Snca-GFP Knock-In Mice Allows Tracking the Endogenous alpha-Synuclein in Action

Non peer reviewe

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

Effects of Neurotrophic Factors in Glial Cells in the Central Nervous System : Expression and Properties in Neurodegeneration and Injury

Astrocytes, oligodendrocytes, and microglia are abundant cell types found in the central nervous system and have been shown to play crucial roles in regulating both normal and disease states. An increasing amount of evidence points to the critical importance of glia in mediating neurodegeneration in Alzheimer's and Parkinson's diseases (AD, PD), and in ischemic stroke, where microglia are involved in initial tissue clearance, and astrocytes in the subsequent formation of a glial scar. The importance of these cells for neuronal survival has previously been studied in co-culture experiments and the search for neurotrophic factors (NTFs) initiated after finding that the addition of conditioned media from astrocyte cultures could support the survival of primary neurons in vitro. This led to the discovery of the potent dopamine neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF). In this review, we focus on the relationship between glia and NTFs including neurotrophins, GDNF-family ligands, CNTF family, and CDNF/MANF-family proteins. We describe their expression in astrocytes, oligodendrocytes and their precursors (NG2-positive cells, OPCs), and microglia during development and in the adult brain. Furthermore, we review existing data on the glial phenotypes of NTF knockout mice and follow NTF expression patterns and their effects on glia in disease models such as AD, PD, stroke, and retinal degeneration.Peer reviewe

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

α-Synuclein Aggregation Inhibitory Prunolides and a Dibrominated β-Carboline Sulfamate from the Ascidian Synoicum prunum

Seven new polyaromatic bis-spiroketal-containing butenolides, the prunolides D–I (4–9) and cis-prunolide C (10), a new dibrominated β-carboline sulfamate named pityriacitrin C (11), alongside the known prunolides A–C (1–3) were isolated from the Australian colonial ascidian Synoicum prunum. The prunolides D–G (4–7) represent the first asymmetrically brominated prunolides, while cis-prunolide C (10) is the first reported with a cis-configuration about the prunolide’s bis-spiroketal core. The prunolides displayed binding activities with the Parkinson’s disease-implicated amyloid protein α-synuclein in a mass spectrometry binding assay, while the prunolides (1–5 and 10) were found to significantly inhibit the aggregation (>89.0%) of α-synuclein in a ThT amyloid dye assay. The prunolides A–C (1–3) were also tested for inhibition of pSyn aggregate formation in a primary embryonic mouse midbrain dopamine neuron model with prunolide B (2) displaying statistically significant inhibitory activity at 0.5 μM. The antiplasmodial and antibacterial activities of the isolates were also examined with prunolide C (3) displaying only weak activity against the 3D7 parasite strain of Plasmodium falciparum. Our findings reported herein suggest that the prunolides could provide a novel scaffold for the exploration of future therapeutics aimed at inhibiting amyloid protein aggregation and the treatment of numerous neurodegenerative diseases.Peer reviewe

Establishment of a lentivirus vector-based neuron-specific CRISPR-Cas9 system to dissect neuroprotective signaling pathways in primary dopaminergic neurons

As a genome editing tool, CRISPR-Cas9 has provided a robust way to generate mutations in the gene of interest, at a certain time point, and in selected cell populations. The impairment of dopaminergic neurons in the substantia nigra is addressed to be one of the main pathologies of Parkinson’s disease. The histopathology of Lewy Bodies, with an undetermined role, accompanies the demise of DA neurons. Development of strategies for the prevention the neurodegeneration has a potential to slow down the progression of Parkinson’s disease. In this study, a novel, neuron-specific CRISPR-Cas9 system was developed for the purpose of dissecting neuroprotective pathways in primary dopaminergic neurons. The optimization of the tool was done by targeting EGFP at TH-positive neurons obtained from transgenic animals expressing EGFP in dopaminergic neurons. Complete loss of EGFP was achieved at day 6 after the introduction of the CRISPR-Cas9 via lentiviral vectors. There were no survival or transduction efficiency differences. Two significant pathways for the survival of dopaminergic neurons, the microRNA biogenesis and GDNF/RET signaling were selected to collect the preliminary data. Dicer, Trbp, Translin, Ago-2 and Ret were targeted with single sgRNAs, which were specifically designed to create indel mutations in these genes, and specific lentivirus vectors were produced with each guide. After transduction with the lentivirus vectors, survival of the TH-positive neurons was unaffected. Data obtained from the quantitative PCR suggested that there was 50-70% decline in transcript levels of Trbp. However, the unchanged transcript levels of the other miRNA-related targets suggest the need for further optimization of the specific guides. Knockdown of Ret was validated by inhibition of pharmacological benefits of GDNF. Overall, this research has shown the further development of this CRISPR-Cas9 tool would be useful to dissect neuroprotective signaling pathways in dopaminergic neurons

GDNF/RET signaling and its downstream pathways eliminate alpha-synuclein pathology in dopamine neurons

Main cellular pathologies of Parkinson’s disease are demise of midbrain dopamine neurons and abnormal inclusions inside neurons and neurites called respectively, Lewy bodies and Lewy neurites. The alpha-synuclein protein was one of the first components identified in Lewy bodies. Though the exact link between alpha-synuclein and neurodegeneration is unclear, it is well-established that alpha-synuclein is important for both hereditary and spontaneous Parkinson’s disease. There is convincing evidence for the spreading of alpha-synuclein pathology between neurons. In agreement with the systemic nature of the disease, the pathology is found in the other organs outside of the central nervous system. Currently, there are no treatments against the progressive neurodegeneration or alpha-synuclein pathology in Parkinson’s disease. Neurotrophic factors hold great promise for preserving the neurons. Glial cell line-derived neurotrophic factor (GDNF) protects dopamine neurons against toxin-based animal models of neurodegeneration. Research related to GDNF and alpha-synuclein has been controversial and limited to genetic models of Parkinson’s disease. However, these genetic models cannot be generalized, especially since Lewy pathology observed in the people’s brains does not depend on the genetic mutations and can occur sporadically. It is crucial to study the relationship between GDNF and alpha-synuclein aggregation with more comprehensive models of alpha-synuclein pathology. To study the effect of GDNF on alpha-synuclein aggregation, we scaled up micro island cultures of primary mouse midbrain cells in 96-well plates. Alpha-synuclein pre-formed fibrils (PFFs) were used to induce Lewy body- and neurite-like inclusions in dopamine neurons. Delivered up to four days after PFFs, GDNF – protein or gene expression – decreased alpha-synuclein aggregation in midbrain dopamine neurons. In vivo, viral vector-mediated overexpression of GDNF in the mouse substantia nigra prevented alpha-synuclein inclusions in this brain region. Primary midbrain cells treated with PFFs were used to inspect the role of GDNF’s main signaling receptor, rearranged during transfection (RET), and its downstream signaling pathways. A neuron-specific CRISPR/Cas9 system was used to knock out RET for nulling GDNF’s protective effect against alpha-synuclein aggregation. Drug-mediated inhibition of SRC led to comparable results as knocking out RET, whereas inhibition of the phosphoinositositide-3-kinase (PI3K)/AKT pathway increased the ratio of inclusion bearing dopamine neurons, without significant loss of GDNF’s effect on alpha-synuclein aggregation. None of the treatments, except specific AKT inhibition, were harmful for the cultured dopamine neurons. To co-study neurodegeneration and alpha-synuclein pathology, the proteasome inhibitor lactacystin was assessed together with PFFs. Although GDNF failed to protect cultured dopamine neurons against lactacystin-induced neurodegeneration, it continued to eliminate alpha-synuclein pathology both in vitro and in vivo. The robust effect of GDNF against alpha-synuclein aggregation signifies the importance of neurotrophic factors and their signaling pathways for neuroprotection. These findings could promote a basis for the development of target-specific treatments against Parkinson’s disease and other disorders with alpha-synuclein pathology.Parkinsonin taudin tärkeimmät solupatologiset löydökset ovat keskiaivojen dopamiinihermosolujen häviäminen ja epänormaalit proteiinisakkaumat hermosolujen sisällä, mitä kutsutaan Lewyn kappaleiksi ja Lewyn neuriiteiksi. Alfa-synukleiini on yksi ensimmäisistä Lewyn kappaleissa tunnistetuista proteiineista. Vaikka tarkka yhteys alfa-synukleiinin ja neurodegeneraation välillä on edelleen epäselvä, tutkijoiden keskuudessa on yleisesti hyväksytty, että alfa-synukleiini on tärkeä sekä perinnölliselle että yleiselle Parkinsonin taudille. Alfa-synukleiinipatologian leviämisestä hermosolujen välillä on vakuuttavia todisteita. Taudin patologisia löydöksiä on myös keskushermoston ulkopuolisissa elimissä. Tällä hetkellä Parkinsonin taudin etenevään hermorappeumaan tai alfa-synukleiinipatologiaan ei ole taudin kulkua hidastavia lääkehoitoja. Hermosolujen kasvutekijät ovat olleet lupaavia molekyylejä uudeksi terapiamuodoksi, koska niillä on hermosolujen eloonjäämistä edistäviä vaikutuksia. Gliasolulinjaperäinen hermokasvutekijä (GDNF) suojaa dopamiinihermosoluja hermomyrkyiltä hermosolujen rappeuttavissa tautimalleissa. GDNF:n vaikutukset alfa-synukleiiniin patologisiin vaikutuksiin ovat olleet kiistanalaisia ja tutkimukset ovat rajoittuneet Parkinsonin taudin geneettisiin malleihin. Tuloksia näistä geneettisistä malleista on vaikea yleistää, koska ihmisten aivoissa havaittu Lewy-patologia ei riipu geneettisistä mutaatioista ja patologiaa voi esiintyä satunnaisesti. On ratkaisevan tärkeää tutkia GDNF:n ja alfa-synukleiiniaggregaation välistä suhdetta lisää ja eri alfa-synukleiinipatologian malleilla. Tutkiaksemme GDNF:n vaikutusta alfa-synukleiinin sakkaamiseen käytimme hiiren primäärisiä keskiaivosolujen viljelmiä 96-kuoppalevyillä ja käytimme uutta mikrosaariviljelytekniikkaa. Alfa-synukleiinin ennalta muodostettuja fibrillejä (PFF:itä) käytettiin aikaansaamaan solujen oman alfa-synukleiinin sakkaaminen ja Lewyn kappaleiden kaltaisten sakkaumien kehittyminen dopamiinihermosoluissa. GDNF annettuna jopa neljä päivää PFF:ien jälkeen vähensi alfa-synukleiinin aggregaatiota keskiaivojen dopamiinihermosoluissa. In vivo virusvektorin välittämä GDNF:n ilmentyminen hiiren mustatumakkeessa esti alfa-synukleiinin sakkaumien kehittymisen tälle aivoalueella. PFF:llä käsiteltyjä primäärisiä keskiaivosoluja käytettiin selvittämään GDNF:n signalointireittejä. Väitöskirjatutkimuksessani havaitsimme, että RET-reseptori välitti GDNF:n vaikutukset Lewyn kappaleen patologian vähentämiseen. Tutkimuksessa kehitin hermosoluspesifisen geenisaksityökalun (CRISPR/Cas9) ja sen avulla poistettiin RET ja GDNF:n suojaavat vaikutukset alfa-synukleiinin sakkaamiseen. Käyttäen pienimolekyylisiä yhdisteitä havaitsimme, että RET:n signaalireiteistä erityisesti SRC:n esto oli tärkeä GDNF:n vasteessa. Neurodegeneraation ja alfa-synukleiinipatologian tutkimiseksi käytimme proteasomi-inhibiittori laktakystiinia ja sitä käytetttiin yhdessä PFF:ien kanssa. Vaikka GDNF ei onnistunut suojaamaan viljeltyjä dopamiinihermosoluja laktakystiinin aiheuttamaa hermoston rappeutumista vastaan, se esti alfa-synukleiinipatologiaa sekä in vitro että in vivo. GDNF:n voimakas vaikutus alfa-synukleiinipatologiaa vastaan osoittaa hermokasvutekijöiden ja niiden signalointireittien tärkeyden hermosolujen suojauksessa. Väitöskirjatutkimukseni havainnot edistävät spesifisten lääkehoitojen kehittämistä Parkinsonin tautia ja muita alfa-synukleiinipatologioita vastaan

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