ORCID Profile
0000-0001-7591-1149
Current Organisation
National Institutes of Health
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Publisher: American Association for the Advancement of Science (AAAS)
Date: 15-05-2019
DOI: 10.1126/SCITRANSLMED.AAX4874
Abstract: α-synuclein oligomer uptake by neurons and oligodendrocytes exploits connexin-32.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 21-02-2018
DOI: 10.1126/SCITRANSLMED.AAT3168
Abstract: α-Synuclein seeding activity can be measured quickly and might prove useful for the diagnosis of α-synucleinopathies.
Publisher: Springer Science and Business Media LLC
Date: 24-03-2018
Publisher: Springer Science and Business Media LLC
Date: 28-10-2017
DOI: 10.1007/S12013-016-0768-Z
Abstract: The ability to model brain tissue in three-dimensions offers new potential for elucidating functional cellular interactions and corruption of such functions during pathogenesis. Many protocols now exist for growing neurones in three-dimensions and these vary in complexity and cost. Herein, we describe a straight-forward method for generating three-dimensional, terminally differentiated central nervous system cultures from adult murine neural stem cells. The protocol requires no specialist equipment, is not labour intensive or expensive and produces mature cultures within 10 days that can survive beyond a month. Populations of functional glutamatergic neurones could be identified within cultures. Additionally, the three dimensional neuronal cultures can be used to investigate tissue changes during the development of neurodegenerative disease where demonstration of hallmark features, such as plaque generation, has not previously been possible using two-dimensional cultures of neuronal cells. Using a prion model of acquired neurodegenerative disease, biochemical changes indicative of prion pathology were induced within 2-3 weeks in the three dimensional cultures. Our findings show that tissue differentiated in this simplified three dimensional culture model is physiologically competent to model central nervous system cellular behaviour as well as manifest the functional failures and pathological changes associated with neurodegenerative disease.
Publisher: Elsevier BV
Date: 11-2014
Publisher: Wiley
Date: 19-06-2006
DOI: 10.1111/J.1471-4159.2006.03906.X
Abstract: The prion protein is a membrane tethered glycoprotein that binds copper. Conversion to an abnormal isoform is associated with neurodegenerative diseases known as prion diseases. Expression of the prion protein has been suggested to prevent cell death caused by oxidative stress. Using cell based models we investigated the potential of the prion protein to protect against copper toxicity. Although prion protein expression effectively protected neurones from copper toxicity, this protection was not necessarily associated with reduction in oxidative damage. We also showed that glycine and the prion protein could both protect neuronal cells from oxidative stress. Only the prion protein could protect these cells from the toxicity of copper. In contrast glycine increased copper toxicity without any apparent oxidative stress or lipid peroxidation. Mutational analysis showed that protection by the prion protein was dependent upon the copper binding octameric repeat region. Our findings demonstrate that copper toxicity can be independent of measured oxidative stress and that prion protein expression primarily protects against copper toxicity independently of the mechanism of cell death.
Publisher: Elsevier BV
Date: 11-2014
Publisher: Springer Science and Business Media LLC
Date: 18-11-2019
DOI: 10.1186/S12974-019-1614-1
Abstract: La Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection. Human cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus. Overall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability. These findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.
Publisher: Elsevier BV
Date: 07-2021
Publisher: Oxford University Press (OUP)
Date: 02-2011
Publisher: IntechOpen
Date: 20-01-2019
Abstract: Prion diseases are transmissible and fatal neurological disorders associated with the misfolding of cellular prion protein (PrPC) into disease-causing isoforms (PrPD) in the central nervous system. The diseases have three etiologies acquired through exposure to the infectious PrPD, sporadic, arising from no known cause, and hereditary due to familial mutations within the PRNP gene. The manifestation of clinical signs is associated with the disruption of neuronal activity and subsequent degeneration of neurons. To generate insight into the mechanisms by which neuronal activity becomes disrupted in prion diseases, electrophysiological techniques have been applied to closely study the electrical signaling properties of neurons that lack functional PrPC as well as neurons that are developing pathological features of prion diseases due to infection or genetic mutation. In this review, we will compile the electrophysiological evidences of neurophysiological roles of PrPC, how those roles are changed in neurons that are developing prion diseases, and how disease-associated effects are exacerbated during the clinical stage of disease.
Publisher: Elsevier BV
Date: 11-2014
Publisher: Public Library of Science (PLoS)
Date: 04-11-2021
DOI: 10.1371/JOURNAL.PONE.0259597
Abstract: Prion diseases are progressive, neurodegenerative diseases affecting humans and animals. Also known as the transmissible spongiform encephalopathies, for the hallmark spongiform change seen in the brain, these diseases manifest increased oxidative damage early in disease and changes in antioxidant enzymes in terminal brain tissue. Superoxide dismutase 2 (SOD2) is an antioxidant enzyme that is critical for life. SOD2 knock-out mice can only be kept alive for several weeks post-birth and only with antioxidant therapy. However, this results in the development of a spongiform encephalopathy. Consequently, we hypothesized that reduced levels of SOD2 may accelerate prion disease progression and play a critical role in the formation of spongiform change. Using SOD2 heterozygous knock-out and litter mate wild-type controls, we examined neuronal long-term potentiation, disease duration, pathology, and degree of spongiform change in mice infected with three strains of mouse adapted scrapie. No influence of the reduced SOD2 expression was observed in any parameter measured for any strain. We conclude that changes relating to SOD2 during prion disease are most likely secondary to the disease processes causing toxicity and do not influence the development of spongiform pathology.
Publisher: Springer Science and Business Media LLC
Date: 30-10-2011
Publisher: Public Library of Science (PLoS)
Date: 07-08-2015
Publisher: Springer Science and Business Media LLC
Date: 28-01-2023
DOI: 10.1007/S00441-022-03589-X
Abstract: Human cerebral organoids are an exciting and novel model system emerging in the field of neurobiology. Cerebral organoids are spheres of self-organizing, neuronal lineage tissue that can be differentiated from human pluripotent stem cells and that present the possibility of on-demand human neuronal cultures that can be used for non-invasively investigating diseases affecting the brain. Compared with existing humanized cell models, they provide a more comprehensive replication of the human cerebral environment. The potential of the human cerebral organoid model is only just beginning to be elucidated, but initial studies have indicated that they could prove to be a valuable model for neurodegenerative diseases such as prion disease. The application of the cerebral organoid model to prion disease, what has been learned so far and the future potential of this model are discussed in this review.
Publisher: MDPI AG
Date: 18-06-2020
Abstract: Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human cerebral cortex. COs are a promising new system for modelling pathological features of neurological disorders, including prion diseases. COs expressing normal prion protein (PrPC) are susceptible to prion infection when exposed to the disease isoforms of PrP (PrPD). This causes the COs to develop aspects of prion disease pathology considered hallmarks of disease, including the production of detergent-insoluble, protease-resistant misfolded PrPD species capable of seeding the production of more misfolded species. To determine whether COs can model aspects of familial prion diseases, we produced COs from donor fibroblasts carrying the E200K mutation, the most common cause of human familial prion disease. The mature E200K COs were assessed for the hallmarks of prion disease. We found that up to 12 months post-differentiation, E200K COs harbored no PrPD as confirmed by the absence of detergent-insoluble, protease-resistant, and seeding-active PrP species. Our results suggest that the presence of the E200K mutation within the prion gene is insufficient to cause disease in neuronal tissue. Therefore, other factors, such as further genetic modifiers or aging processes, may influence the onset of misfolding.
Publisher: Elsevier BV
Date: 06-2015
DOI: 10.1016/J.BBRC.2015.04.048
Abstract: The protein misfolding cyclic lification (PMCA) technique has become a widely-adopted method for lifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-10-2019
DOI: 10.1126/SCITRANSLMED.AAZ3719
Abstract: Combination therapy may offer a way to slow the progression of prion diseases.
Publisher: Public Library of Science (PLoS)
Date: 27-10-2022
DOI: 10.1371/JOURNAL.PONE.0277051
Abstract: Prion diseases are a group of rare, transmissible, and invariably fatal neurodegenerative diseases that affect both humans and animals. The cause of these diseases is misfolding of the prion protein into pathological isoforms called prions. Of all human prion diseases, 10–15% of cases are genetic and the E200K mutation, which causes familial Creutzfeldt-Jakob disease (CJD), is the most prevalent. For both sporadic and genetic disease, it remains uncertain as to how initial protein misfolding is triggered. Prior studies have linked protein misfolding with oxidative stress insults, deregulated interactions with cellular cofactors, and viral infections. Our previous work developed a cerebral organoid (CO) model using human induced pluripotent stem cells containing the E200K mutation. COs are three-dimensional human neural tissues that permit the study of host genetics and environmental factors that contribute to disease onset. Isogenically matched COs with and without the E200K mutation were used to investigate the propensity of E200K PrP to misfold following cellular insults associated with oxidative stress. Since viral infections have also been associated with oxidative stress and neurodegenerative diseases, we additionally investigated the influence of Herpes Simplex Type-1 virus (HSV1), a neurotropic virus that establishes life-long latent infection in its host, on E200K PrP misfolding. While COs proved to be highly infectable with HSV1, neither acute nor latent infection, or direct oxidative stress insult, resulted in evidence of E200K prion misfolding. We conclude that misfolding into seeding-active PrP species is not readily induced by oxidative stress or HSV1 in our organoid system.
Publisher: Public Library of Science (PLoS)
Date: 26-10-2023
Publisher: Elsevier BV
Date: 03-2001
Publisher: Medknow
Date: 2016
Publisher: Public Library of Science (PLoS)
Date: 19-01-2023
DOI: 10.1371/JOURNAL.PGEN.1010565
Abstract: Fatal familial insomnia (FFI) is a rare neurodegenerative disease caused by a dominantly inherited single amino acid substitution (D178N) within the prion protein (PrP). No in vitro human brain tissue model for this disease has previously been available. Consequently, how this mutation exerts its damaging effect on brain cells is still unknown. Using CRISPR-Cas9 engineered induced pluripotent stem cells, we made D178N cerebral organoids and compared these with isotype control organoids. We found that, in the absence of other hallmarks of FFI, the D178N organoids exhibited astrogliosis with cellular oxidative stress. Abnormal post-translational processing of PrP was evident but no tissue deposition or propagation of mis-folded PrP isoforms were observed. Neuronal electrophysiological function was compromised and levels of neurotransmitters, particularly acetylcholine and GABA, altered. Underlying these dysfunctions were changes in cellular energy homeostasis, with substantially increased glycolytic and Krebs cycle intermediates, and greater mitochondrial activity. This increased energy demand in D178N organoids was associated with increased mitophagy and depletion of lipid droplets, in turn resulting in shifts of cellular lipid composition. Using a double mutation (178NN) we could confirm that most changes were caused by the presence of the mutation rather than interaction with PrP molecules lacking the mutation. Our data strongly suggests that shifting biosynthetic intermediates and oxidative stress, caused by an imbalance of energy supply and demand, results in astrogliosis with compromised neuronal activity in FFI organoids. They further support that many of the disease associated changes are due to a corruption of PrP function and do not require propagation of PrP mis-folding.
Publisher: American Chemical Society (ACS)
Date: 30-09-2010
DOI: 10.1021/CN100068X
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-07-2019
DOI: 10.1126/SCITRANSLMED.AAY3567
Abstract: The prion protein functions as a carrier for delivering Aβ to exosomes during Alzheimer’s disease.
Publisher: Elsevier BV
Date: 08-2011
DOI: 10.1016/J.FREERADBIOMED.2011.03.035
Abstract: Neuronal loss is a pathological feature of prion diseases for which increased reactive oxygen species (ROS) and consequent oxidative stress is one proposed mechanism. The processes underlying ROS production in prion disease and the precise relationship to misfolding of the prion protein remain obscure. Using cell culture models of prion infection we found that cells demonstrate a rapid, prion protein (PrP) dependent, increase in intracellular ROS following exposure to infectious inoculum. ROS production correlated with internalisation and increased intracellular protease resistant PrP (PrP(Res)). The ROS increase was predominantly lysosomal in origin but not sustained, with cells adapting within 48 hours. Overall ROS levels remained normal in the chronically prion infected cell population however a subpopulation characterised by loss of membrane phosphatidylserine asymmetry exhibited highly peroxidised intracellular aggregates that localised with PrP and intense caspase activation. These apoptotic cells showed increased ROS closely correlating with increased PrP(Res). Our findings demonstrate that a PrP-dependent, transient, increase in intracellular ROS is characteristic of acute cellular prion infection, while chronic phases of prion infection in vitro are associated with a significant subpopulation manifesting apoptosis accompanying heightened oxidative stress and increased PrP(Res) burden. Such observations strengthen the direct links between heightened ROS and ongoing prion propagation with eventual cellular demise.
Publisher: The Company of Biologists
Date: 15-05-2009
DOI: 10.1242/JCS.043604
Abstract: Beta-cleavage of the neurodegenerative disease-associated prion protein (PrP) protects cells from death induced by oxidative insults. The beta-cleavage event produces two fragments, designated N2 and C2. We investigated the role of the N2 fragment (residues 23-89) in cellular stress response, determining mechanisms involved and regions important for this reaction. The N2 fragment differentially modulated the reactive oxygen species (ROS) response induced by serum deprivation, with amelioration when copper bound. Amino acid residues 23-50 alone mediated a ROS reduction response. PrP23-50 ROS reduction was not due to copper binding or direct antioxidant activity, but was instead mediated through proteoglycan binding partners localised in or interacting with cholesterol-rich membrane domains. Furthermore, mutational analyses of both PrP23-50 and N2 showed that their protective capacity requires the sterically constraining double proline motif within the N-terminal polybasic region. Our findings show that N2 is a biologically active fragment that is able to modulate stress-induced intracellular ROS through interaction of its structurally defined N-terminal polybasic region with cell-surface proteoglycans.
Publisher: Springer Science and Business Media LLC
Date: 21-10-2021
DOI: 10.1038/S41564-021-00968-Y
Abstract: La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml
Publisher: Springer Science and Business Media LLC
Date: 11-10-2021
DOI: 10.1186/S13041-021-00864-W
Abstract: The neuro-physiological properties of in iduals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of in iduals with confirmed genetic mutations including PRNP E200K , trisomy 21 ( T21) , and LRRK2 G2019S , which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson’s disease. We utilized no known disease/healthy COs ( HC ) as normal function controls. At 3–4 and 6–10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6–10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with the HC , including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the litudes of gamma oscillations. Such changes were linked with the detection of hypersynchronous events like spike-and-wave discharges. These dysfunctions were associated with altered production and release of neurotransmitters, compromised activity of excitatory ionotropic receptors including receptors of kainate, AMPA, and NMDA, and changed levels and function of excitatory glutamatergic synapses and inhibitory GABAergic synapses. Neuronal properties that modulate GABAergic inhibition including the activity of Na–K-Cl cotransport 1 (NKCC1) in Cl − homeostasis and the levels of synaptic and extra-synaptic localization of GABA receptors (GABARs) were altered in the T21 COs only. The neurosteroid allopregnanolone, a positive modulator of GABARs, was downregulated in all the dCOs. Treatment with this neurosteroid significantly improved the neuronal communication in the dCOs, possibly through improving the GABAergic inhibition. Overall, without the manifestation of any disease-related pathology, the genetic mutations PRNP E200K , T21 , and LRRK2 G2019S significantly altered the neuronal network communication in dCOs by disrupting the excitatory-to-inhibitory balance.
Publisher: Wiley
Date: 26-06-2006
DOI: 10.1111/J.1471-4159.2006.03981.X
Abstract: Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning.
Publisher: Humana Press
Date: 2008
DOI: 10.1007/978-1-59745-234-2_2
Abstract: Prion protein (PrP)(C) expression levels and protein localization are known to be affected by factors such as metal ions and oxidative stress. By the development of a green fluorescent protein (GFP)-PrP(C) fusion protein, the movement of PrP can be followed in real time. Furthermore, alterations in cellular metabolism can be detected while cells are still viable. The internalization response of PrP to 20 microM manganese (Mn) in alent metal ion-depleted media is used to demonstrate the movement of GFP-tagged proteins in live cells and real time. A live cell microtiter plate assay shows that PrP null cells are less capable of dealing with Mn-induced oxidative stress. In addition, this chapter outlines several complementary techniques for studying live cells and GFP fusion proteins.
Publisher: Springer Science and Business Media LLC
Date: 13-11-2015
DOI: 10.1007/S00018-014-1777-Y
Abstract: The prion protein (PrP(C)) when mis-folded is causally linked with a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies or prion diseases. PrP(C) normal function is still incompletely defined with such investigations complicated by PrP(C) post-translational modifications, such as internal cleavage, which feasibly could change, activate, or deactivate the function of this protein. Oxidative stress induces β-cleavage and the N-terminal product of this cleavage event, N2, demonstrates a cellular protective response against oxidative stress. The mechanisms by which N2 mediates cellular antioxidant protection were investigated within an in vitro cell model. N2 protection was regulated by copper binding to the octarepeat domain, directing the route of internalisation, which stimulated MEK1 signalling. Precise membrane interactions of N2, determined by copper saturation, and involving both the copper-co-ordinating octarepeat region and the structure conferred upon the N-terminal polybasic region by the proline motif, were essential for the correct engagement of this pathway. The phenomenon of PrP(C) post-translational modification, such as cleavage and copper co-ordination, as a molecular "switch" for activation or deactivation of certain functions provides new insight into the apparent multi-functionality of PrP(C).
Publisher: Oxford University Press (OUP)
Date: 10-2009
Publisher: Springer Science and Business Media LLC
Date: 14-06-2019
Publisher: Springer Science and Business Media LLC
Date: 11-04-2015
Publisher: Wiley
Date: 14-07-2015
Abstract: Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.
Publisher: Springer Science and Business Media LLC
Date: 21-06-2016
DOI: 10.1007/S12013-016-0747-4
Abstract: Eight-hydroxyquinolines (8HQs) are a class of compounds that have been identified as potential therapeutics for a number of neurodegenerative diseases. Understanding the influence of structural modifications to the 8HQ scaffold on cellular behaviour will aid the identification of compounds that might be effective in treating dementias. In this study, we describe the action of 2-[(dimethylamino)methyl]-8-hydroxyquinoline (DMAMQ) on adult murine neural stem cells (NSCs) cultured in vitro. Treatment of NSCs with DMAMQ resulted in enhanced self-renewal and increased neurite outgrowth. Concurrent with the positive growth effects was an increase in intracellular reactive oxygen species, with the growth being inhibited by inactivation of the NADPH oxidase (Nox) enzyme family. Our results indicate that DMAMQ can stimulate neurogenesis via the Nox signalling pathway, which may provide therapeutic benefit in treating dementias of various types by replenishing neurones using the brain's own reserves. The narrow concentration range over which these effects were observed, however, suggests that there may exist only a small therapeutic window for neuro-regenerative applications.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 28-08-2019
DOI: 10.1126/SCITRANSLMED.AAZ0303
Abstract: Neurons from the olfactory mucosa in combination with a highly sensitive lification assay may offer a monitoring paradigm for α-synucleinopathies.
Publisher: Springer Science and Business Media LLC
Date: 26-04-2012
Abstract: Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrP res ) conformers of the normal cellular prion protein (PrP C ), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrP res and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrP res levels and infectivity titres at a subcellular resolution. Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrP res . Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication. This study authenticates a subcellular disparity in PrP res and infectivity levels, and eliminates simultaneous ergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrP res levels.
Publisher: Springer Science and Business Media LLC
Date: 28-07-2010
Publisher: Public Library of Science (PLoS)
Date: 21-07-2021
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-7244-9_11
Abstract: Traditional primary and secondary cell cultures have been used for the investigation of prion biology and disease for many years. While both types of cultures produce highly valid and immensely valuable results, they also have their limitations traditional cell lines are often derived from cancers, therefore subject to numerous DNA changes, and primary cultures are labor-intensive and expensive to produce requiring sacrifice of many animals. Neural stem cell (NSC) cultures are a relatively new technology to be used for the study of prion biology and disease. While NSCs are subject to their own limitations-they are generally cultured ex vivo in environments that artificially force their growth-they also have their own unique advantages. NSCs retain the ability for self-renewal and can therefore be propagated in culture similarly to secondary cultures without genetic manipulation. In addition, NSCs are multipotent they can be induced to differentiate into mature cells of central nervous system (CNS) linage. The combination of self-renewal and multipotency allows NSCs to be used as a primary cell line over multiple generations saving time, costs, and animal harvests, thus providing a valuable addition to the existing cell culture repertoire used for investigation of prion biology and disease. Furthermore, NSC cultures can be generated from mice of any genotype, either by embryonic harvest or harvest from adult brain, allowing gene expression to be studied without further genetic manipulation. This chapter describes a standard method of culturing adult NSCs and assays for monitoring NSC growth, migration, and differentiation and revisits basic reactive oxygen species detection in the context of NSC cultures.
Publisher: IMR Press
Date: 2010
DOI: 10.2741/3663
Abstract: Recently, understanding of many molecular interactions has progressed appreciably and cellular events once thought to be by-products of more important reactions or to be detrimental to cellular function are now known to be part of complex interactions of the cell with its environment. Numerous proteins can elicit differing effects depending upon post-translational modification events such as complex glycosylation and endoproteolytic cleavage or through binding co-factors including metal ions the prion protein (PrP) is likely one such ex le. Its absolute requirement for pathogenesis has made the function of PrP an area of intense study but with apparently inconsistent results. This may, in part, stem from the ability of PrP to undergo different modifications to varying extents depending upon precise cellular circumstances. Specific modifications may promote altered association with binding partners resulting in apparent promiscuity of PrP interactions and activation of different signalling pathways, producing the ersity of functions suggested for this protein. This review discusses how modification of PrP by internal cleavage and metal ion co-ordination might influence, or be influenced by, signal transduction cascades. 2.
Publisher: Springer Science and Business Media LLC
Date: 14-07-2009
DOI: 10.1038/CR.2009.86
Abstract: The copper-binding, membrane-anchored, cellular prion protein (PrP(C)) has two constitutive cleavage sites producing distinct N- and C-terminal fragments (N1/C1 and N2/C2). Using RK13 cells expressing either human PrP(C), mouse PrP(C) or mouse PrP(C) carrying the 3F4 epitope, this study explored the influence of the PrP(C) primary sequence on endoproteolytic cleavage and one putative PrP(C) function, MAP kinase signal transduction, in response to exogenous copper with or without a perturbed membrane environment. PrP(C) primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrP(C) showed increased N1/C1 cleavage in response to copper alone, accompanied by specific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrP(C)-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrP(C) harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrP(C) primary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence appears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of PrP(C)-related signal transduction in response to exogenous stimuli.
Publisher: IMR Press
Date: 2010
DOI: 10.2741/3662
Abstract: The prion protein (PrP) has been implicated in many erse functions, making it difficult to pinpoint its basic physiological role. Our most recent studies in zebrafish, mammalian and invertebrate cells indicate that PrP regulates cell-cell communication, as well cell-matrix interactions at focal adhesions. In addition, we previously have shown that upon antibody-mediated cross-linking, PrP can be induced to cluster in the preformed T-cell cap. Here we review these data and discuss how the spatial link between PrP and the microdomain-forming proteins reggie-1 (flotillin-2) and reggie-2 (flotillin-1) may contribute to PrP signaling, leading to the local assembly of membrane protein complexes at sites involved in cellular communication, such as cell-cell contacts, focal adhesions, the T-cell cap, and synapses.
Publisher: Elsevier BV
Date: 07-2023
Publisher: Elsevier BV
Date: 10-2023
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-7244-9_17
Abstract: In vivo near-infrared (NIR) imaging of molecular processes at the preclinical stage promises to provide more valuable mechanistic information about pathological pathways involved in neurodegeneration. NIR imaging has the potential to improve in vivo therapeutic screening protocols by enabling noninvasive monitoring of presymptomatic responses to treatment. We have developed new NIR fluorescent contrast agents conjugated to markers of cell death, and using these agents we have identified molecular pathways associated with prion-induced neurodegeneration and determined the optimal window for meaningful therapeutic intervention in prion disease. This chapter provides a description of the synthesis and purification of our NIR cell Death (NIRD) contrast agent and the application of in vivo NIRD (iNIRD) imaging to a prion model of neurodegeneration.
Publisher: Public Library of Science (PLoS)
Date: 30-06-2023
Publisher: Elsevier BV
Date: 11-2014
Publisher: Medknow
Date: 2016
Publisher: American Association for the Advancement of Science (AAAS)
Date: 18-12-2019
DOI: 10.1126/SCITRANSLMED.AAZ9768
Abstract: α-synuclein strains may be responsible for the differing disease etiologies of the α-Synucleinopathies.
Publisher: Medknow
Date: 2020
Publisher: The Company of Biologists
Date: 2013
DOI: 10.1242/DMM.010678
Abstract: Oxidative stress as a contributor to neuronal death during prion infection is supported by the fact that various oxidative damage markers accumulate in the brain during the course of this disease. The normal cellular substrate of the causative agent, the prion protein, is also linked with protective functions against oxidative stress. Our previous work has found that, in chronic prion infection, an apoptotic subpopulation of cells exhibit oxidative stress and the accumulation of oxidised lipid and protein aggregates with caspase recruitment. Given the likely failure of antioxidant defence mechanisms within apoptotic prion-infected cells, we aimed to investigate the role of the crucial antioxidant pathway components, superoxide dismutases (SOD) 1 and 2, in an in vitro model of chronic prion infection. Increased total SOD activity, attributable to SOD1, was found in the overall population coincident with a decrease in SOD2 protein levels. When apoptotic cells were separated from the total population, the induction of SOD activity in the infected apoptotic cells was lost, with activity reduced back to levels seen in mock-infected control cells. In addition, mitochondrial superoxide production was increased and mitochondrial numbers decreased in the infected apoptotic subpopulation. Furthermore, a pan-caspase probe colocalised with SOD2 outside of mitochondria within cytosolic aggregates in infected cells and inhibition of caspase activity was able to restore cellular levels of SOD2 in the whole unseparated infected population to those of mock-infected control cells. Our results suggest that prion propagation exacerbates an apoptotic pathway whereby mitochondrial dysfunction follows mislocalisation of SOD2 to cytosolic caspases, permitting its degradation. Eventually, cellular capacity to maintain oxidative homeostasis is overwhelmed, thus resulting in cell death.
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.JMB.2007.02.086
Abstract: Expression of the cellular prion protein is necessary for the transmission and propagation of prion diseases. Increasing the level of prion protein expression decreases the incubation period for these diseases. Therefore, understanding the regulation of prion protein expression could be critical for treating or preventing these diseases. We investigated the regulation of prion protein expression by the promoter and noncoding regions of the bovine and murine Prnp genes. We determined that expression is modulated by intron 1 and exon 1. In the absence of intron1, exon 1 inhibited activity of the promoter. However, intron 1 demonstrated promoter-like activity and possessed a TATA box. In addition, we identified an alternative transcript present in the brains of cattle and mice that lacks exon 1. Taken together, these results show that intron 1 and exon 1 play a critical role in the regulation of prion protein expression. Because switching off prion protein expression has been shown to arrest prion disease, these regions present novel targets for intervention in the disease process.
Publisher: ScopeMed
Date: 2013
Publisher: Elsevier BV
Date: 10-2010
Publisher: Springer Science and Business Media LLC
Date: 22-09-2022
DOI: 10.1038/S41598-022-19631-5
Abstract: Cardiomyopathy is a co-morbidity of some prion diseases including genetic disease caused by mutations within the PrP gene ( PRNP ). Although the cellular prion protein (PrP) has been shown to protect against cardiotoxicity caused by oxidative stress, it is unclear if the cardiomyopathy is directly linked to PrP dysfunction. We differentiated cardiomyocyte cultures from donor human induced pluripotent stem cells and found a direct influence of the PRNP E200K mutation on cellular function. The PRNP E200K cardiomyocytes showed abnormal function evident in the irregularity of the rapid repolarization a phenotype comparable with the dysfunction reported in Down Syndrome cardiomyocytes. PRNP E200K cardiomyocyte cultures also showed increased mitochondrial superoxide accompanied by increased mitochondrial membrane potential and dysfunction. To confirm that the changes were due to the E200K mutation, CRISPR-Cas9 engineering was used to correct the E200K carrier cells and insert the E200K mutation into control cells. The isotype matched cardiomyocytes showed that the lysine expressing allele does directly influence electrophysiology and mitochondrial function but some differences in severity were apparent between donor lines. Our results demonstrate that cardiomyopathy in hereditary prion disease may be directly linked to PrP dysfunction.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 13-03-2019
DOI: 10.1126/SCITRANSLMED.AAX0769
Abstract: The brain lymphatic system contributes to TAU clearance in mice.
Publisher: Springer Science and Business Media LLC
Date: 20-04-2020
DOI: 10.1038/S41598-020-63472-Z
Abstract: Microglia act as the protective immune cell of the brain. By surveying the tissue to identify and rectify problems, they function to maintain the health of brain cells. The prion protein N-terminal cleavage fragment, N1, has demonstrated neuroprotective activities in vitro and in vivo . This study aimed to elucidate whether N1 could modulate microglial function and, if so, determine the consequences for the surrounding tissue. Using a mixed neuronal lineage and microglia co-culture system, we showed that N1 stimulation changed overall morphology and metabolism, suggesting enhanced cellular viability. Furthermore, N1 induced an increase in Cxcl10 secretion in the co-cultures. Recombinant Cxcl10, administered exogenously, mediated the changes in the mixed neuronal lineage culture morphology and metabolism in the absence of microglia, but no effect of Cxcl10 was observed on microglia cultured on their own. Direct cell-to-cell contact was required for N1 to influence microglia in the co-cultures, and this was linked with restructuring of microglial membrane composition to include a higher GM1 content at interaction sites with surrounding cells. Our findings show that N1 can play a regulatory role in microglial function in the context of an inter-connected network of cells by changing both cellular interaction sites and cytokine secretion.
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.MCN.2005.07.001
Abstract: The cellular isoform of the prion protein (PrP(c)) is located at the cell membrane, anchored externally by a glycosylphosphatidylinositol (GPI) anchor. It is a copper (Cu) binding glycoprotein with a rapid basal turnover. Previous studies have shown that exposure of cells to Cu causes internalisation of PrP(c) in vitro. In this study, we show that physiological levels of Cu promote internalisation of PrP(c). Interaction between PrP(c) and Cu was found to be the overriding factor in stimulating the internalisation response with other metals showing no effect. Deletion mutation studies have shown that two domains are essential for copper-induced internalisation to occur. These two domains are the octameric repeat region, encompassing amino acids 51-89, and the palindromic region, amino acids 112-119 with the sequence AGAAAAGA. The decrease in detectable levels of PrP(c) at the cell surface following Cu treatment was found to be the result of rapid internalisation rather than loss into the surrounding environment. These results have implications for both normal metabolism of PrP(c) and the possible mechanism of conversion of PrP(c) to PrP(sc).
Publisher: Springer Science and Business Media LLC
Date: 14-02-2023
DOI: 10.1186/S40478-023-01512-1
Abstract: Human cerebral organoids (COs) are three-dimensional self-organizing cultures of cerebral brain tissue differentiated from induced pluripotent stem cells. We have recently shown that COs are susceptible to infection with different subtypes of Creutzfeldt–Jakob disease (CJD) prions, which in humans cause different manifestations of the disease. The ability to study live human brain tissue infected with different CJD subtypes opens a wide array of possibilities from differentiating mechanisms of cell death and identifying neuronal selective vulnerabilities to testing therapeutics. However, the question remained as to whether the prions generated in the CO model truly represent those in the infecting inoculum. Mouse models expressing human prion protein are commonly used to characterize human prion disease as they reproduce many of the molecular and clinical phenotypes associated with CJD subtypes. We therefore inoculated these mice with COs that had been infected with two CJD subtypes (MV1 and MV2) to see if the original subtype characteristics (referred to as strains once transmitted into a model organism) of the infecting prions were maintained in the COs when compared with the original human brain inocula. We found that disease characteristics caused by the molecular subtype of the disease associated prion protein were similar in mice inoculated with either CO derived material or human brain material, demonstrating that the disease associated prions generated in COs shared strain characteristics with those in humans. As the first and only in vitro model of human neurodegenerative disease that can faithfully reproduce different subtypes of prion disease, these findings support the use of the CO model for investigating human prion diseases and their subtypes.
Publisher: Springer Science and Business Media LLC
Date: 09-03-2021
DOI: 10.1038/S41598-021-84689-6
Abstract: Creutzfeldt–Jakob Disease (CJD) is a fatal, currently incurable, neurodegenerative disease. The search for candidate treatments would be greatly facilitated by the availability of human cell-based models of prion disease. Recently, an induced pluripotent stem cell derived human cerebral organoid model was shown to take up and propagate human CJD prions. This model offers new opportunities to screen drug candidates for the treatment of human prion diseases in an entirely human genetic background. Here we provide the first evidence that human cerebral organoids can be a viable model for CJD drug screening by using an established anti-prion compound, pentosan polysulfate (PPS). PPS delayed prion propagation in a prophylactic-like treatment paradigm and also alleviated propagation when applied following establishment of infection in a therapeutic-like treatment paradigm. This study demonstrates the utility of cerebral organoids as the first human 3D cell culture system for screening therapeutic drug candidates for human prion diseases.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Cathryn Haigh.