NMDA receptor-independent processes in brain function and disease. Our study provides an initial characterisation of a new mouse model for studying the role of unedited GluA2(Q) in synaptic and dendritic spine plasticity in disorders where unedited GluA2(Q), synapse loss, neurodegeneration, behavioural impairments and/or seizures are observed, such as ischemia, seizures and epilepsy, Huntington's disease, amyotrophic lateral sclerosis, astrocytoma, cocaine seeking behaviour and Alzheimer's disease.

A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice.

The intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice and rats has been plagued by inconsistency, owing in part to the multitude of variables requiring control. In this study we investigated the impact of several major variables on survival rate, lesion volume, neurological scores, cerebral blood flow (CBF) and body weight including filament width, time after reperfusion, occlusion time and the choice of surgical method. Using the Koizumi method, we found ischemic injury can be detected as early as 30 min after reperfusion, to a degree that is not statistically different from 24 h post-perfusion, using 2,3,5-Triphenyltetrazolium chloride (TTC) staining. We also found a distinct increase in total lesion volume with increasing occlusion time, with 30-45 min a critical time for the development of large, reproducible lesions. Furthermore, although we found no significant difference in total lesion volume generated by the Koizumi and Longa methods of MCAO, nor were survival rates appreciably different between the two at 4 h after reperfusion, the Longa method produces significantly greater reperfusion. Finally, we found no statistical evidence to support the exclusion of data from animals experiencing a CBF reduction of <70% in the MCA territory following MCAO, using laser-Doppler flowmetry. Instead we suggest the main usefulness of laser-Doppler flowmetry is for guiding filament placement and the identification of subarachnoid haemorrhages and premature reperfusion. In summary, this study provides detailed evaluation of the Koizumi method of intraluminal filament MCAO in mice and a direct comparison to the Longa method.

Inconsistencies and controversies surrounding the amyloid hypothesis of Alzheimer's disease.

The amyloid hypothesis has driven drug development strategies for Alzheimer's disease for over 20 years. We review why accumulation of amyloid-beta (Aß) oligomers is generally considered causal for synaptic loss and neurodegeneration in AD. We elaborate on and update arguments for and against the amyloid hypothesis with new data and interpretations, and consider why the amyloid hypothesis may be failing therapeutically. We note several unresolved issues in the field including the presence of Aß deposition in cognitively normal individuals, the weak correlation between plaque load and cognition, questions regarding the biochemical nature, presence and role of Aß oligomeric assemblies in vivo, the bias of pre-clinical AD models toward the amyloid hypothesis and the poorly explained pathological heterogeneity and comorbidities associated with AD. We also illustrate how extensive data cited in support of the amyloid hypothesis, including genetic links to disease, can be interpreted independently of a role for Aß in AD. We conclude it is essential to expand our view of pathogenesis beyond Aß and tau pathology and suggest several future directions for AD research, which we argue will be critical to understanding AD pathogenesis.