Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson’s disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.Peer reviewe

Domain-Independent Inhibition of CBP/p300 Attenuates alpha-Synuclein Aggregation

Neurodegenerative diseases are associated with failed proteostasis and accumulation of insoluble protein aggregates that compromise neuronal function and survival. In Parkinson's disease, a major pathological finding is Lewy bodies and neurites that are mainly composed of phosphorylated and aggregated alpha-synuclein and fragments of organelle membranes. Here, we analyzed a series of selective inhibitors acting on multidomain proteins CBP and p300 that contain both lysine acetyltransferase and bromodomains and are responsible for the recognition and enzymatic modification of lysine residues. By using high-affinity inhibitors, A-485, GNE-049, and SGC-CBP30, we explored the role of two closely related proteins, CBP and p300, as promising targets for selective attenuation of alpha-synuclein aggregation. Our data show that selective CBP/p300 inhibitors may alter the course of pathological alpha-synuclein accumulation in primary mouse embryonic dopaminergic neurons. Hence, drug-like CBP/p300 inhibitors provide an effective approach for the development of high-affinity drug candidates preventing alpha-synuclein aggregation via systemic administration.Peer reviewe

Cell Culture Media, Unlike the Presence of Insulin, Affect α-Synuclein Aggregation in Dopaminergic Neurons

There are several links between insulin resistance and neurodegenerative disorders such as Parkinson’s disease. However, the direct influence of insulin signaling on abnormal α-synuclein accumulation—a hallmark of Parkinson’s disease—remains poorly explored. To our best knowledge, this work is the first attempt to investigate the direct effects of insulin signaling on pathological α-synuclein accumulation induced by the addition of α-synuclein preformed fibrils in primary dopaminergic neurons. We found that modifying insulin signaling through (1) insulin receptor inhibitor GSK1904529A, (2) SHIP2 inhibitor AS1949490 or (3) PTEN inhibitor VO-OHpic failed to significantly affect α-synuclein aggregation in dopaminergic neurons, in contrast to the aggregation-reducing effects observed after the addition of glial cell line-derived neurotrophic factor. Subsequently, we tested different media formulations, with and without insulin. Again, removal of insulin from cell culturing media showed no effect on α-synuclein accumulation. We observed, however, a reduced α-synuclein aggregation in neurons cultured in neurobasal medium with a B27 supplement, regardless of the presence of insulin, in contrast to DMEM/F12 medium with an N2 supplement. The effects of culture conditions were present only in dopaminergic but not in primary cortical or hippocampal cells, indicating the unique sensitivity of the former. Altogether, our data contravene the direct involvement of insulin signaling in the modulation of α-synuclein aggregation in dopamine neurons. Moreover, we show that the choice of culturing media can significantly affect preformed fibril-induced α-synuclein phosphorylation in a primary dopaminergic cell culture

Cell Culture Media, Unlike the Presence of Insulin, Affect alpha-Synuclein Aggregation in Dopaminergic Neurons

There are several links between insulin resistance and neurodegenerative disorders such as Parkinson's disease. However, the direct influence of insulin signaling on abnormal alpha-synuclein accumulation-a hallmark of Parkinson's disease-remains poorly explored. To our best knowledge, this work is the first attempt to investigate the direct effects of insulin signaling on pathological alpha-synuclein accumulation induced by the addition of oc-synuclein preformed fibrils in primary dopaminergic neurons. We found that modifying insulin signaling through (1) insulin receptor inhibitor GSK1904529A, (2) SHIP2 inhibitor AS1949490 or (3) PTEN inhibitor VO-OHpic failed to significantly affect alpha-synuclein aggregation in dopaminergic neurons, in contrast to the aggregation-reducing effects observed after the addition of glial cell line-derived neurotrophic factor. Subsequently, we tested different media formulations, with and without insulin. Again, removal of insulin from cell culturing media showed no effect on alpha-synuclein accumulation. We observed, however, a reduced alpha-synuclein aggregation in neurons cultured in neurobasal medium with a B27 supplement, regardless of the presence of insulin, in contrast to DMEM/F12 medium with an N2 supplement. The effects of culture conditions were present only in dopaminergic but not in primary cortical or hippocampal cells, indicating the unique sensitivity of the former. Altogether, our data contravene the direct involvement of insulin signaling in the modulation of alpha-synuclein aggregation in dopamine neurons. Moreover, we show that the choice of culturing media can significantly affect preformed fibril-induced oc-synuclein phosphorylation in a primary dopaminergic cell culture.Peer reviewe

Cell Culture Media, Unlike the Presence of Insulin, Affect α-Synuclein Aggregation in Dopaminergic Neurons

There are several links between insulin resistance and neurodegenerative disorders such as Parkinson’s disease. However, the direct influence of insulin signaling on abnormal α-synuclein accumulation—a hallmark of Parkinson’s disease—remains poorly explored. To our best knowledge, this work is the first attempt to investigate the direct effects of insulin signaling on pathological α-synuclein accumulation induced by the addition of α-synuclein preformed fibrils in primary dopaminergic neurons. We found that modifying insulin signaling through (1) insulin receptor inhibitor GSK1904529A, (2) SHIP2 inhibitor AS1949490 or (3) PTEN inhibitor VO-OHpic failed to significantly affect α-synuclein aggregation in dopaminergic neurons, in contrast to the aggregation-reducing effects observed after the addition of glial cell line-derived neurotrophic factor. Subsequently, we tested different media formulations, with and without insulin. Again, removal of insulin from cell culturing media showed no effect on α-synuclein accumulation. We observed, however, a reduced α-synuclein aggregation in neurons cultured in neurobasal medium with a B27 supplement, regardless of the presence of insulin, in contrast to DMEM/F12 medium with an N2 supplement. The effects of culture conditions were present only in dopaminergic but not in primary cortical or hippocampal cells, indicating the unique sensitivity of the former. Altogether, our data contravene the direct involvement of insulin signaling in the modulation of α-synuclein aggregation in dopamine neurons. Moreover, we show that the choice of culturing media can significantly affect preformed fibril-induced α-synuclein phosphorylation in a primary dopaminergic cell culture

α-Synuclein aggregation inhibitory activity of the bromotyrosine derivatives aerothionin and aerophobin-2 from the subtropical marine sponge Aplysinella sp

The neuronal protein α-synuclein (α-syn) is one of the main constituents of intracellular amyloid aggregations found in the post-mortem brains of Parkinson’s disease (PD) patients. Recently, we screened the MEOH extracts obtained from 300 sub-tropical marine invertebrates for α-syn binding activity using affinity MS and this resulted in the extract of the Verongida marine sponge Aplysinella sp. 1194, (QM G339263) displaying molecules that bind to the protein. The subsequent bioassay-guided separation of the Aplysinella sp. extract led to the isolation of the known bromotyrosine derivatives (+)-aerothionin (1) and (+)-aerophobin-2 (2). Both compounds bind to α-syn as detected by a MS affinity assay and inhibit α-syn aggregation in an assay that uses the fluorescence probe, thioflavin T, to detect aggregation. (+)-Aerothionin (1) was toxic to primary dopaminergic neurons at its expected α-syn aggregation inhibitory concentration and so could not be tested for pSyn aggregates in this functional assay. (+)-Aerophobin-2 (2) was not toxic and shown to weakly inhibit pSyn aggregation in primary dopaminergic neurons at 10 µM.Peer reviewe

Hesperine, a new imidazole alkaloid and α-synuclein binding activity of 1-methyl-1,2,7,8-tetrahydro-2,8-dioxoadenosine from the marine sponge Clathria (Thalysias) cf. hesperia

During a high-throughput screen of 300 Australian marine invertebrate extracts, the extract of the marine sponge Clathria (Thalysias) cf. hesperia was identified with α-synuclein binding activity. The bioassay-guided purification of this extract resulted in the isolation of 1-methyl-1,2,7,8-tetrahydro-2,8-dioxoadenosine (2) as the α-syn binder along with one new compound, hesperine (1), and five known compounds, indole-3-carboxaldehyde (3), (Z)-2'-demethylaplysinopsin (4), 2-amino-4'-hydroxyacetophenone (5), 4-hydroxybenzoic acid (6) and 4-hydroxybenzaldehyde (7). Herein, we report the structure elucidation of hesperine (1) and α-syn binding activity of 1-methyl-1,2,7,8-tetrahydro-2,8-dioxoadenosine (2).Peer reviewe

GDNF/RET Signaling Pathway Activation Eliminates Lewy Body Pathology in Midbrain Dopamine Neurons

Background Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded alpha-synuclein (alpha-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded alpha-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. Objectives We studied the effects of GDNF on misfolded alpha-syn accumulation in DA neurons. Methods Lewy pathology progression was modeled by the application of alpha-syn preformed fibrils in cultured DA neurons and in the adult mice. Results We discovered that GDNF prevented accumulation of misfolded alpha-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced alpha-syn accumulation. Conclusions For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded alpha-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. (c) 2020 International Parkinson and Movement Disorder SocietyPeer reviewe