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 binding activity of the plant growth promoter asterubine.

Preventing the aggregation of certain amyloid proteins has the potential to slow down the progression of diseases like Alzheimer's, Parkinson's, and type 2 diabetes mellitus. During a high-throughput screen of 300 Australian marine invertebrate extracts, the extract of the marine sponge Thorectandra sp. 4408 displayed binding activity to the Parkinson's disease-associated protein, α-synuclein. Isolation of the active component led to its identification as the known plant growth promoter asterubine (1). This molecule shares distinct structural similarities with potent amyloid beta aggregation inhibitors tramiprosate (homotaurine) and ALZ-801. Herein we report the isolation, NMR data acquired in DMSO and α-synuclein binding activity of asterubine (1).

Domain-Independent Inhibition of CBP/p300 Attenuates α-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 α-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 α-synuclein aggregation. Our data show that selective CBP/p300 inhibitors may alter the course of pathological α-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 α-synuclein aggregation via systemic administration.

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 α-synuclein (α-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 α-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 α-syn accumulation in DA neurons. METHODS: Lewy pathology progression was modeled by the application of α-syn preformed fibrils in cultured DA neurons and in the adult mice. RESULTS: We discovered that GDNF prevented accumulation of misfolded α-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 α-syn accumulation. CONCLUSIONS: For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded α-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. © 2020 International Parkinson and Movement Disorder Society.

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.