Welcome to the AD/PD™ 2022 Interactive Program
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INTEGRATED GENOMICS FOR THE IDENTIFICATION OF NOVEL FACTORS CONTROLLING MIDBRAIN DOPAMINERGIC NEURON DIFFERENTIATION
Abstract
Aims
Objective: In this project, we are studying the gene regulatory program underlying midbrain dopaminergic neurons (mDANs) to identify novel factors controlling their lineage commitment with the aim to facilitate improved mDAN reprogramming in the future.
Methods
Methods: Using human induced pluripotent stem cell (hiPSC) technology and a tyrosine hydroxylase reporter cell line, we have generated time-series data on chromatin accessibility and transcriptome changes during neural precursor differentiation towards mDANs. Integration of the transcription factor (TF) binding profiles with the corresponding transcriptome data across differentiation time points was performed using our EPIC-DREM pipeline. The generated time-point-specific gene regulatory networks were used to identify putative key TFs controlling mDAN differentiation. To further prioritize the identified TFs, we performed low input ChIP-seq for histone H3 lysine 27 acetylation (H3K27ac) to identify TFs controlled by super-enhancers in mDANs.
Results
Results: LBX1, NHLH1 and NR2F1/2 were identified as novel factors and selected for functional validation. RNAi assays showed the selected TFs to be essential for mDAN differentiation. Transcriptome profiling upon TF depletion was performed to characterize the main pathways and targets of each candidate in this process.
Conclusions
Conclusions: First, our data set provides an integrated multi-omic profile of iPSC-derived mDANs. Second, our integrative analysis identified novel TFs controlling mDAN differentiation and predictions were validated in vitro. Third, our results could be exploited to improve in vitro protocols. Lastly, the candidates identified have been found to be deregulated in iPSC from Parkinson’s disease (PD) patients. Therefore, our results could also be used to better understand PD pathological mechanisms.
VULNERABILITIES OF MIDBRAIN DOPAMINERGIC NEURONS TO PARKINSON'S DISEASE REVEALED BY SINGLE-CELL GENOMICS
Abstract
Aims
The loss of some dopamine (DA) neurons within the substantia nigra pars compacta (SNpc) is a defining pathological hallmark of Parkinson’s Disease (PD). Yet, the molecular features associated with DA neuron vulnerability have not yet been fully identified.
Methods
We developed an antibody-based enrichment method, in combination with single-nucleus RNA-sequencing (snRNA-seq), to transcriptionally profile human midbrain DA neurons. We used Slide-seq (Stickels et al., 2021) and in situ hybridization (ISH) to localize these populations within the SNpc. Finally, we applied MAGMA and stratified LD score to nominate which cell types harbor the strongest enrichment of expression of common variants of PD.
Results
We performed snRNA-seq on 387,483 nuclei from 10 individuals with either PD or Lewy body disease (LBD) and 8 age-matched controls, identifying 68 transcriptionally-defined populations. We obtained 22,048 DA neuron profiles, a 180-fold enrichment over previous studies, and identified 10 distinct DA subtypes. With Slide-seq, we localized these 10 populations to the dorsal and ventral tier, and confirmed the localization of a representative set with in situ hybridization in the human midbrain. A single subtype, marked by the expression of the gene AGTR1, was highly susceptible to degeneration and showed the strongest upregulation of TP53 and NR2F2 and their targets, nominating altered pathways associated with degeneration in vivo. This same vulnerable population was uniquely enriched for the heritable risk associated with sporadic PD.
Conclusions
These results highlight the importance of cell-intrinsic pathways in determining the differential vulnerability of DA neurons to degeneration in PD.
EXCESSIVE SOMATIC MUTATIONS WITH DISTINCTIVE PATTERNS IN ALZHEIMER’S DISEASE NEURONS REVEALED BY NOVEL SINGLE-CELL WHOLE GENOME SEQUENCING METHOD
Abstract
Aims
Alzheimer’s disease (AD) is characterized pathologically by deposition of misfolded amyloid-β and tau proteins. However, protein-directed therapeutic strategies have shown limited clinical benefit, pointing to the need to examine pathogenesis from a broader lens.
Neurons each harbor somatic single nucleotide variants (sSNV) in their genomes, which increase with age, at a rate of ~15-20 sSNV per year. In AD, DNA damage is increased, with potentially significant effects on the genome of each cell.
Methods
We performed single-cell whole genome sequencing on neurons from AD and age-matched controls, using two independent genome amplification methods, and analyzed the burden of somatic mutations and associated nucleotide change signatures for mutational patterns.
Results
We found significantly increased sSNV in AD, with hundreds of additional somatic mutations per neuron, and a distinct mutation pattern. AD neurons show an increase in Signature C, which contains distinct nucleotide changes including C>A variants. We find elevated 8-Oxoguanine DNA lesions, evidence that these mutations may result from oxidative DNA damage. Mutations also show influences of transcription in sSNV generation. Somatic mutations are predicted to produce deleterious effects on the neuron, including gene inactivation and neoantigen-stimulated immune attack.
Conclusions
AD neurons show increased somatic mutations, with mutagenic causes that illuminate upstream components of disease pathogenesis, including DNA oxidation and transcription-coupled DNA repair. Somatic mutations further predispose neurons for dysfunction and death. The identification of somatic mutation accumulation as a novel element of neurodegeneration will enable further dissection of the cascade of events in disease pathogenesis.
GENERATION OF A PATIENT IPSC-INDUCED 3D SPHEROID MODEL TO STUDY PATHOPHYSIOLOGY OF ALZHEIMER’S DISEASE AND INVESTIGATE NOVEL THERAPEUTICS
Abstract
Aims
Exploration of the pathophysiology of Alzheimer’s disease (AD) has been hampered by lack of systems that accurately recapitulate full profile of disease progression. We have developed a 3-dimentional assembloid model with iPSC induced neurons, astrocyte and microglia derived from AD subjects to investigate the pathophysiology, protein-protein interactions, cellular mechanism and interventional strategies for AD.
Methods
Lines of APP V717I iPSC and isogenic controls were differentiated into neuronal, astrocytic and microglial progenitor cells in 2D culture, and the neuronal cells were seeded with tau oligomers (oTau). The three cell types of progenitor cells were then mixed together to generate 3D Microglia-Astrocyte-Neuronal spheroids (MAstAD).
Results
Analysis of the MAstAD cultures after 1-3 weeks of culture revealed abundant b-amyloid deposition, tau pathology, neurodegeneration, astrogliosis and microglial activation. Immunofluorescence labeling demonstrated abundant Ab deposits (observed by Thiazine red or 4G8 antibody labeling), tau phosphorylation and aggregation, microglial activation (Iba1 labeling and TNF-a- secretion) and neurodegeneration quantified using FluoroJade B, cleaved caspase 3 and LDH measurements. APP V717I mutation and oTau seeding synergistically exacerbated all the phenotypes. Coincident single cell RNA sequencing and mass spectrometry were also performed to explore the cellular and molecular signal pathways in the disease progression.
Conclusions
We have generated a novel 3D assembloid model of AD, which we term MAstAD. The MAstAD system recapitulates many features of AD pathology including microglia/astrocyte gliosis, Ab and tau pathology, and neurodegeneration. This innovative AD model offers an advanced platform to study the cellular and molecular mechanisms of disease progression and develop effective therapeutic strategies for AD.
REPEATED INFUSION OF AUTOLOGOUS ADIPOSE TISSUE-DERIVED STEM CELLS FOR MULTIPLE SYSTEM ATROPHY
Abstract
Aims
Autologous adipose tissue-derived stem cells (ADSCs) have been shown to have the ability to repair nerve cells and myelin sheaths, have anti-inflammatory effects, and improve blood flow, as well as the potential to degrade and remove insoluble proteins. We have confirmed the safety of repeated intravenous administration of ADSCs for Alzheimer's disease, Parkinson's disease, and ALS, and have experienced multiple cases of symptomatic improvements. We hypothesize that ADSCs treatment is effective for neurodegenerative diseases including MSA.
Methods
ADSCs were administered intravenously to eight patients with multiple system atrophy (MSA; MSA consisted of one MSA-p and seven MSA-c) six times approximately every month. Patients were evaluated by neurological findings, modified Rankin Scale (mRS), ALSFRS-R (ALS Functional Rating Scale), and interviews with themselves and their caregivers.
Results
No adverse reactions were observed. There was no deterioration in vital signs, no significant worsening of blood chemistry, and no evidence of Amyloid Related Imaging Abnormalities (ARIA) on MRI. The mRS and ALSFRS scores were maintained during the observation period, and three patients showed improvement in subjective symptoms, including less swelling, ability to handle a pen, and opening the cap of a plastic bottle.
Conclusions
Intravenous administration of ADSCs appears to be promising for MSA, as well as for neurodegenerative diseases such as Alzheimer's, Parkinson's, and ALS. In neurodegenerative diseases, abnormal protein deposits occur in the brain, which correlates with aging, and ADSCs may have a mechanism to prevent these deposits.
SETTING UP THE MODEL OF ALZHEIMER’S DISEASE USING STEM CELLS-DERIVED CEREBRAL ORGANOIDS
Abstract
Aims
The advancements in stem cell technology and the possibility of obtaining stem cells directly from patients represent a great promise of modeling complex neurodegenerative diseases, including Alzheimer’s disease, in vitro. Thus, our study aimed to create an in vitro model of Alzheimer’s disease (AD) directly from patients’ cells and subsequently to study the development of AD pathogenesis.
Methods
Using cell reprogramming, we successfully created induced pluripotent stem cells from cells of three patients with familial AD as well as from three healthy controls. We subsequently created 3D cerebral organoids (miniature organs resembling the human brain). We tested the presence of AD-specific pathology (i.e., accumulation of Aβ and p-Tau) using numerous methods including ELISA, qPCR, Western blot, and targeted MS.
Results
Our results show that AD-iPSC-derived cerebral organoids mimic the development of AD pathology in vitro. They also show the signs of synaptic loss, cellular stress, and senescence. Additionally, by collecting several consecutive time points of the cerebral organoid development, we were able to map the development of these pathologies in vitro. Lastly, using this cell system, we are currently testing several hypotheses on the primary cause of Aβ accumulation.
Conclusions
Altogether, our data will not only help to elucidate the development of AD pathology but will also allow the testing of substances that may prevent its development under in vitro conditions.
ANTISENSE OLIGONUCLEOTIDES TARGETING SNCA REDUCE ALPHA-SYNUCLEIN AND ASSOCIATED CELLULAR PATHOLOGY IN PARKINSON’S PATIENT IPSC-DERIVED MIDBRAIN DOPAMINERGIC NEURONS
Abstract
Aims
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and the fastest growing neurological disease. With no approved curative or disease-modifying therapies available, PD presents a major unmet clinical need. Overwhelming evidence suggests the abnormal aggregation of the protein α-synuclein, encoded by the SNCA gene, is the causative agent of PD. Multiplications of the SNCA gene are known to cause familial PD, whilst PD risk loci identified through genome-wide association studies have been shown to regulate SNCA expression. Here, we aim to validate a SNCA-targeting antisense oligonucleotide (ASO) approach in a human model system of PD.
Methods
We use targeted long-read RNA sequencing of SNCA to reveal the transcriptional landscape of the SNCA gene in midbrain dopaminergic (mDA) neurons generated from PD patient-derived induced pluripotent stem cells. We then use novel antisense oligonucleotides (ASOs) targeted to SNCA to examine the effect of reduced α-synuclein expression on cellular pathology.
Results
In these neurons, we find a number of novel SNCA isoforms, including those with alternative 5′ start sites and variable 3′ UTR lengths, and we show that relative transcript expression of isoforms differs across genotypes. Importantly, we demonstrate that an SNCA-targeting ASO-induced reduction in α-synuclein expression reverses established α-synuclein pathology and rescues cell death in these neurons. We further demonstrate that this improvement in neuronal health may be mediated through an amelioration of mitochondrial function.
Conclusions
In summary, our data supports the continued exploration of ASO technology targeted to SNCA as a novel disease-modifying therapy for PD and other related synucleinopathies.
RECIPROCAL EFFECTS OF ALPHA-SYNUCLEIN AGGREGATION AND LYSOSOMAL HOMEOSTASIS IN IPSC-DERIVED SYNUCLEINOPATHY MODELS
Abstract
Aims
Lysosomal dysfunction has been implicated in a number of neurodegenerative diseases such as Parkinson’s disease (PD). Various molecular, clinical and genetic studies emphasize a central role of lysosomal pathways and proteins contributing to the pathogenesis of PD. Within PD pathology the synaptic protein alpha-synuclein (aSyn) converts from a soluble monomer into insoluble amyloid fibrils. The aim of our study is to unravel the effect of aSyn aggregates on lysosomal turnover, in particular focusing on lysosomal homeostasis and cathepsins. Since these enzymes have been shown to be directly involved in the lysosomal degradation of aSyn, impairment of their enzymatic capacity has extensive consequences.
Methods
In our study we are applied state-of-the-art technology of patient-derived induced pluripotent stem cells (iPSCs) to examine the effect of intracellular, pathological aSyn conformers, on cell homeostasis and lysosomal function on dopaminergic neurons by biochemical analyses.
Results
We showed in patient-derived dopaminergic neurons with synuclein aggregation an impaired lysosomal trafficking of cathepsins resulting in reduced proteolytic activity of cathepsins in the lysosome. Additionally, by using a small compound boosting trafficking towards the lysosome, we were able to rescue cathepsin function.
Conclusions
Our findings demonstrate a strong interplay between aSyn aggregation pathways and function of lysosomal cathepsins. It appears that aSyn directly interferes with enzymatic activity of cathepsins which might lead to a vicious cycle of impaired alpha-synuclein degradation.