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- Almandoz Gil, Leire, 1988-
(författare)
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Characterization of Physiological and Pathological Alpha-Synuclein : Implications for Parkinson’s Disease and Related Disorders
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aggregated alpha-synuclein is the main component of Lewy bodies and Lewy neurites, intraneuronal inclusions found in the brains of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) patients (synucleinopathies). Alpha-synuclein is a presynaptic protein, which is most commonly an unfolded monomer in its physiological state. However, under pathological conditions it can start to misfold and enter an aggregation pathway that will lead to the formation of oligomers of increasing size and finally insoluble fibrils. The oligomers have been hypothesized to be the most neurotoxic species, but studies of their properties have been hindered by their heterogeneity and kinetic instability. The overall aim of this thesis was to characterize and compare physiological and pathological forms of alpha-synuclein from different sources: recombinant monomers, oligomers formed in vitro through exposure to oxidative stress related reactive aldehydes, aggregates from a synucleinopathy mouse model and from synucleinopathy patients.In paper I we studied the effect of low molar excess of two lipid peroxidation products, 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE), on the oligomerization of alpha-synuclein. Through biophysical methods we observed that, although both aldehydes bound to alpha-synuclein directly, ONE produced SDS-stable oligomers more rapidly than HNE. Moreover, ONE induced oligomerization at both acidic and neutral pH, while HNE only formed oligomers at neutral pH.In paper II we mapped the surface exposed epitopes of in vitro and in vivo generated alpha-synuclein species by using immunoglobulin Y antibodies raised against short linear peptides covering most of the alpha-synuclein sequence. Monomers were found to react with most antibodies, while the latter part of the N-terminus and mid-region of HNE oligomers and fibrils was found to be occluded in oligomers and fibrils. Through immunohistochemistry we compared alpha-synuclein aggregates in brain tissue from patients with synucleinopathies as well as from a mouse model expressing A30P human alpha-synuclein. Although the exposed epitopes were found to be similar overall, subtle differences were detected in the C-terminus.An additional aim of this thesis was to characterize synaptic aggregates of alpha-synuclein. In paper III we obtained synaptosomal preparations of the A30P mouse model and found that a subset of the alpha-synuclein present in the synaptosomes was proteinase K resistant and therefore aggregated. Further biochemical analyses showed that the aggregated alpha-synuclein mainly was of human, i.e. transgenic, origin and that Ser 129 was not phosphorylated, which otherwise is a common post translational modification of alpha-synuclein in Lewy bodies.It has been suggested that alpha-synuclein plays a role in neurotransmitter release by binding to the SNARE protein VAMP-2 and thereby chaperoning the SNARE complex assembly. In paper IV we used proximity ligation assay to visualize the co-localization of alpha-synuclein and the SNARE proteins in primary neurons from non-transgenic and A30P transgenic mice.In conclusion, in this thesis we have characterized a variety of alpha-synuclein species and shed light on the diversity of alpha-synuclein aggregates. Additionally, we have characterized synaptic species of alpha-synuclein and analyzed the co-localization between alpha-synuclein and SNARE proteins in neurons.
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- Behere, Anish, et al.
(författare)
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A proximity ligation assay recognizing phosphorylated α-syn reveals previously undetected α-syn pathology in the brains of synucleinopathy patients and mouse model.
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Aim: To enhance detection sensitivity of phosphorylated α-synuclein (pSynS129) on post mortem synucleinopathy brains using the newly developed PLA and characterize possible ‘strain’-specific differences in the synucleinopathy brains.Experimental plan: Four different antibodies detecting different epitopes from N- to C- terminal of α-syn were paired systematically with an antibody detecting pSynS129 to reveal patho-morphological features of α-syn aggregates on post mortem brain tissue. In addition, we tested the application of our novel PLA technique in the A30P-tg mouse model that shows different types of pSynS129 aggregates in different stages of PD.Results: The PLA experiments revealed a wide distribution of pSynS129 aggregates in post mortem synucleinopathy-patient brains. We observed unique staining patterns on the brain tissue sections using only certain antibody combinations in a PLA setup, which could not be visualized using regular immunohistochemistry. In A30P-tg mice, the morphological pattern of PLA signal indicated an age-progressive, intracellular shift of pSynS129 aggregation species from periphery towards soma in the prefrontal cortex.Significance: Here we demonstrate that employing PLA with certain α-syn antibodies pair combinations can enhance detection sensitivity and specificity of α-syn pathology in the respective synucleinopathies. Additionally, it could be a useful tool to monitor the ‘strain’-specific aggregation and intracellular morphology of α-syn on post mortem brain tissue.
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- Behere, Anish, et al.
(författare)
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Alpha synuclein pre-formed fibrils trigger astrocytic activation prior to intra-neuronal deposition in a seeding mouse model of Parkinson’s disease
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Aim: To monitor temporal evolution of glial and peripheral events occurring, prior to pSynS129 inclusion formation, after a single intra-cranial injection of pre-formed fibrils (PFFs) in the wild-type (wt) mice.Experimental plan: Here we perform intracerebral inoculations with mouse PFFs in wt mice (n=30) to study early pathological and inflammatory events from 1 to 30 days post-injections (dpi) at regular time intervals. The paraffin-fixed brain sections were stained against pSynS129 species with the in house developed proximity ligation assay. Furthermore, studies using different glial and inflammatory markers revealed more information regarding the early cellular interactions involving formation and propagation pSynS129 species.Results: Already after 1 dpi, we observe strong pSynS129 immunoreactivity close the striatal injection site. Intriguingly, this type of staining disappeared with the concurrent formation of peri-nuclear pSynS129 inclusions in motor and piriform cortex, amygdala and periventricular hypothalamus after 14 dpi. Concomitantly, we observed astrocytic activation as early event happening prior to intracellular formation and propagation pSynS129 inclusions in the brain and peripheral organs.Significance: Our study elucidates the temporal relationship regarding inflammation and formation of pSynS129 inclusions. Our results indicate that a single PFF injection is enough to induce astrocytic activation and neuro-inflammatory response that occur prior to intra-neuronal accumulation of misfolded α-syn.
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- Behere, Anish, 1993-
(författare)
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Ex‘PLA’ining the progression of pathological proteins in Alzheimer’s and Parkinson’s diseases : see(d)ing is believing
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common forms of neurodegenerative disorders affecting approximately 50 million people worldwide. The underlying neuropathological processes leading to AD and PD share many similarities, i.e. aberrant protein aggregation of tau and alpha-synuclein (αSyn) in the brain. Monitoring tau and αSyn aggregation is challenging, due to morphological heterogeneity of the aggregating species and problems in preserving the antigen conformation ex vivo.In paper-I, we validated the usefulness of proximity ligation assay (PLA), a technique that enabled us to visualize previously undetected early αSyn pathology in the A30P-tg mouse model of PD. We observed an age-progressive increase in the levels of phosphorylated αSyn (pSynS129) and the compactness of aggregates in the brain. Although loss of dopaminergic neurons was not found, a subtle dysregulation of other catecholamines was recorded in the older mice.In paper-II, we revealed a wide distribution of pSynS129 aggregates in alpha-synucleinopathy-patient brains. By using a PLA setup with certain antibody pair combinations on brain sections, we observed unique staining patterns, which could not be visualized using regular immunohistochemistry (IHC). In A30P-tg mice, the morphological pattern of the PLA signals indicated an intracellular shift of pSynS129 from the periphery towards the neuronal soma.In Paper-III, we demonstrated that multiplex pTauS202,T205-pTauT231, singleplex pTauT231 and singleplex pSynS129 PLAs can recognize an extensive tau and αSyn pathology compared to regular IHC. We found that using our PLA approach we could differentiate between pTauS202,T205 and pTauT231 pathology in AD brains, whereas IHC could not. Similarly, in the PD brain, singleplex pSynS129 PLA detected novel structures, i.e. apparent thick intercellular tunnelling nanotubes and early aggregates; whereas pSynS129 IHC was limited to the detection of mature pathology. Lastly, we demonstrated that our multiplex PLA approach detected co-aggregates of pSynS129-pTau.In Paper-IV, in an αSyn seeding mouse model we observed pSynS129 immunoreactivity close to the striatal injection site one day post-injection (dpi). Intriguingly, this type of staining disappeared with the concurrent formation of peri-nuclear pSynS129 inclusions in specific brain regions after 14 dpi. In parallel, astrocytic activation prior to pSynS129 inclusion formation was observed.In conclusion, we have developed several novel PLAs that detect both tau and αSyn pathology with a higher ex vivo sensitivity and specificity than currently used immunostaining methods. This thesis work provides valuable insights that potentially could be used for the development of future biomarkers for tauopathies and synucleinopathies.
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- Behere, Anish, et al.
(författare)
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Novel visualization of phosphorylated tau and alpha-synuclein aggregates in the Alzheimer’s disease and Parkinson’s disease brain
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Several neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), display deposits of phosphorylated tau (pTau) and/or alpha-synuclein (pSyn) in affected parts of the brain. However, the pathological and morphological properties of these protein aggregates remain poorly characterized, due to lack of specificity and sensitivity of in situ detection techniques. The aim of this study was to investigate the patho-morphological properties of phosphorylated tau and α-syn aggregates on AD and PD brain tissues with a novel sensitive in situ proximity ligation assay (PLA) technique. We took advantage of the sensitivity and <40 nm resolution of PLA, along with the selectivity of different antibodies directed against pTau and pSyn epitopes. Most notably, multiplex pTauS202, T205-pTauT231, singleplex pTauT231 and pSynS129 PLA recognized more extensive phosphorylated tau and αSyn pathology, compared to conventional immunohistochemistry (IHC) using the same antibodies on adjacent brain sections. Furthermore, singleplex pTauT231 PLA captured additional pathological aggregates compared to the singleplex pTauS202, T205 PLA in late Braak stage AD brains, where traditional IHC failed to distinguish between pTauS202, T205 and pTauT231 pathology. Similarly, in PD brains, singleplex pSynS129 PLA detected novel pathological structures, such as intercellular thick tunneling nanotubes and pre-Lewy body intracytoplasmic aggregates, whereas pSynS129 IHC was limited to the detection of mature Lewy body/neurite pathology. Lastly, we could demonstrate that our dual PLA approach also can be applied to detect co-aggregates of pSyn-pTau.
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