Neuronal activity modulates alpha-synuclein aggregation and spreading in organotypic brain slice cultures and in vivo.
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ABSTRACT:
SUBMITTER: Wu Q
PROVIDER: S-EPMC8030660 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.
| S-EPMC6924077 | biostudies-literature
Project description:Tau pathology extends throughout the brain in a prion-like fashion through connected brain regions. However, the details of the underlying mechanisms are incompletely understood. The present study aims to examine the spreading of P301S aggregated tau, a mutation that is implicated in tauopathies, using organotypic slice cultures. Coronal hippocampal organotypic brain slices (170 µm) were prepared from postnatal (day 8-10) C57BL6 wild-type mice. Collagen hydrogels loaded with P301S aggregated tau were applied to slices and the spread of tau was assessed by immunohistochemistry after 8 weeks in culture. Collagen hydrogels prove to be an effective protein delivery system subject to natural degradation in 14 days and they release tau proteins up to 8 weeks. Slices with un- and hyperphosphorylated P301S aggregated tau demonstrate significant spreading to the ventral parts of the hippocampal slices compared to empty collagen hydrogels after 8 weeks. Moreover, the spread of P301S aggregated tau occurs in a time-dependent manner, which was interrupted when the neuroanatomical pathways are lesioned. We illustrate that the spreading of tau can be investigated in organotypic slice cultures using collagen hydrogels to achieve a localized application and slow release of tau proteins. P301S aggregated tau significantly spreads to the ventral areas of the slices, suggesting that the disease-relevant aggregated tau form possesses spreading potential. Thus, the results offer a novel experimental approach to investigate tau pathology.
| S-EPMC9496515 | biostudies-literature
Project description:Overexposure to manganese has been known to promote alpha-synuclein oligomerization and enhance cellular toxicity. However, the exact mechanism of Mn-induced alpha-synuclein oligomerization is unclear. To explore whether alpha-synuclein oligomerization was associated with the cleavage of alpha-synuclein by calpain, we made a rat brain slice model of manganism and pretreated slices with calpain inhibitor II, a cell-permeable peptide that restricts the activity of calpain. After slices were treated with 400 ?M Mn for 24 h, there were significant increases in the percentage of apoptotic cells, lactate dehydrogenase release, intracellular [Ca2+]i, calpain activity, and the mRNA and protein expression of calpain 1 and alpha-synuclein. Moreover, the number of C- and N-terminal fragments of alpha-synuclein and the amount of alpha-synuclein oligomerization also increased. These results also showed that calpain inhibitor II pretreatment could reduce Mn-induced nerve cell injury and alpha-synuclein oligomerization. Additionally, there was a significant decrease in the number of C- and N-terminal fragments of alpha-synuclein in calpain inhibitor II-pretreated slices. These findings revealed that Mn induced the cleavage of alpha-synuclein protein via overactivation of calpain and subsequent alpha-synuclein oligomerization in cultured slices. Moreover, the cleavage of alpha-synuclein by calpain 1 is an important signaling event in Mn-induced alpha-synuclein oligomerization.
| S-EPMC4355489 | biostudies-literature
Project description:Both genetic and environmental factors increase risk for Parkinson's disease. Many of the known genetic factors influence α-synuclein aggregation or degradation, whereas most of the identified environmental factors produce oxidative stress. Studies using in vitro approaches have identified mechanisms by which oxidative stress can accelerate the formation of α-synuclein aggregates, but there is a paucity of evidence supporting the importance of these processes over extended time periods in brain. To assess this issue, we evaluated α-synuclein aggregates in brains of three transgenic mouse strains: hSyn mice, which overexpress human α-synuclein in neurons and spontaneously develop α-synuclein aggregates; EAAT3-/- mice, which exhibit a neuron-specific impairment in cysteine uptake and resultant neuron-selective chronic oxidative stress; and double-transgenic hSyn/EAAT3-/- mice. Aggregate formation was evaluated by quantitative immunohistochemistry for phosphoserine 129 α-synuclein and by an α-synuclein proximity ligation assay. Both methods showed that the double transgenic hSyn/EAAT3-/- mice exhibited a significantly higher α-synuclein aggregate density than littermate hSyn mice in each brain region examined. Negligible aggregate formation was observed in the EAAT3-/- mouse strain, suggesting a synergistic rather than additive interaction between the two genotypes. A similar pattern of results was observed in assessments of motor function: the pole test and rotarod test. Together, these observations indicate that chronic, low-grade neuronal oxidative stress promotes α-synuclein aggregate formation in vivo. This process may contribute to the mechanism by which environmentally induced oxidative stress contributes to α-synuclein pathology in idiopathic Parkinson's disease.
| S-EPMC9774295 | biostudies-literature
Project description:Interneuronal transfer and brain spreading of pathogenic proteins are features of neurodegenerative diseases. Pathophysiological conditions and mechanisms affecting this spreading remain poorly understood. This study investigated the relationship between neuronal activity and interneuronal transfer of α-synuclein, a Parkinson-associated protein, and elucidated mechanisms underlying this relationship. In a mouse model of α-synuclein brain spreading, hyperactivity augmented and hypoactivity attenuated protein transfer. Important features of neuronal hyperactivity reported here were an exacerbation of oxidative and nitrative reactions, pronounced accumulation of nitrated α-synuclein, and increased protein aggregation. Data also pointed to mitochondria as key targets and likely sources of reactive oxygen and nitrogen species within hyperactive neurons. Rescue experiments designed to counteract the increased burden of reactive oxygen species reversed hyperactivity-induced α-synuclein nitration, aggregation, and interneuronal transfer, providing first evidence of a causal link between these pathological effects of neuronal stimulation and indicating a mechanistic role of oxidant stress in hyperactivity-induced α-synuclein spreading.
| S-EPMC9432848 | biostudies-literature
Project description:BackgroundSynucleinopathy is a group of neurodegenerative disorders characterized by neurodegeneration and accumulation of alpha-synuclein (α-syn) aggregates in various brain regions. The detailed mechanism of α-syn-caused neurotoxicity remains obscure, which is partly due to the lack of a suitable model that retains the in vivo three-dimensional cellular network and allows a convenient dissection of the neurotoxic pathways. Recent studies revealed that the pre-formed recombinant α-syn amyloid fibrils (PFFs) induce a robust accumulation of pathogenic α-syn species in cultured cells and animals.ObjectiveOur goal is to determine whether PFFs are able to induce the pathogenic α-syn accumulation and neurotoxicity in organotypic brain slice culture, an ex vivo system that retains the in vivo three-dimensional cell-cell connections.Methods/resultsAdding PFFs to cultured wild-type rat or mouse brain slices induced a time-dependent accumulation of pathogenic α-syn species, which was indicated by α-syn phosphorylated at serine 129 (pα-syn). The PFF-induced pα-syn was abolished in brain slices prepared from α-syn null mice, suggesting that the pα-syn is from the phosphorylation of endogenous α-syn. Human PFFs also induced pα-syn in brain slices prepared from mice expressing human α-syn on a mouse α-syn-null background. Furthermore, the synaptophysin immunoreactivity was inversely associated with pα-syn accumulation and an increase of neuronal loss was detected.ConclusionPFF-treatment of brain slices is able to induce key pathological features of synucleinopathy: pα-syn accumulation and neurotoxicity. This model will be useful for investigating the neurotoxic mechanism and evaluating efficacy of therapeutic approaches.
| S-EPMC7683096 | biostudies-literature
Project description:α-Synuclein (α-syn) and tau aggregates are the neuropathological hallmarks of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively, although both pathologies co-occur in patients with these diseases, suggesting possible crosstalk between them. To elucidate the interactions of pathological α-syn and tau, we sought to model these interactions. We show that increased accumulation of tau aggregates occur following simultaneous introduction of α-syn mousepreformed fibrils (mpffs) and AD lysate-derived tau seeds (AD-tau) both in vitro and in vivo. Interestingly, the absence of endogenous mouse α-syn in mice reduces the accumulation and spreading of tau, while the absence of tau did not affect the seeding or spreading capacity of α-syn. These in vivo results are consistent with our in vitro data wherein the presence of tau has no synergistic effects on α-syn. Our results point to the important role of α-syn as a modulator of tau pathology burden and spreading in the brains of AD, PDD, and DLB patients.
| S-EPMC7588140 | biostudies-literature
Project description:BackgroundProteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression.MethodsAlpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed.ResultsTo study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions.ConclusionThe successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments.
| S-EPMC8356412 | biostudies-literature
Project description:Domoic acid is a neurotoxin produced by algae and is found in seafood during harmful algal blooms. As a glutamate agonist, domoic acid inappropriately stimulates excitatory activity in neurons. At high doses, this leads to seizures and brain lesions, but it is unclear how lower, asymptomatic exposures disrupt neuronal activity. Domoic acid has been detected in an increasing variety of species across a greater geographical range than ever before, making it critical to understand the potential health impacts of low-level exposure on vulnerable marine mammal and human populations. To determine whether prolonged domoic acid exposure altered neuronal activity in hippocampal networks, we used a custom-made 512 multi-electrode array with high spatial and temporal resolution to record extracellular potentials (spikes) in mouse organotypic brain slice cultures. We identified individual neurons based on spike waveform and location, and measured the activity and functional connectivity within the neuronal networks of brain slice cultures. Domoic acid exposure significantly altered neuronal spiking activity patterns, and increased functional connectivity within exposed cultures, in the absence of overt cellular or neuronal toxicity. While the overall spiking activity of neurons in domoic acid-exposed cultures was comparable to controls, exposed neurons spiked significantly more often in bursts. We also identified a subset of neurons that were electrophysiologically silenced in exposed cultures, and putatively identified those neurons as fast-spiking inhibitory neurons. These results provide evidence that domoic acid affects neuronal activity in the absence of cytotoxicity, and suggest that neurodevelopmental exposure to domoic acid may alter neurological function in the absence of clinical symptoms.
| S-EPMC5433861 | biostudies-literature
Project description:Organotypic slice cultures of brain or spinal cord have been a longstanding tool in neuroscience research but their utility for understanding Alzheimer's disease (AD) and other neurodegenerative proteinopathies has only recently begun to be evaluated. Organotypic brain slice cultures (BSCs) represent a physiologically relevant three-dimensional model of the brain. BSCs support all the central nervous system (CNS) cell types and can be produced from brain areas involved in neurodegenerative disease. BSCs can be used to better understand the induction and significance of proteinopathies underlying the development and progression of AD and other neurodegenerative disorders, and in the future may serve as bridging technologies between cell culture and in vivo experiments for the development and evaluation of novel therapeutic targets and strategies. We review the initial development and general use of BSCs in neuroscience research and highlight the advantages of these cultures as an ex vivo model. Subsequently we focus on i) BSC-based modeling of AD and other neurodegenerative proteinopathies ii) use of BSCs to understand mechanisms underlying these diseases and iii) how BSCs can serve as tools to screen for suitable therapeutics prior to in vivo investigations. Finally, we will examine i) open questions regarding the use of such cultures and ii) how emerging technologies such as recombinant adeno-associated viruses (rAAV) may be combined with these models to advance translational research relevant to neurodegenerative disorders.
| S-EPMC6889333 | biostudies-literature