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.

A panel of TDP-43-regulated splicing events verifies loss of TDP-43 function in amyotrophic lateral sclerosis brain tissue.

TDP-43 dysfunction is a molecular hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major hypothesis of TDP-43 dysfunction in disease is the loss of normal nuclear function, resulting in impaired RNA regulation and the emergence of cryptic exons. Cryptic exons and differential exon usage are emerging as promising markers of lost TDP-43 function in addition to revealing biological pathways involved in neurodegeneration in ALS/FTD. In this brief report, we identified markers of TDP-43 loss of function by depleting TARDBP from post-mortem human brain pericytes, a manipulable in vitro primary human brain cell model, and identifying differential exon usage events with bulk RNA-sequencing analysis. We present these data in an interactive database (https://www.scotterlab.auckland.ac.nz/research-themes/tdp43-lof-db-v2/) together with seven other TDP-43-depletion datasets we meta-analysed previously, for user analysis of differential expression and splicing signatures. Differential exon usage events that were validated by qPCR were then compiled into a 'differential exon usage panel' with other well-established TDP-43 loss-of-function exon markers. This differential exon usage panel was investigated in ALS and control motor cortex tissue to verify whether, and to what extent, TDP-43 loss of function occurs in ALS. We find that profiles of TDP-43-regulated cryptic exons, changed exon usage and changed 3' UTR usage discriminate ALS brain tissue from controls, verifying that TDP-43 loss of function occurs in ALS. We propose that TDP-43-regulated splicing events that occur in brain tissue will have promise as predictors of disease.

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.

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.

Cell Type-Specific Nuclei Markers: The Need for Human Brain Research to Go Nuclear.

Identifying and interrogating cell type-specific populations within the heterogeneous milieu of the human brain is paramount to resolving the processes of normal brain homeostasis and the pathogenesis of neurological disorders. While brain cell type-specific markers are well established, most are localized on cellular membranes or within the cytoplasm, with limited literature describing those found in the nucleus. Due to the complex cytoarchitecture of the human brain, immunohistochemical studies require well-defined cell-specific nuclear markers for more precise and efficient quantification of the cellular populations. Furthermore, efficient nuclear markers are required for cell type-specific purification and transcriptomic interrogation of archived human brain tissue through nuclei isolation-based RNA sequencing. To sate the growing demand for robust cell type-specific nuclear markers, we thought it prudent to comprehensively review the current literature to identify and consolidate a novel series of robust cell type-specific nuclear markers that can assist researchers across a range of neuroscientific disciplines. The following review article collates and discusses several key and prospective cell type-specific nuclei markers for each of the major human brain cell types; it then concludes by discussing the potential applications of cell type-specific nuclear workflows and the power of nuclear-based neuroscientific research.

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.

Analyzing pericytes under mild traumatic brain injury using 3D cultures and dielectric elastomer actuators.

Traumatic brain injury (TBI) is defined as brain damage due to an external force that negatively impacts brain function. Up to 90% of all TBI are considered in the mild severity range (mTBI) but there is still no therapeutic solution available. Therefore, further understanding of the mTBI pathology is required. To assist with this understanding, we developed a cell injury device (CID) based on a dielectric elastomer actuator (DEA), which is capable of modeling mTBI via injuring cultured cells with mechanical stretching. Our injury model is the first to use patient-derived brain pericyte cells, which are ubiquitous cells in the brain involved in injury response. Pericytes were cultured in our CIDs and mechanically strained up to 40%, and by at least 20%, prior to gene expression analysis. Our injury model is a platform capable of culturing and stretching primary human brain pericytes. The heterogeneous response in gene expression changes in our result may suggest that the genes implicated in pathological changes after mTBI could be a patient-dependent response, but requires further validation. The results of this study demonstrate that our CID is a suitable tool for simulating mTBI as an in vitro stretch injury model, that is sensitive enough to induce responses from primary human brain pericytes due to mechanical impacts.

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.

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.

Human pericytes degrade diverse α-synuclein aggregates.

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

N-terminal mutant huntingtin deposition correlates with CAG repeat length and symptom onset, but not neuronal loss in Huntington's disease.

Huntington's disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake an immunohistochemical screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs). Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabeled human brain tissue. Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with a customized automated image analysis pipeline on the TMAs. A 2.5-12 fold increase in the number of Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. MAB5374, MW1, and EPR5526 Htt aggregate numbers were positively correlated with CAG repeat length, and negatively correlated with the age of symptom onset in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and greater Htt cortical aggregate deposition is associated with an earlier age of symptom onset in HD. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD do not specifically immunolabel Htt aggregates in HD human brain tissue exclusively, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

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.

Serum biomarkers of neuroinflammation and blood-brain barrier leakage in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an incurable and rapidly progressive neurological disorder. Biomarkers are critical to understanding disease causation, monitoring disease progression and assessing the efficacy of treatments. However, robust peripheral biomarkers are yet to be identified. Neuroinflammation and breakdown of the blood-brain barrier (BBB) are common to familial and sporadic ALS and may produce a unique biomarker signature in peripheral blood. Using cytometric bead array (n = 15 participants per group (ALS or control)) and proteome profiling (n = 6 participants per group (ALS or control)), we assessed a total of 106 serum cytokines, growth factors, and BBB breakdown markers in the serum of control and ALS participants. Further, primary human brain pericytes, which maintain the BBB, were used as a biosensor of inflammation following pre-treatment with ALS serum. Principal components analysis of all proteome profile data showed no clustering of control or ALS sera, and no individual serum proteins met the threshold for statistical difference between ALS and controls (adjusted P values). However, the 20 most changed proteins between control and ALS sera showed a medium effect size (Cohen's d = 0.67) and cluster analysis of their levels together identified three sample subsets; control-only, mixed control-ALS, and ALS-only. These 20 proteins were predominantly pro-angiogenic and growth factors, including fractalkine, BDNF, EGF, PDGF, Dkk-1, MIF and angiopoietin-2. S100ß, a protein highly concentrated in glial cells and therefore a marker of BBB leakage when found in blood, was unchanged in ALS serum, suggesting that serum protein profiles were reflective of peripheral rather than CNS biofluids. Finally, primary human brain pericytes remained proliferative and their secretome was unchanged by chronic exposure to ALS serum. Our exploratory study suggests that individual serum cytokine levels may not be robust biomarkers in small studies of ALS, but that larger studies using multiplexed analysis of pro-angiogenic and growth factors may identify a peripheral signature of ALS pathogenesis.

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

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

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

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

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.

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) locate 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 25 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.

Blood-spinal cord barrier leakage is independent of motor neuron pathology in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving progressive degeneration of upper and lower motor neurons. The pattern of lower motor neuron loss along the spinal cord follows the pattern of deposition of phosphorylated TDP-43 aggregates. The blood-spinal cord barrier (BSCB) restricts entry into the spinal cord parenchyma of blood components that can promote motor neuron degeneration, but in ALS there is evidence for barrier breakdown. Here we sought to quantify BSCB breakdown along the spinal cord axis, to determine whether BSCB breakdown displays the same patterning as motor neuron loss and TDP-43 proteinopathy. Cerebrospinal fluid hemoglobin was measured in living ALS patients (n = 87 control, n = 236 ALS) as a potential biomarker of BSCB and blood-brain barrier leakage. Cervical, thoracic, and lumbar post-mortem spinal cord tissue (n = 5 control, n = 13 ALS) were then immunolabelled and semi-automated imaging and analysis performed to quantify hemoglobin leakage, lower motor neuron loss, and phosphorylated TDP-43 inclusion load. Hemoglobin leakage was observed along the whole ALS spinal cord axis and was most severe in the dorsal gray and white matter in the thoracic spinal cord. In contrast, motor neuron loss and TDP-43 proteinopathy were seen at all three levels of the ALS spinal cord, with most abundant TDP-43 deposition in the anterior gray matter of the cervical and lumbar cord. Our data show that leakage of the BSCB occurs during life, but at end-stage disease the regions with most severe BSCB damage are not those where TDP-43 accumulation is most abundant. This suggests BSCB leakage and TDP-43 pathology are independent pathologies in ALS.