2,160 research outputs found
Generation of in vitro and in vivo models to study pain perception disorders using genome editing
Several heritable disorders alter an individual's perception of pain. In the present work, two independent models for in vitro and in vivo study of pain-related genes were successfully generated using the CRISPR-Cas system for the exploration of pain mechanisms. First, novel putatively pathogenic mutations in the NTRK1 gene were identified in patients with hereditary sensory and autonomic neuropathy type IV (HSAN-IV). Functional characterisation of the mutations was done using CRISPR-Cas-edited PC12 cells in which Ntrk1 was disrupted. Western blot analysis of cells overexpressing the mutant NTRK1 proteins revealed altered activation of signalling pathways as well as transport defects. Moreover, neurite outgrowth was impaired in Ntrk1-KO PC12 cells overexpressing the mutant proteins, and localisation studies showed altered expression patterns in one of the mutants. Taken together, these results indicate that this cellular system is a valuable tool to investigate the pathogenicity of NTRK1 protein variants and suggests that different perturbations of the downstream signalling cascade can result in loss of nociceptor function. Second, a double-knockout (DKO) mouse model disrupting the genetically linked genes Scn10A and Scn11a, encoding the ion channels NaV1.8 and NaV1.9, respectively, was generated. This mouse model will be used shortly to address the function of these two sodium channels in pain perception. Moreover, DKO-dorsal root ganglion neurones (DRGs) can be used as a cellular model for the functional characterisation of NaV1.8 and NaV1.9 disease-causing protein variants
Defining the functional role of NaV1.7 in human nociception
Loss-of-function mutations in NaV1.7 cause congenital insensitivity to pain (CIP); this voltage-gated sodium channel is therefore a key target for analgesic drug development. Utilizing a multi-modal approach, we investigated how NaV1.7 mutations lead to human pain insensitivity. Skin biopsy and microneurography revealed an absence of C-fiber nociceptors in CIP patients, reflected in a reduced cortical response to capsaicin on fMRI. Epitope tagging of endogenous NaV1.7 revealed the channel to be localized at the soma membrane, axon, axon terminals, and the nodes of Ranvier of induced pluripotent stem cell (iPSC) nociceptors. CIP patient-derived iPSC nociceptors exhibited an inability to properly respond to depolarizing stimuli, demonstrating that NaV1.7 is a key regulator of excitability. Using this iPSC nociceptor platform, we found that some NaV1.7 blockers undergoing clinical trials lack specificity. CIP, therefore, arises due to a profound loss of functional nociceptors, which is more pronounced than that reported in rodent models, or likely achievable following acute pharmacological blockade
Perspective: Quality Versus Quantity; Is It Important to Assess the Role of Enhancers in Complex Disease from an In Vivo Perspective?
AMcE was funded by BBSRC project grant (BB/N017544/1).Peer reviewedPublisher PD
Mapping the Ethical Issues of Brain Organoid Research and Application
脳オルガノイドの研究と臨床応用での倫理問題を体系化. 京都大学プレスリリース. 2021-04-07.Society is not ready to make human brains. 京都大学プレスリリース. 2021-03-26.In 2008, researchers created human three-dimensional neural tissue – known as the pioneering work of “brain organoids.” In recent years, some researchers have transplanted human brain organoids into animal brains for applicational purposes. With these experiments have come many ethical concerns. It is thus an urgent task to clarify what is ethically permissible and impermissible in brain organoid research. This paper seeks (1) to sort out the ethical issues related to brain organoid research and application and (2) to propose future directions for additional ethical consideration and policy debates in the field. Toward (1), this paper first outlines the current state of brain organoid research, and then briefly responds to previously raised related ethical concerns. Looking next at anticipated scientific developments in brain organoid research, we will discuss (i) ethical issues related to in vitro brain organoids, (ii) ethical issues raised when brain organoids form complexes or have relationships with other entities, and (iii) ethical issues of research ethics and governance. Finally, in pursuit of (2), we propose research policies that are mindful of the ethics of brain organoid research and application and also suggest the need for an international framework for research and application of brain organoids
Psychiatric Disorders and lncRNAs: A Synaptic Match
Psychiatric disorders represent a heterogeneous class of multifactorial mental diseases whose origin entails a pathogenic integration of genetic and environmental influences. Incidence of these pathologies is dangerously high, as more than 20% of the Western population is affected. Despite the diverse origins of specific molecular dysfunctions, these pathologies entail disruption of fine synaptic regulation, which is fundamental to behavioral adaptation to the environment. The synapses, as functional units of cognition, represent major evolutionary targets. Consistently, fine synaptic tuning occurs at several levels, involving a novel class of molecular regulators known as long non-coding RNAs (lncRNAs). Non-coding RNAs operate mainly in mammals as epigenetic modifiers and enhancers of proteome diversity. The prominent evolutionary expansion of the gene number of lncRNAs in mammals, particularly in primates and humans, and their preferential neuronal expression does represent a driving force that enhanced the layering of synaptic control mechanisms. In the last few years, remarkable alterations of the expression of lncRNAs have been reported in psychiatric conditions such as schizophrenia, autism, and depression, suggesting unprecedented mechanistic insights into disruption of fine synaptic tuning underlying severe behavioral manifestations of psychosis. In this review, we integrate literature data from rodent pathological models and human evidence that proposes the biology of lncRNAs as a promising field of neuropsychiatric investigation
Genetics of neuropathic pain:the emerging role of variants in ion channels and pain-related genes
Adeno-Associated Viral Vectors in Neuroscience Research
Adeno-associated viral vectors (AAVs) are increasingly useful preclinical tools in neuroscience research studies for interrogating cellular and neurocircuit functions and mapping brain connectivity. Clinically, AAVs are showing increasing promise as viable candidates for treating multiple neurological diseases. Here, we briefly review the utility of AAVs in mapping neurocircuits, manipulating neuronal function and gene expression, and activity labeling in preclinical research studies as well as AAV-based gene therapies for diseases of the nervous system. This review highlights the vast potential that AAVs have for transformative research and therapeutics in the neurosciences
G protein biased signaling by non-catechol dopamine D1 receptor agonists
Dopamine is a catecholamine neurotransmitter with essential roles in voluntary movement, working memory, attention, and reward. Dopamine acts through five G protein coupled receptors with the D1 and D5 receptors (D1R) stimulating Galphas/olf activation and increasing neuronal excitability. Deficits in D1R signaling are implicated in Parkinson\u2019s disease motor deficits as well as cognitive deficits in schizophrenia and attention deficit hyperactivity disorder. For more than 40 years, academic and industry scientists have been searching for a drug-like D1R agonist, but this has remained elusive. The challenge in developing D1R selective agonists is that all previous agonists possess a common problematic chemical moiety, a catechol. Catechols are associated with poor oral bioavailability, poor brain penetration, and rapid metabolism in the serum. Very recently, the breakthrough discovery of the first non-catechol D1R selective agonists overcame the pitfalls associated with the catechols. Unexpectedly, the non-catechol agonists also selectively activate G protein signaling without engaging beta-arrestin indicating that they are G protein biased. The primary goals for this study were to characterize novel signaling by non-catechol agonists and elucidate a mechanism of action for the G protein biased non-catechol agonists. First, the role of beta-arrestin in D1R agonist-induced endocytosis was established in HEK293 cells that had beta-arrestin1/2 knocked out by CRISPR/Cas9 genome editing. The knockout of beta-arrestin1/2 eliminated D1R agonist-induced endocytosis. beta-arrestin1/2 knockout significantly reduced D1R agonist-induced endocytosis in an ELISA assay that measures cell surface D1R. Furthermore, re-expressing either beta-arrestin1 or 2 rescued D1R endocytosis in confocal imaging and cell surface ELISA assays. Together, these results indicate that beta-arrestin1/2 are required for D1R agonist-induced endocytosis. Next, catechol and non-catechol D1R agonists were tested in cAMP Glosensor and beta-arrestin Tango assays to investigate potential biased signaling. The unbiased catechol D1R full agonist SKF-81297 was used as the reference compound in all following studies. The non-catechol D1R agonists dose-dependently increased cAMP production in HEK293 cells similar to the full agonist SKF-81297 (Emax 100%), but did not engage beta-arrestin. Interestingly, one non-catechol agonist (PW441) robustly activated both cAMP (Emax = 92%, EC50 = 4.4 nM) and also fully recruited beta-arrestin (Emax = 100%, EC50 = 100 nM). The catechol agonist A-77636 dose-dependently increased full cAMP production (Emax = 104%, EC50 = 3.1 nM) but was a super agonist for beta-arrestin recruitment (Emax = 130%, EC50 = 35 nM). To determine the effect of G protein biased agonists on D1R endocytosis, the catechol and non-catechol D1R agonists were tested in imaging and cell surface ELISA assays. The non-catechol G protein biased agonists all induced significantly less total D1R endocytosis than the catechol agonist SKF-81297. The pure G protein biased agonists PF-1119 and PW464 maximally induced 5% and 11% loss of cell surface D1R, respectively. In contrast, the catechol A-77636 maximally induced 47% loss of cell surface D1R and induced significantly more total endocytosis than SKF-81297. Moreover, the efficacy for beta-arrestin recruitment strongly correlates to the maximum receptor endocytosis in Spearman\u2019s correlation analysis (r = 0.96, p<0.05). Collectively, this study demonstrates the essential role of beta-arrestin in D1R agonist-induced endocytosis and characterizes novel non-catechol agonists. In addition, the discovery of the first balanced non-catechol D1R selective agonist adds a unique tool for future in vitro and in vivo studies. These results further elucidate a mechanism of action for the G protein biased non-catechol agonists in which agonist binding induces G protein activation without also inducing D1R endocytosis. These results provide insights into the molecular mechanism of the G protein biased non-catechol agonists. While the clinical efficacy of the non-catechol agonists is currently being explored, the mechanism of action is not fully understood. This study explored novel derivatives and their downstream effects on D1R endocytosis.Dopamine is a catecholamine neurotransmitter with essential roles in voluntary movement, working memory, attention, and reward. Dopamine acts through five G protein coupled receptors with the D1 and D5 receptors (D1R) stimulating Galphas/olf activation and increasing neuronal excitability. Deficits in D1R signaling are implicated in Parkinson\u2019s disease motor deficits as well as cognitive deficits in schizophrenia and attention deficit hyperactivity disorder. For more than 40 years, academic and industry scientists have been searching for a drug-like D1R agonist, but this has remained elusive. The challenge in developing D1R selective agonists is that all previous agonists possess a common problematic chemical moiety, a catechol. Catechols are associated with poor oral bioavailability, poor brain penetration, and rapid metabolism in the serum. Very recently, the breakthrough discovery of the first non-catechol D1R selective agonists overcame the pitfalls associated with the catechols. Unexpectedly, the non-catechol agonists also selectively activate G protein signaling without engaging beta-arrestin indicating that they are G protein biased. The primary goals for this study were to characterize novel signaling by non-catechol agonists and elucidate a mechanism of action for the G protein biased non-catechol agonists. First, the role of beta-arrestin in D1R agonist-induced endocytosis was established in HEK293 cells that had beta-arrestin1/2 knocked out by CRISPR/Cas9 genome editing. The knockout of beta-arrestin1/2 eliminated D1R agonist-induced endocytosis. beta-arrestin1/2 knockout significantly reduced D1R agonist-induced endocytosis in an ELISA assay that measures cell surface D1R. Furthermore, re-expressing either beta-arrestin1 or 2 rescued D1R endocytosis in confocal imaging and cell surface ELISA assays. Together, these results indicate that beta-arrestin1/2 are required for D1R agonist-induced endocytosis. Next, catechol and non-catechol D1R agonists were tested in cAMP Glosensor and beta-arrestin Tango assays to investigate potential biased signaling. The unbiased catechol D1R full agonist SKF-81297 was used as the reference compound in all following studies. The non-catechol D1R agonists dose-dependently increased cAMP production in HEK293 cells similar to the full agonist SKF-81297 (Emax 100%), but did not engage beta-arrestin. Interestingly, one non-catechol agonist (PW441) robustly activated both cAMP (Emax = 92%, EC50 = 4.4 nM) and also fully recruited beta-arrestin (Emax = 100%, EC50 = 100 nM). The catechol agonist A-77636 dose-dependently increased full cAMP production (Emax = 104%, EC50 = 3.1 nM) but was a super agonist for beta-arrestin recruitment (Emax = 130%, EC50 = 35 nM). To determine the effect of G protein biased agonists on D1R endocytosis, the catechol and non-catechol D1R agonists were tested in imaging and cell surface ELISA assays. The non-catechol G protein biased agonists all induced significantly less total D1R endocytosis than the catechol agonist SKF-81297. The pure G protein biased agonists PF-1119 and PW464 maximally induced 5% and 11% loss of cell surface D1R, respectively. In contrast, the catechol A-77636 maximally induced 47% loss of cell surface D1R and induced significantly more total endocytosis than SKF-81297. Moreover, the efficacy for beta-arrestin recruitment strongly correlates to the maximum receptor endocytosis in Spearman\u2019s correlation analysis (r = 0.96, p<0.05). Collectively, this study demonstrates the essential role of beta-arrestin in D1R agonist-induced endocytosis and characterizes novel non-catechol agonists. In addition, the discovery of the first balanced non-catechol D1R selective agonist adds a unique tool for future in vitro and in vivo studies. These results further elucidate a mechanism of action for the G protein biased non-catechol agonists in which agonist binding induces G protein activation without also inducing D1R endocytosis. These results provide insights into the molecular mechanism of the G protein biased non-catechol agonists. While the clinical efficacy of the non-catechol agonists is currently being explored, the mechanism of action is not fully understood. This study explored novel derivatives and their downstream effects on D1R endocytosis
Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation
Poster number: P-T099
Theme: Neurodegenerative disorders & ageing
Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation
Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London
Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10)
were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling
responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation.
References
Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7.
Cunningham C (2013). Glia 61: 71-90.
Heneka MT et al. (2015). Lancet Neurol 14: 388-40
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