32 results found
Bengoa-Vergniory N, Velentza-Almpani E, Silva AM, et al., 2021, Tau-proximity ligation assay reveals extensive previously undetected pathology prior to neurofibrillary tangles in preclinical Alzheimer’s disease, Acta Neuropathologica Communications, Vol: 9, ISSN: 2051-5960
BackgroundMultimerization is a key process in prion-like disorders such as Alzheimer’s disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau–tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers.MethodsHere, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold.ResultsOur novel tau-proximity ligation assay (tau-PLA) directly visualized tau–tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large les
Agarwal D, Sandor C, Volpato V, et al., 2020, A human single-cell atlas of the Substantia nigra reveals novel cell-specific pathways associated with the genetic risk of Parkinson's disease and neuropsychiatric disorders., 53rd European Society of Human Genetics (ESHG) Conference: Oral Presentations, Publisher: Springer Nature [academic journals on nature.com], Pages: 71-71, ISSN: 1018-4813
Sutton-Smith M, Yasin Z, Boharoon H, et al., 2020, A case of cyclic cushing’s disease – cortisol surfing; catching the cortisol wave!, Endocrine Abstracts
Agarwal D, Sandor C, Volpato V, et al., 2020, A single-cell atlas of the human substantia nigra reveals cell-specific pathways associated with neurological disorders, Nature Communications, Vol: 11, ISSN: 2041-1723
We describe a human single-nuclei transcriptomic atlas for the substantia nigra (SN), generated by sequencing approximately 17,000 nuclei from matched cortical and SN samples. We show that the common genetic risk for Parkinson's disease (PD) is associated with dopaminergic neuron (DaN)-specific gene expression, including mitochondrial functioning, protein folding and ubiquitination pathways. We identify a distinct cell type association between PD risk and oligodendrocyte-specific gene expression. Unlike Alzheimer's disease (AD), we find no association between PD risk and microglia or astrocytes, suggesting that neuroinflammation plays a less causal role in PD than AD. Beyond PD, we find associations between SN DaNs and GABAergic neuron gene expression and multiple neuropsychiatric disorders. Conditional analysis reveals that distinct neuropsychiatric disorders associate with distinct sets of neuron-specific genes but converge onto shared loci within oligodendrocytes and oligodendrocyte precursors. This atlas guides our aetiological understanding by associating SN cell type expression profiles with specific disease risk.
Velentza-Almpani E, Bengoa-Vergniory N, Silva A, et al., 2020, Understanding the pathophysiological role of early tau aggregates in Alzheimer's disease by their direct visualisation in situ, 121st Meeting of the British-Neuropathological-Society / Developmental Neuropathology Symposium, Publisher: WILEY, Pages: 42-42, ISSN: 0305-1846
Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are neurodegenerative disorders resulting in progressive motor/cognitive deficits among other symptoms. They are characterised by stereotypical brain cell loss accompanied by the formation of proteinaceous aggregations of the protein α-synuclein (α-syn), being, therefore, termed α-synucleinopathies. Although the presence of α-syn inclusions is a common hallmark of these disorders, the exact nature of the deposited protein is specific to each disease. Different neuroanatomical regions and cellular populations manifest a differential vulnerability to the appearance of protein deposits, cell dysfunction, and cell death, leading to phenotypic diversity. The present review describes the multiple factors that contribute to the selective vulnerability in α-synucleinopathies. We explore the intrinsic cellular properties in the affected regions, including the physiological and pathophysiological roles of endogenous α-syn, the metabolic and genetic build-up of the cells and their connectivity. These factors converge with the variability of the α-syn conformational strains and their spreading capacity to dictate the phenotypic diversity and regional vulnerability of each disease. Finally, we describe the exogenous and environmental factors that potentially contribute by igniting and modulating the differential pathology in α-synucleinopathies. In conclusion, we think that it is the confluence of this disruption of the cellular metabolic state and α-syn structural equilibrium through the anatomical connectivity which appears to initiate cascades of pathological processes triggered by genetic, environmental, or stochastic events that result in the "death by a thousand cuts" profile of α-synucleinopathies.
Hunn BHM, Vingill S, Threlfell S, et al., 2019, Impairment of macroautophagy in dopamine neurons has opposing effects on Parkinsonian pathology and behavior, Cell Reports, Vol: 29, Pages: 920-e7, ISSN: 2211-1247
Parkinson’s disease (PD) is characterized by the death of dopamine neurons in the substantia nigra pars compacta (SNc) and accumulation of α-synuclein. Impaired autophagy has been implicated and activation of autophagy proposed as a treatment strategy. We generate a human α-synuclein-expressing mouse model of PD with macroautophagic failure in dopamine neurons to understand the interaction between impaired macroautophagy and α-synuclein. We find that impaired macroautophagy generates p62-positive inclusions and progressive neuron loss in the SNc. Despite this parkinsonian pathology, motor phenotypes accompanying human α-synuclein overexpression actually improve with impaired macroautophagy. Real-time fast-scan cyclic voltammetry reveals that macroautophagy impairment in dopamine neurons increases evoked extracellular concentrations of dopamine, reduces dopamine uptake, and relieves paired-stimulus depression. Our findings show that impaired macroautophagy paradoxically enhances dopamine neurotransmission, improving movement while worsening pathology, suggesting that changes to dopamine synapse function compensate for and conceal the underlying PD pathogenesis, with implications for therapies that target autophagy.
Roberts RF, Bengoa-Vergniory N, Alegre-Abarrategui J, 2019, Alpha-Synuclein Proximity Ligation Assay (AS-PLA) in brain sections to probe for alpha-synuclein oligomers, Methods in Molecular Biology, Vol: 1948, Pages: 69-76, ISSN: 1940-6029
Alpha-synuclein oligomers are thought to be toxic mediators of Parkinson's disease and other alpha-synucleinopathies, but their histological detection in situ in diseased brain has been a challenge in the field for some time. Here we describe a method, the alpha-synuclein proximity ligation assay (AS-PLA), to detect alpha-synuclein oligomers in paraffin-embedded brain sections. Using AS-PLA previously unobserved alpha-synuclein oligomeric pathology is revealed.
Gordon D, Dafinca R, Scaber J, et al., 2019, Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction, Neurobiology of Disease, Vol: 121, Pages: 148-162, ISSN: 0969-9961
Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of 'sporadic' cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.
Ruffmann C, Bengoa-Vergniory N, Poggiolini I, et al., 2018, Detection of alpha-synuclein conformational variants from gastro-intestinal biopsy tissue as a potential biomarker for Parkinson's disease, Neuropathology and Applied Neurobiology, Vol: 44, Pages: 722-736, ISSN: 0305-1846
AIMS: Gastrointestinal (GI) α-synuclein (aSyn) detection as a potential biomarker of Parkinson's disease (PD) is challenged by conflicting results of recent studies. To increase sensitivity and specificity, we applied three techniques to detect different conformations of aSyn in GI biopsies obtained from a longitudinal, clinically well-characterized cohort of PD patients and healthy controls (HC). METHODS: With immunohistochemistry (IHC), we used antibodies reactive for total, phosphorylated and oligomeric aSyn; with aSyn proximity ligation assay (AS-PLA), we targeted oligomeric aSyn species specifically; and with paraffin-embedded tissue blot (AS-PET-blot) we aimed to detect fibrillary, synaptic aSyn. RESULTS: A total of 163 tissue blocks were collected from 51 PD patients (113 blocks) and 21 HC (50 blocks). In 31 PD patients, biopsies were taken before the PD diagnosis (Prodromal); while in 20 PD patients biopsies were obtained after diagnosis (Manifest). The majority of tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four staining patterns were detected (neuritic, ganglionic, epithelial and cellular), while two distinct staining patterns were detected both with AS-PLA (cellular and diffuse signal) and with AS-PET-blot (aSyn-localized and pericrypt signal). The level of agreement between different techniques was low and no single technique or staining pattern reliably distinguished PD patients (Prodromal or Manifest) from HC. CONCLUSIONS: Our study suggests that detection of aSyn conformational variants currently considered pathological is not adequate for the diagnosis or prediction of PD. Future studies utilizing novel ultrasensitive amyloid aggregation assays may increase sensitivity and specificity.
Smith AM, Depp C, Ryan BJ, et al., 2018, Mitochondrial dysfunction and increased glycolysis in prodromal and early Parkinson's blood cells, Movement Disorders, Vol: 33, Pages: 1580-1590, ISSN: 0885-3185
Background: Although primarily a neurodegenerative process, there is increasing awareness of peripheral disease mechanisms in Parkinson's disease. To investigate disease processes in accessible patient cells, we studied peripheral blood mononuclear cells in recently diagnosed PD patients and rapid eye movement‐sleep behavior disorder patients who have a greatly increased risk of developing PD. We hypothesized that peripheral blood mononuclear cells may recapitulate cellular pathology found in the PD brain and investigated these cells for mitochondrial dysfunction and oxidative stress.Methods: Peripheral blood mononuclear cells were isolated and studied from PD patients, rapid eye movement‐sleep behavior disorder patients and age‐ and sex‐matched control individuals from the well‐characterized Oxford Discovery cohort. All participants underwent thorough clinical assessment.Results: Initial characterization showed that PD patients had elevated levels of CD14 + monocytes and monocytes expressing C‐C motif chemokine receptor 2. Mitochondrial dysfunction and oxidative stress were increased in PD patient peripheral blood mononuclear cells, with elevated levels of mitochondrial reactive oxygen species specifically in patient monocytes. This was combined with reduced levels of the antioxidant superoxide dismutase in blood cells from PD patients and, importantly, also in rapid eye movement‐sleep behavior disorder patients. This mitochondrial dysfunction was associated with a concomitant increase in glycolysis in both PD and rapid eye movement‐sleep behavior disorder patient blood cells independent of glucose uptake or monocyte activation.Conclusions: This work demonstrates functional bioenergetic deficits in PD and rapid eye movement‐sleep behavior disorder patient blood cells during the early stages of human disease. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Socie
Scaber J, Gordon D, Mutihac R, et al., 2016, Absence of wide-spread mis-splicing in the preclinical phase of a native promoter driven TDP-43 mouse model of ALS, Publisher: WILEY, Pages: 797-797, ISSN: 1351-5101
Ruffmann C, Poggiolini I, Bengoa-Vergniory N, et al., 2016, Colonic alpha-synuclein: A potential diagnostic biomarker in Parkinson's disease, 20th International Congress of Parkinson's Disease and Movement Disorders, Publisher: WILEY-BLACKWELL, Pages: S584-S584, ISSN: 0885-3185
Farrimond L, Gordon D, Mutihac R, et al., 2016, TDP-43 is cytoplasmically mislocalized and associated with impaired stress responses and survival of primary neurons from symptomatic amyotrophic lateral sclerosis (ALS) mice, Publisher: WILEY, Pages: 390-391, ISSN: 1351-5101
Scaber J, Gordon D, Mutihac R, et al., 2016, Absence of wide-spread mis-splicing in the preclinical phase of a native promoter driven TDP-43 mouse model of ALS, Publisher: PERGAMON-ELSEVIER SCIENCE LTD, Pages: S31-S31, ISSN: 0960-8966
Sloan M, Alegre-Abarrategui J, Potgieter D, et al., 2016, LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function, Human Molecular Genetics, Vol: 25, Pages: 951-963, ISSN: 0964-6906
Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18–21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.
Walton H, Morley S, Alegre-Abarrategui J, 2015, A rare case of atypical skull base meningioma with perineural spread, Journal of Radiology Case Reports, Vol: 9
Atypical meningioma is a rare cause of perineural tumour spread. In this report, we present the case of a 46-year-old female with an atypical meningioma of the skull base demonstrating perineural tumour spread. We describe the imaging features of this condition and its distinguishing features from other tumours exhibiting perineural spread.
Roberts RF, Wade-Martins R, Alegre-Abarrategui J, 2015, Direct visualization of alpha-synuclein oligomers reveals previously undetected pathology in Parkinson's disease brain, Brain, Vol: 138, Pages: 1642-1657, ISSN: 1460-2156
Oligomeric forms of alpha-synuclein are emerging as key mediators of pathogenesis in Parkinson’s disease. Our understanding of the exact contribution of alpha-synuclein oligomers to disease is limited by the lack of a technique for their specific detection. We describe a novel method, the alpha-synuclein proximity ligation assay, which specifically recognizes alpha-synuclein oligomers. In a blinded study with post-mortem brain tissue from patients with Parkinson’s disease (n = 8, age range 73–92 years, four males and four females) and age- and sex-matched controls (n = 8), we show that the alpha-synuclein proximity ligation assay reveals previously unrecognized pathology in the form of extensive diffuse deposition of alpha-synuclein oligomers. These oligomers are often localized, in the absence of Lewy bodies, to neuroanatomical regions mildly affected in Parkinson’s disease. Diffuse alpha-synuclein proximity ligation assay signal is significantly more abundant in patients compared to controls in regions including the cingulate cortex (1.6-fold increase) and the reticular formation of the medulla (6.5-fold increase). In addition, the alpha-synuclein proximity ligation assay labels very early perikaryal aggregates in morphologically intact neurons that may precede the development of classical Parkinson’s disease lesions, such as pale bodies or Lewy bodies. Furthermore, the alpha-synuclein proximity ligation assay preferentially detects early-stage, loosely compacted lesions such as pale bodies in patient tissue, whereas Lewy bodies, considered heavily compacted late lesions are only very exceptionally stained. The alpha-synuclein proximity ligation assay preferentially labels alpha-synuclein oligomers produced in vitro compared to monomers and fibrils, while stained oligomers in human brain display a distinct intermediate proteinase K resistance, suggesting the detection of a conformer that is different from both physiological, presynapt
Mutihac R, Alegre-Abarrategui J, Gordon D, et al., 2015, TARDBP pathogenic mutations increase cytoplasmic translocation of TDP-43 and cause reduction of endoplasmic reticulum Ca2+ signaling in motor neurons, NEUROBIOLOGY OF DISEASE, Vol: 75, Pages: 64-77, ISSN: 0969-9961
Lufino MMP, Silva AM, Nemeth AH, et al., 2013, A GAA repeat expansion reporter model of Friedreich's ataxia recapitulates the genomic context and allows rapid screening of therapeutic compounds, HUMAN MOLECULAR GENETICS, Vol: 22, Pages: 5173-5187, ISSN: 0964-6906
Thomas M, Alegre-Abarrategui J, Wade-Martins R, 2013, RNA dysfunction and aggrephagy at the centre of an amyotrophic lateral sclerosis/frontotemporal dementia disease continuum, BRAIN, Vol: 136, Pages: 1345-1360, ISSN: 0006-8950
Sloan M, Alegre-Abarrategui J, Wade-Martins R, 2012, Insights into LRRK2 function and dysfunction from transgenic and knockout rodent models, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 40, Pages: 1080-1085, ISSN: 0300-5127
Klionsky DJ, Abdalla FC, Abeliovich H, et al., 2012, Guidelines for the use and interpretation of assays for monitoring autophagy, AUTOPHAGY, Vol: 8, Pages: 445-544, ISSN: 1554-8627
Phadwal K, Alegre-Abarrategui J, Watson AS, et al., 2012, A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells, Autophagy, Vol: 8, Pages: 677-689, ISSN: 1554-8627
Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8+ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.
Thomas M, Alegre-Abarrategui J, Ansorge O, et al., 2012, Dissecting the Role of FUS Mutations on Subcellular Protein Localisation and Function Using a Novel Genomic Expression System, 8th International Conference on Frontotemporal Dementias, Publisher: KARGER, Pages: 66-67, ISSN: 1420-8008
Alegre-Abarrategui J, Wade-Martins R, 2009, Parkinson disease, LRRK2 and the endocytic-autophagic pathway, AUTOPHAGY, Vol: 5, Pages: 1208-1210, ISSN: 1554-8627
Alegre-Abarrategui J, Christian H, Lufino MMP, et al., 2009, LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model, HUMAN MOLECULAR GENETICS, Vol: 18, Pages: 4022-4034, ISSN: 0964-6906
Lufino MMP, Alegre-Abarrategui J, Lim F, et al., 2009, A Novel Genomic DNA-Reporter Gene Model for the Study of Friedreich's Ataxia in Neuronal Cells, 12th Annual Meeting of the American Society of Gene Therapy, Publisher: NATURE PUBLISHING GROUP, Pages: S325-S326, ISSN: 1525-0016
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