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Mutations in theUBQLN2gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The neuropathology of suchUBQLN2-linked cases of ALS/FTD is characterised by aggregates of the ubiquilin 2 protein in addition to aggregates of the transactive response DNA-binding protein of 43 kDa (TDP-43). ALS and FTD withoutUBQLN2mutations are also characterised by TDP-43 aggregates, that may or may not colocalise with wildtype ubiquilin 2. Despite this, the relative contributions of TDP-43 and ubiquilin 2 to disease pathogenesis remain largely under-characterised, as does their relative deposition as aggregates across the central nervous system (CNS). Here we conducted multiplex immunohistochemistry of threeUBQLN2p.T487I-linked ALS/FTD cases, three non-UBQLN2-linked (sporadic) ALS cases, and eight non-neurodegenerative disease controls, covering 40 CNS regions. We then quantified ubiquilin 2 aggregates, TDP-43 aggregates, and aggregates containing both proteins in regions of interest to determine howUBQLN2-linked and non-UBQLN2-linked proteinopathy differ. We find that ubiquilin 2 aggregates that are negative for TDP-43 are predominantly small and punctate, and are abundant in the hippocampal formation, spinal cord, all tested regions of neocortex, medulla, and substantia nigra inUBQLN2-linked ALS/FTD but not sporadic ALS. Curiously, the striatum harboured small punctate ubiquilin 2 aggregates in all cases examined, while large diffuse striatal ubiquilin 2 aggregates were specific toUBQLN2-linked ALS/FTD. While ubiquilin 2 deposition in frontotemporal regions may enhance cognitive risk inUBQLN2-linked cases, ubiquilin 2 is deposited mainly in clinically unaffected regions throughout the CNS such that overall symptomology in these cases maps best to the aggregation of TDP-43.

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), PDGFRβ (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.

Transactive response DNA binding protein 43 (TDP-43) is an RNA processing protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear TDP-43 mislocalizes in patients to the cytoplasm, where it forms ubiquitin-positive inclusions in affected neurons and glia. Physiologically, cytoplasmic TDP-43 is associated with stress granules (SGs). Here, we explored TDP-43 cytoplasmic accumulation and stress granule formation following osmotic and oxidative stress. We show that sorbitol drives TDP-43 redistribution to the cytoplasm, while arsenite induces the recruitment of cytoplasmic TDP-43 to TIA-1 positive SGs. We demonstrate that inducing acute oxidative stress after TDP-43 cytoplasmic relocalization by osmotic shock induces poly (ADP-ribose) polymerase (PARP) cleavage, which triggers cellular toxicity. Recruitment of cytoplasmic TDP-43 to polyribosomes occurs in an SH-SY5Y cellular stress model and is observed in FTD brain lysate. Moreover, the processing body (P-body) marker DCP1a is detected in TDP-43 granules during recovery from stress. Overall, this study supports a central role for cytoplasmic TDP-43 in controlling protein translation in stressed cells.

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.

TDP-43 proteinopathy is the major pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal dementia (FTD). Mounting evidence implicates a loss of normal TDP-43 function in neurodegeneration, either resultant from or independent of TDP-43 aggregation. TDP-43 knockdown is therefore a common paradigm for modelling ALS and FTD. However, because TDP-43 can interact directly with thousands of mRNA targets and regulate the function of other RNA binding proteins, the phenotype of TDP-43 depletion is likely to differ depending on the proteomic and transcriptomic profile of the model cell type. Here, we conducted a meta-analysis of publicly available RNA-sequencing datasets that utilized TDP-43 knockdown to model ALS or FTD, and validated these against RNA-sequencing data from TDP-43-immunonegative neuronal nuclei from ALS/FTD brain. We present these analyses as easy-to-use interactive graphical databases. Of 9 TDP-43-knockdown datasets identified, 4 showed significant depletion of TARDBP (human HeLa and SH-SY5Y cell lines, induced human motor neurons, and mouse striatal tissue). There was little overlap in differentially expressed genes between TDP-43-knockdown model cell types, but PFKP, RANBP1, KIAA1324, ELAVL3, and STMN2 were among the common TDP-43 targets. Similarly, there were few genes that showed common patterns of differential exon usage between cell types and which validated in TDP-43-immunonegative neurons, but these included well-known targets POLDIP3, RANBP1, STMN2, and UNC13A, and novel targets EXD3, CEP290, KPNA4, and MMAB. Enrichment analysis showed that TDP-43 knockdown in different cell types affected a unique range of biological pathways. Together, these data identify novel TDP-43 targets, validate known TDP-43 targets, and show that TDP-43 plays both conserved and cell-type-specific roles in the regulation of gene expression and splicing. Identification of cell-type-specific TDP-43 targets will enable sensitive mapping of cell-autonomous TDP-43 dysfunction beyond just neurons, while shared TDP-43 targets are likely to have therapeutic value across myriad cell types.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving progressive degeneration of upper and lower motor neurons. Both lower motor neuron loss and the deposition of phosphorylated TDP-43 inclusions display regional patterning along the spinal cord. The blood-spinal cord barrier (BSCB) ordinarily restricts entry into the spinal cord parenchyma of blood components that are neurotoxic, but in ALS there is evidence for barrier breakdown. Here we sought to examine whether BSCB breakdown, motor neuron loss, and TDP-43 proteinopathy display the same regional patterning across and along the spinal cord.MethodsWe measured cerebrospinal fluid (CSF) hemoglobin in living ALS patients (n=87 controls, n=236 ALS) as a potential biomarker of BSCB and blood-brain barrier leakage. We then immunostained cervical, thoracic, and lumbar post mortem spinal cord tissue (n=5 controls, n=13 ALS) and employed semi-automated imaging and analysis to quantify and map lower motor neuron loss and phosphorylated TDP-43 inclusion load against hemoglobin leakage.ResultsMotor 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 ventral grey (lamina IX) of the cervical and lumbar cord. In contrast, hemoglobin leakage was observed along the ALS spinal cord axis but was most severe in the dorsal grey and white matter in the thoracic spinal cord.ConclusionsOur data show that leakage of the blood-spinal cord barrier occurs during life but at end-stage its distribution is independent from the major motor neuron pathology and is unlikely to be a major contributor to pathogenesis in ALS.

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-1β 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.

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., Jansson et al. design a high content screening system to target inflammation in human brain cells of the blood–brain barrier (pericytes and endothelial cells) to identify inflammatory modifiers. They identify cardiac glycosides as powerful regulators of neuroinflammatory pathways in brain barrier tissues such as vasculature, meninges and choroid plexus.

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.

Hannu Laaksovirta konsortion jäsenenä. The Genomics England Research Consortium, The International ALS/FTD Genomics Consortium (iAFGC), The International FTD Genetics Consortium (IFGC), The International LBD Genomics Consortium (iLBDGC), The NYGC ALS Consortium, The PROSPECT Consortium,17 James B. Rowe,17 Luisa Benussi,18 Giuliano Binetti,18,19 Roberta Ghidoni,18 Edwin Jabbari,20,21 Coralie Viollet,22 Jonathan D. Glass,23 Andrew B. Singleton,24 Vincenzo Silani,25,26 Owen A. Ross,27 Mina Ryten,8,28,29 Ali Torkamani,30 Toshiko Tanaka,31 Luigi Ferrucci,31 Susan M. Resnick,32 We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered. We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

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.

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-throughput screening system to target inflammation in cells of the blood-brain barrier (primary human pericytes and endothelia) and microglia enabling us to target human disease-specific inflammatory modifiers. Screening an FDA-approved drug library we identified digoxin and lanatoside C, members of the cardiac glycoside family as inflammatory modulating drugs that work in blood-brain barrier cells. A novelex vivoassay of leptomeningeal and choroid plexus explants further confirmed that these drugs maintain their function in 3D cultures of brain border tissues. While current therapeutic strategies for the treatment of neurodegenerative diseases are missing the mark in terms of targets, efficacy and translatability, our innovative approach usingin vitroandex vivohuman barrier cells and tissues to target neuroinflammatory pathways is a step forward in drug development and testing, and brings us closer to translatable treatments for human neurodegenerative disease.One Sentence SummaryWe have identified cardiac glycosides as powerful regulators of neuroinflammatory pathways in brain-barrier tissues such as vasculature, meninges and choroid plexus.

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-β (PDGFRβ), α-smooth muscle actin (αSMA), CD13, neural/glial antigen-2 (NG2), CD146 and desmin was examined. We determined that PDGFRβ, 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 (PDGFRβ, 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.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in exon 1 of the huntingtin gene. Emerging evidence shows that additional epigenetic factors can modify disease phenotypes. Harnessing the ability of the epigenome to modify the disease for therapeutic purposes is therefore of interest. Epigenome modifiers, such as histone deacetylase inhibitors (HDACi), have improved pathology in a range of HD models. Yet in clinical trials, HDACi have failed to alleviate HD symptoms in patients. This study investigated potential reasons for the lack of translation of the therapeutic benefits of HDACi from lab to clinic. We analysed histone acetylation patterns of immuno-positive nuclei from brain sections and tissue microarrays from post-mortem human control and HD cases alongside several well-established HD models (OVT73 transgenic HD sheep, YAC128 mice, and an in vitro cell model expressing 97Q mutant huntingtin). Significant increases in histone H4 acetylation were observed in post-mortem HD cases, OVT73 transgenic HD sheep and in vitro models; these changes were absent in YAC128 mice. In addition, nuclear labelling for acetyl-histone H4 levels were inversely proportional to mutant huntingtin aggregate load in HD human cortex. Our data raise concerns regarding the utility of HDACi for the treatment of HD when regions of pathology exhibit already elevated histone acetylation patterns and emphasize the importance of searching for alternative epigenetic targets in future therapeutic strategies aiming to rescue HD phenotypes.

BackgroundMuscle weakness and muscle wasting can be a consequence of aging (sarcopenia) and neuromuscular disorders (NMD). Genome-wide association (GWA) studies have identified genetic variants associated with grip strength (GS, an inverse measure of muscle weakness) and NMD (multiple sclerosis (MS), myasthenia gravis (MG) and amyotrophic lateral sclerosis (ALS)). However, how these variants contribute to the muscle weakness caused by aging or NMD remains obscure.MethodsWe have integrated GS and NMD associated SNPs in a multimorbid analysis that leverages high-throughput chromatin interaction (Hi-C) data and expression quantitative trait loci (eQTL) data to identify allele-specific gene regulation (i.e. eGenes). Pathways and shared drug targets that are enriched by colocalised eGenes were then identified using pathway and drug enrichment analysis.ResultsWe identified gene regulatory mechanisms (eQTL-eGene effects) associated with GS, MG, MS and ALS. The eQTLs associated with GS regulate a subset of eGenes that are also regulated by the eQTLs of MS, MG and ALS. Yet, we did not find any eGenes commonly regulated by all four phenotypes associated eQTLs. By contrast, we identified three pathways (mTOR signaling, axon guidance, and alcoholism) that are commonly affected by the gene regulatory mechanisms associated with all four phenotypes. 13% of the eGenes we identified were known drug targets, and GS shares at least one druggable eGene and pathway with each of the NMD phenotypes.ConclusionsCollectively, these findings identify significant biological overlaps between GS and NMD, demonstrating the potential for spatial genetic analysis to identify mechanisms underlying muscle weakness due to aging and NMD.

Amyotrophic lateral sclerosis (ALS) is a disease with highly heterogenous causes, most of which remain unknown, a multitude of possible disease mechanisms, and no therapy currently available that can halt disease progression. However, recent advances in antisense oligonucleotides have made them a viable option for targeted therapeutics for patients. These molecules offer a method of targeting RNA that is highly specific, adaptable, and does not require viral delivery. Antisense oligonucleotides are therefore being developed for several genetic causes of ALS. Furthermore, biological pathways involved in the pathogenesis of disease also offer tantalizing targets for intervention using antisense oligonucleotides. Here we detail existing and potential targets for antisense oligonucleotides in ALS and briefly examine the requirements for these drugs to reach and be effective in clinic.

There are only limited treatments currently available for Motor Neurone Disease, each with modest benefits. However, there is a large amount of research and drug discovery currently underway worldwide. The New Zealand Motor Neurone Disease Registry was established in 2017 to facilitate participation in research and clinical trials, and to aid researchers in planning and recruitment. The NZ MND Registry is an opt in patient registry which collects demographic, contact and clinical data for those who choose to enrol. We report anonymised aggregated data from the first year’s enrolment. 12th July 2018, there were 142 participants enrolled in the NZ MND Registry. Participant sex distribution reflects the demographics reported worldwide, but ethnicity is divergent from what is seen in New Zealand overall, with an over-representation of people who identify as New Zealand European. 85.5% of participants are diagnosed with sporadic MND and 6.1% with familial MND. The remainder were participants who have not been diagnosed but have a family history, or positive genetic test for a MND-causing mutation. Levels of disability are reported using ALSFRS-R scores, and show that the majority of participants are within the higher range of the scale. The registry has facilitated entry of patients into three studies to date. The establishment of the NZ MND Registry illustrates a swift launch of a rare disease patient registry. The role of patient registries is an ever changing one, but with clear utility at every point of along the pathway to drug discovery.

Background: We determined the mortality rates of motor neuron disease (MND) in New Zealand over 22 years from 1992 to 2013. Previous studies have found an unusually high and/or increasing incidence of MND in certain regions of New Zealand; however, no studies have examined MND rates nationwide to corroborate this. Methods: Death certificate data coded G12.2 by International Classification of Diseases (ICD)-10 coding, or 335.2 by ICD-9 coding were obtained. These codes specify amyotrophic lateral sclerosis, progressive bulbar palsy, or other motor neuron diseases as the underlying cause of death. Mortality rates for MND deaths in New Zealand were age-standardized to the European Standard Population and compared with rates from international studies that also examined death certificate data and were age-standardized to the same standard population. Results and Conclusion: The age-standardized mortality from MND in New Zealand was 2.3 per 100,000 per year from 1992–2007 and 2.8 per 100,000 per year from 2008–2013. These rates were 3.3 and 4.0 per 100,000 per year, respectively, for the population 20 years and older. The increase in rate between these two time periods was likely due to changes in MND death coding from 2008. Contrary to a previous regional study of MND incidence, nationwide mortality rates did not increase steadily over this time period once aging was accounted for. However, New Zealand MND mortality rate was higher than comparable studies we examined internationally (mean 1.67 per 100,000 per year), suggesting that further analysis of MND burden in New Zealand is warranted.

Therapeutic options for patients with amyotrophic lateral sclerosis (ALS) are currently limited. However, recent studies show that almost all cases of ALS, as well as tau-negative frontotemporal dementia (FTD), share a common neuropathology characterized by the deposition of TAR-DNA binding protein (TDP)-43-positive protein inclusions, offering an attractive target for the design and testing of novel therapeutics. Here we demonstrate how diverse environmental stressors linked to stress granule formation, as well as mutations in genes encoding RNA processing proteins and protein degradation adaptors, initiate ALS pathogenesis via TDP-43. We review the progressive development of TDP-43 proteinopathy from cytoplasmic mislocalization and misfolding through to macroaggregation and the addition of phosphate and ubiquitin moieties. Drawing from cellular and animal studies, we explore the feasibility of therapeutics that act at each point in pathogenesis, from mitigating genetic risk using antisense oligonucleotides to modulating TDP-43 proteinopathy itself using small molecule activators of autophagy, the ubiquitin-proteasome system, or the chaperone network. We present the case that preventing the misfolding of TDP-43 and/or enhancing its clearance represents the most important target for effectively treating ALS and frontotemporal dementia. Electronic supplementary material The online version of this article (doi:10.1007/s13311-015-0338-x) contains supplementary material, which is available to authorized users.

Background Brain 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. Methods Pericytes 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. Results P-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. Conclusions Despite 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. Electronic supplementary material The online version of this article (10.1186/s12868-018-0405-4) contains supplementary material, which is available to authorized users.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

Summary The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration., Graphical Abstract, Highlights • Longer G4C2 transcripts form neurotoxic RNA foci in cells and zebrafish • Longer G4C2 foci sequester RNA binding proteins hnRNP-H, SC35, and SF2 • G4C2 RNA foci and hnRNP-H knockdown similarly affect TARBP2 splicing • Seventy percent of G4C2 RNA foci in C9ORF72 ALS/FTD brains colocalize with hnRNP-H, In this study, Shaw and colleagues explore mechanisms underlying toxicity of the expanded G4C2 hexanucleotide intronic repeat in C9ORF72, the most common known cause of ALS and FTD. Pathologically expanded G4C2 RNA transcripts form intranuclear foci, sequester specific RNA-binding proteins, and are potently toxic in transfected cells and zebrafish embryos. One protein, hnRNP-H, is detected in 70% of foci in C9ORF72 brain tissues, and loss of hnRNP-H leads to aberrant RNA splicing that could contribute to neurodegeneration.

Background Neuroinflammation and blood-brain barrier (BBB) disruption are common features of many brain disorders, including Alzheimer’s disease, epilepsy, and motor neuron disease. Inflammation is thought to be a driver of BBB breakdown, but the underlying mechanisms for this are unclear. Brain pericytes are critical cells for maintaining the BBB and are immunologically active. We sought to test the hypothesis that inflammation regulates the BBB by altering pericyte biology. Methods We exposed primary adult human brain pericytes to chronic interferon-gamma (IFNγ) for 4 days and measured associated functional aspects of pericyte biology. Specifically, we examined the influence of inflammation on platelet-derived growth factor receptor-beta (PDGFRβ) expression and signalling, as well as pericyte proliferation and migration by qRT-PCR, immunocytochemistry, flow cytometry, and western blotting. Results Chronic IFNγ treatment had marked effects on pericyte biology most notably through the PDGFRβ, by enhancing agonist (PDGF-BB)-induced receptor phosphorylation, internalization, and subsequent degradation. Functionally, chronic IFNγ prevented PDGF-BB-mediated pericyte proliferation and migration. Conclusions Because PDGFRβ is critical for pericyte function and its removal leads to BBB leakage, our results pinpoint a mechanism linking chronic brain inflammation to BBB dysfunction. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0722-4) contains supplementary material, which is available to authorized users.

Background and purpose: The therapeutic potential of cannabinoids in Huntington's disease (HD) has been investigated by several groups with complex and sometimes contrasting results. We sought to examine key points of intersection between cannabinoid receptor 1 (CB1) signalling, survival and the formation of mutant huntingtin aggregates in HD. Experimental approach: Using a simplified pheochromocytoma (PC12) cell model of HD expressing exon 1 of wild-type or mutant huntingtin, we assayed cell death and aggregate formation using high-throughput cytotoxicity and image-based assays respectively. Key results: CB1 activation by HU210 conferred a small but significant level of protection against mutant huntingtin-induced cell death. Pertussis toxin uncoupled HU210 from the inhibition of cAMP, preventing rescue of cell death. Phosphorylation of extracellular signal-regulated kinase (ERK) was also critical to CB1-mediated rescue. Conversely, treatments that elevated cAMP exacerbated mutant huntingtin-induced cell death. Despite opposing effects on HD cell survival, both HU210 and compounds that elevated cAMP increased the formation of mutant huntingtin aggregates. The increase in aggregation by HU210 was insensitive to Pertussis toxin and UO126, suggesting a G-protein alpha subtype s (Gs)-linked mechanism. Conclusions and implications: We suggest that the CB1 receptor, through G-protein alpha subtype i/o (Gi/o)-linked, ERK-dependent signal transduction, is a therapeutic target in HD. However the protective potential of CB1 may be limited by promiscuous coupling to Gs, the stimulation of cAMP formation and increased aggregate formation. This may underpin the poor therapeutic efficacy of cannabinoids in more complex model systems and suggest that therapies that are selective for the Gi/o, ERK pathway may be of most benefit in HD. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x

The Cannabis sativa plant has been exploited for medicinal, agricultural and spiritual purposes in diverse cultures over thousands of years. Cannabis has been used recreationally for its psychotropic properties, while effects such as stimulation of appetite, analgesia and anti-emesis have lead to the medicinal application of cannabis. Indeed, reports of medicinal efficacy of cannabis can been traced back as far as 2700 BC, and even at that time reports also suggested a neuroprotective effect of the cultivar. The discovery of the psychoactive component of cannabis resin, Δ9-tetrahydrocannabinol (Δ9-THC) occurred long before the serendipitous identification of a G-protein coupled receptor at which Δ9-THC is active in the brain. The subsequent finding of endogenous cannabinoid compounds, the synthesis of which is directed by neuronal excitability and which in turn served to regulate that excitability, further widened the range of potential drug targets through which the endocannabinoid system can be manipulated. As a result of this, alterations in the endocannabinoid system have been extensively investigated in a range of neurodegenerative disorders. In this review we examine the evidence implicating the endocannabinoid system in the cause, symptomatology or treatment of neurodegenerative disease. We examine data from human patients and compare and contrast this with evidence from animal models of these diseases. On the basis of this evidence we discuss the likely efficacy of endocannabinoid-based therapies in each disease context. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x

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.

Neuro2a cells endogenously express cannabinoid type 1 (CB1) receptors. CB1 stimulation with HU210 activated ERK and induced the transcription factor Krox-24. A functional MEK-ERK pathway is an important requirement for CB1-mediated Krox-24 induction as blockade of MEK signaling by UO126 reduces both basal and CB1-mediated activation of Krox-24. CB1 receptor stimulation did not activate either JNK or p38 MAPK pathways or the pro-proliferation phosphatidylinositol 3-kinase (PI3K)-Akt pathway. However, serum removal or blockade of PI3K signaling by LY294002 transiently stimulated basal Krox-24 expression and increased CB1-mediated induction of Krox-24. This was consistent with a transient increase in pMEK, pERK, and pCREB levels following PI3K blockade. These data demonstrate that CB1-mediated activation of the Krox-24 transcription factor is negatively regulated through the PI3K-Akt pathway and reveals several points of signaling cross-talk between these two important kinase pathways.

TDP-43 and TDP-43 Proteinopathy, Paragraph 1: BIn the nucleus, TDP-43 plays a critical role in regulating RNA splicing, as well as modulating microRNA biogenesis [8, 9].^ Reference 9 should be: Kawahara Y, Mieda-Sato A. TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes. Proc Natl Acad Sci U S A. 2012;109(9):3347-52. doi:10.1073/pnas.1112427109. TDP-43 and TDP-43 Proteinopathy, Paragraph 1: BIn addition to TDP-43 RNA, TDP-43 regulates the splicing and stability of a large number of other transcripts [10, 12–15], and thus influences diverse cellular processes.^ Reference 13 should be: Strong MJ, Volkening K, Hammond R, Yang W, Strong W, Leystra-Lantz C et al. TDP-43 is a human low molecular weight neurofilament (hNFL) mRNAbinding protein. Mol Cell Neurosci. 2007;35(2):320-327. Reference 14 should be: Buratti E, Dork T, Zuccato E, Pagani F, Romano M, Baralle FE. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO J. 2001;20(7):1774-84. TDP-43 and TDP-43 Proteinopathy, Paragraph 2: BAlthough mostly nuclear, up to ~30 % of TDP-43 protein can be found in the cytoplasm [16], with nuclear efflux regulated by both activity and stress [17].^ Reference 17 should be: Wang IF, Wu LS, Chang HY, Shen CK. TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor. J Neurochem. 2008;105(3):797806. doi:10.1111/j.1471-4159.2007.05190.x. Protein degradation gene mutations, Paragraph 1: BTDP43 proteostasis is normally maintained by the coordinated action of the UPS and autophagy, which is particularly important for clearing TDP-43 oligomers and aggregates [81–87].^ This should be references 83-87 Protein degradation gene mutations, Paragraph 2: BNotably, VCP and p62 are required for the formation of Baggresomes^ [84, 88, 89], which are large perinuclear inclusions decorated with ubiquitin, ubiquilin, and p 62.^ Reference 84 should be: Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated prote in aggregates by autophagy. J Biol Chem. 2007;282(33):24131-45 (originally designated reference 79) Protein degradation gene mutations, Paragraph 2: BThe TDP43-positive aggregates that are the hallmark ALS pathology are likely aggresomes [76, 90].^ Reference 76 should be: Scotter EL, Vance C, Nishimura AL, et al. Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species. J Cell Sci 2014;127: 1263–1278. (originally designated reference 83) Protein degradation gene mutations, Paragraph 4: BCertainly, small molecule activators of the UPS or autophagy The online version of the original article can be found at http://dx.doi.org/ 10.1007/s13311-015-0338-x. E. L. Scotter :H.

TAR DNA-binding protein (TDP-43, also known as TARDBP) is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients post mortem. TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways.

SummaryExome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis.Video Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, which causes progressive and eventually fatal loss of motor function. Here, we describe genetic and pathologic characterization of brain tissue banked from 19 ALS patients over nearly 20 years at the Department of Anatomy and the Centre for Brain Research, University of Auckland, New Zealand. We screened for mutations in SOD1, TARDBP, FUS, and C9ORF72 genes and for neuropathology caused by phosphorylated TDP-43, dipeptide repeats (DPRs), and ubiquilin. We identified 2 cases with C9ORF72 repeat expansions. Both harbored phosphorylated TDP-43 and DPR inclusions. We show that DPR inclusions can incorporate or occur independently of ubiquilin. We also identified 1 case with a UBQLN2 mutation, which showed phosphorylated TDP-43 and characteristic ubiquilin protein inclusions. This is the first study of ALS genetics in New Zealand, adding New Zealand to the growing list of countries in which C9ORF72 repeat expansion and UBQLN2 mutations are detected in ALS cases.

Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein–protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.

Huntington's disease is a late-onset autosomal dominant inherited neurodegenerative disease characterised by increased symptom severity over time and ultimately premature death. An expanded CAG repeat sequence in the huntingtin gene leads to a polyglutamine expansion in the expressed protein, resulting in complex dysfunctions including cellular excitotoxicity and transcriptional dysregulation. Symptoms include cognitive deficits, psychiatric changes and a movement disorder often referred to as Huntington's chorea, which involves characteristic involuntary dance-like writhing movements. Neuropathologically Huntington's disease is characterised by neuronal dysfunction and death in the striatum and cortex with an overall decrease in cerebral volume (Ho et al., 2001). Neuronal dysfunction begins prior to symptom presentation, and cells of particular vulnerability include the striatal medium spiny neurons. Huntington's is a devastating disease for patients and their families and there is currently no cure, or even an effective therapy for disease symptoms. G-protein coupled receptors are the most abundant receptor type in the central nervous system and are linked to complex downstream pathways, manipulation of which may have therapeutic application in many neurological diseases. This review will highlight the potential of G-protein coupled receptor drug targets as emerging therapies for Huntington's disease.

The cannabinoid receptors CB1 and CB2 each couple to intracellular G proteins in order to transduce agonist binding into a cellular response. Signalling by the two receptors can differ markedly, as indeed can signal transduction through each individual receptor in response to various ligands. The divergence of signalling is regulated at various stages—from G protein coupling to activation of effectors, and in many cases appears to be cell type specific. This review will initially examine the ability of CB1 and CB2 to activate G proteins, and then look at downstream pathways, both G protein dependent and independent, that are activated by these receptors.

The study of endogenous cannabinoid CB1 receptor proteins in neuronal tissues and cells relies on the availability of highly specific antibodies. We have tested the ability of a series of CB1 antibodies to detect endogenous receptors in brain as well as hemagglutinin (HA)-tagged receptors transfected into HEK-293 cells using a combination of immunological methods. An initial comparison of several CB1 antibodies in mouse brain revealed substantial differences in staining pattern to ligand binding by autoradiography. Antibodies were then tested immunocytochemically against HEK cells expressing HA-tagged rat and human CB1 receptors. None of the commercial antibodies tested were able to detect the receptor in this context. All antibodies were then screened by Western blotting using lysates from the HEK cells and rodent brain homogenates. Again, none of the commercially available antibodies detected proteins of the correct molecular weight in transfected cell lines or brain homogenates, although all recognized multiple proteins in brain tissues. We conclude that the commercially available antibodies we tested failed to detect CB1 receptors abundantly expressed in HEK cells or native receptors in brain slices or homogenates. As such, comprehensive validation of the specificity of these CB1 antibodies for a particular application is essential before use.

Mutant protein aggregates are an important biomarker in Huntington's and other neurodegenerative diseases however their quantification has typically relied on manual imaging and counting, or cell-free assays, which do not allow for concurrent analysis of cell viability. Here we describe four automated high throughput image analysis methods, developed using Metamorph™ software, to quantify mutant huntingtin aggregates in a cellular context. Imaging of aggregate-forming cells was also automated, using a Discovery-1™ automated fluorescence microscope. All four analysis methods measured aggregate formation accurately in relation to manual counting, but with differing throughput. Our in-house PolyQ assay gave the highest throughput, processing images at 0.31 s per image. The Cell Scoring assay gave lower throughput, at 19.5 s per image, but offered accurate quantification of the proportion of cells which formed aggregates, without bias from cell death. These image analysis tools provide rapid and objective alternatives to manual counting in studies of aggregate formation, to facilitate the discovery of drugs to treat Huntington's and related neurodegenerative diseases.