Aims

Alzheimer’s disease (AD) is the most common cause of dementia, with no current cure. Familial AD (fAD) can be caused by mutations in APP or PSEN1 and PSEN2. Our human induced pluripotent stem cell (hiPSC) model consists of patient hiPSC with mutations in PSEN1 (A79V and P150L) and their respective isogenic controls generated via CRISPR-Cas9 precision gene editing.

Methods

The respective hiPSC have been differentiated into cortical glutamatergic neurons and displayed disease characteristic phenotypes such as increased Abeta secretion, Tau hyperphosphorylation and mitochondrial and synaptic deficits. Intriguingly, these neurons displayed Golgi fragmentation, indicating impairments in protein processing and post-translational modifications. RNA sequencing showed differentially expressed genes involved in glycosylation and glycan patterns.

Results

Such processes take mainly place in the Golgi apparatus, linking Golgi fragmentation and impaired processing. These results were further supported by lectin assays as well as glycan profiling. Golgi fragmentation appears to be an early event in AD pathogenesis, observed prior to mitochondrial and synaptic dysfunctions. Additionally, Golgi fragmentation could be induced in control neurons via Abeta treatment, suggesting a potential cascade of Abeta accumulation, triggering Golgi fragmentation, causing impaired processing of key proteins and metabolites, resulting in neurodegeneration.

Conclusions

Our research highlights the potential of iPSC disease modelling, as well as Golgi processing and glycosylation as possible entry points for AD intervention.

Aims

Parkinson’s disease (PD) is neuropathologically characterized by the deposition of aggregated alpha-synuclein (aSyn) and the loss of midbrain dopaminergic neurons (mDANs), preceded by neuritic degeneration. Point mutations and multiplications of aSyn gene (SNCA) have been associated with the familial forms of PD (fPD), indicating a crucial role of aSyn in the pathogenesis of PD. In contrast to age-related sporadic PD (sPD), fPDs are frequently linked to an early onset, a rapid progression and a severe aSyn pathology. In this study, we aimed to prove the hypothesis that SNCA dosage increase is the primary trigger of aSyn pathology and mDAN degeneration in fPD with SNCA multiplication.

Methods

We generated mDANs from human-induced pluripotent stem cells (hiPSC) derived from fPD patients carrying a SNCA duplication (SNCA^Dupl) and compared their phenotypes to those of mDANs from sPD patients and healthy controls.

Results

We observed a significant increase of aSyn levels and aggregation in SNCA^Dupl-mDANs, accompanied by neurite impairments. Biochemical analyses further revealed SNCA^Dupl-mediated perturbations in microtubule organization. Moreover, an increased basal apoptotic rate was specifically detected in SNCA^Dupl-mDANs. By contrast, mDANs from sPD patients did not exhibit marked changes in aSyn homeostasis, neuronal morphology, and neuritic activity. sPD-mDANs showed deficits in neurite outgrowth only, when exposed to exogenous stress, such as autophagy inhibition.

Conclusions

Our studies using the identical human mDAN model system indicate that mDANs from SNCA^Dupl PD patients are severely impaired even under steady-state conditions. Moreover, SNCA^Dupl-induced aSyn overload is sufficient to cause PD-related aSyn pathology and neurodegeneration.

Aims

DYRK1A triplication in Down Syndrome and its overexpression in Alzheimer Disease (AD) support a synergistic neurodegenerative effect in the abnormal metabolism of APP. Transport defects are early phenotypes in the progression of AD, which leads to APP processing impairments. However, whether DYRK1A regulates the intracellular transport and delivery of APP in mature neurons remains unknown. We propose to unravel the molecular mechanisms mediated by DYRK1A that control axonal transport dynamics of APP.

Methods

A protocol to obtain highly polarised human derived neurons in culture is used to pharmacologically inhibit DYRK1A activity or to overexpress DYRK1A kinase in oreder to perform high resolution/speed images to register the axonal transport of fluorescently labeled vesicles using the amyloid precursor protein fused to YFP. Matlab algortihms and routines were used to extract deep axonal transport parameters.

Results

Live-imaging in harmine-treated human-derived neurons showed reduced distal loading of APP vesicles and increased its stochastic retrograde axonal transport. Contrarily, DYRK1A overexpression increased the retrograde loading and processivity of APP vesicles. DYRK1A inhibition or overexpression showed no changes in vesicle speed transitions, however, modified the fraction and speed of retrograde segmental velocities suggesting a selective modulation of dynein motor activity.

Conclusions

We propose DYRK1A as a relevant modulator of APP metabolism through the control of its transport towards the soma and stress DYRK1A inhibition as a therapeutic intervention to restore axonal transport in AD

Aims

Acetylcholinesterase (AChE) is the enzyme that hydrolyses acetylcholine at cholinergic synapses. In Alzheimer’s Disease (AD), despite the decrease in enzymatic activity it has been described maintained levels of AChE protein in cortical areas. Furthermore, non-cholinergic roles have been described for AChE like favouring neurite outgrowth or amyloid beta deposition. In order to reflect changes in AD brain, culture of human induced pluripotent stem cells (iPS)-derived cortical neurons provide a great cellular model. In this context, the aim of this study is to characterise expression of AChE in patient-derived iPS and neurons when cortical neurons are cultivated alone, co-cultured with astrocytes or/and microglia.

Methods

Differentiations are carried out up to 70 days, maintained in neural maintenance media or BrainPhys, which supposedly favours neuronal maturation. Co-cultures with microglia are performed 14 days before deadline. Then, they are collected for imaging, enzymatic activity, protein and transcript levels.

Results

AChE enzymatic activity increases during differentiation from iPS to cortical neurons. In addition, cortical neurons co-cultured with microglia or when their maturation has been favoured, display an increased AChE activity compared to control conditions. Also, most of the cortical neurons display cholinergic phenotype as shown by immunostaining.

Conclusions

Differentiation of iPS to cortical neurons shifts AChE towards the cholinergic phenotype. Furthermore, there seem to be a more cholinergic form of AChE when iPS-derived cortical neurons are co-cultured with microglia. Therefore, iPS-derived cortical neurons provide a useful cellular model to characterise AChE in AD.

Aims

The APOE4 allele is the major genetic risk factor for AD while APOE3 is defined as average risk and APOE2 is protective. Despite recent advances, the fundamental role of different APOE alleles in brain homeostasis is still poorly understood. Here, we aim to uncover the functional role of APOE2, E3 and E4 in human astrocytes.

Methods

We differentiated human APOE-isogenic iPSCs (E4, E3, E2 and APOE-knockout (KO)) to functional astrocytes (hereafter “iAstrocytes”). iAstrocytes at baseline and after activation with Interleukin-1β were analyzed for proteomic profiles using unlabelled mass spectrometry. iAstrocyte functions and properties, such as uptake of glutamate and beta-amyloid, release of cytokine and lipid/cholesterol metabolism were characterized by various assays.

Results

APOE4 iAstrocytes showed lowest capacity for uptake of beta-amyloid and glutamate, while uptake was highest in APOE2 and APOE-KO cells. We observed a genotype-dependent reduction of cholesterol and lipid metabolic and biosynthetic proteomic pathways, and an increase in immunoregulatory pathways at baseline (E4>E3>E2). Cholesterol efflux and biosynthesis were reduced in E4 iAstrocytes, and subcellular localization of cholesterol in lysosomes was increased. APOE4 iAstrocytes showed an increase in inflammatory proteomic pathway, accompanied by highest release of cytokines at baseline, while APOE2 and APOE-KO iAstrocytes show lowest cytokine release (APOE4>E3>E2>KO).

Conclusions

We show that APOE plays a major role in several physiological and metabolic processes in human astrocytes with APOE4 pushing iAstrocytes to a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signalling and reduced β-amyloid uptake while APOE2 iAstrocytes show opposing (protective) effects.

Aims

Brain research heavily depends on models recapitulating key aspects of human brain physiology and disease pathology. Human iPSCs have great potential to complement existing disease models, as they allow directly studying affected human cell types. In addition, recent developments in CRISPR genome editing revolutionized how impacts of genetic alterations on disease formation can be investigated. Co-culture of disease-relevant iPSC-derived cells with disease-relevant mutations enables studying complex phenotypes involving cellular crosstalk.

Methods

Combining these technologies we established a new generation of iPSC-based human brain tissue models for neurodegenerative and neurovascular brain diseases. We successfully established a multicellular brain tissue model containing iPSC-derived cortical neurons, astrocytes, microglia, and recently also oligodendrocytes.

Results

Our technology provides highly controllable and reproducible 3-dimensional tissues with typical cell morphologies and functional features, including complex somatodendritic morphology of neurons, widespread synapse formation, spontaneous and induced electrical activity, network formation, microglial ramification and tiling etc. Tissues can be cultured for over 12 months in a postmitotic state without signs of proliferation or cell death, thus providing a more controllable, reproducible, and long-lived alternative to cortical organoids currently used for 3D disease modelling. To establish human models of AD, FTD-Tau, or neurovascular diseases, we used our efficient CRISPR pipeline to introduce synergistic disease-associated mutations to accelerate naturally occurring disease processes and promote pathology. We will present a first phenotypic characterization of our multicellular tissue models.

Conclusions

We expect that these models will support studies elucidating novel, potentially human-specific pathomechanisms and provide a human framework for translation and screening.

Aims

Parkinson’s disease (PD) is a disorder in which the degeneration of dopaminergic neurons (DAns) in the nigrostriatal pathway leads to debilitating motor symptoms. There remains to be a unifying hypothesis for how this degeneration is initiated and because of this, no disease-modifying therapeutics are available. Evidence from several animal models of familial PD indicates that defective dopamine release is an early cardinal feature of PD, preceding both neurodegeneration and symptom onset. Therefore, we aim to address whether dopamine release is dysfunctional in human dopamine neurons from PD-affected individuals and the molecular mechanisms by which this occurs.

Methods

We produced induced pluripotent stem cell (iPSC)-derived dopamine neurons from patients with PD-associated mutation SNCA-triplication using a modified Krik’s protocol. KCl-evoked dopamine release and total intracellular dopamine content were measured using high performance liquid chromatography electrochemical detection (HPLC-ECD) and synaptic defects were measured using whole-cell patch-clamp electrophysiology.

Results

We observed a ninety percent decrease in evoked dopamine release from iPSC-DAns harbouring SNCA-triplication and found that this coincides with a decrease in total intracellular dopamine content of the same magnitude. Both defective release and content were restored by acute L-DOPA treatment. Further data supports that these defects are not due to defective dopamine synthesis, but rather alterations in its handling. These defects in dopamine release and content coincide with electrophysiological dysfunction.

Conclusions

Combined, these data provide support from human models that synaptic dysfunction occurs early in PD. This study will be critical to providing novel targets for the development of effective disease-modifying therapeutics.

Abstract Body

Parkinson’s disease (PD) is one of the most promising target diseases for cell therapy based on the history of fetal nigral transplantation in clinics. Due to the shortage of donor supply of fetal tissue and ethical problems, fetal nigral transplantation has not been a standard treatment. The technology of pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) offer a limitless donor supply and more advantageous donor source.

After many experiments of non-clinical studies with PD models of mice, rats, and cynomolgus monkeys, several groups have started or are about to start clinical trials.

Kyoto University has started a clinical trial for Parkinson’s disease that transplants dopaminergic progenitors generated from iPSCs since 2018; Kyoto Trial to Evaluate the Safety and Efficacy of iPSC-derived dopaminergic progenitors in the treatment of Parkinson's Disease (Phase I/II). The study is ongoing without any serious adverse event. In this trial, donor iPSCs from healthy volunteer are used with immune suppression to avoid immune rejection. There are several options to reduce the risk of immune rejection by iPSCs beside the immune suppression for the future; 1) autologous transplantation with patient's own iPSCs, 2) matching human leukocyte antigens (HLAs) between donor and recipient with HLA-homo iPSCs, and 3) HLA-depleted iPSCs by gene-editing so called "universal iPSCs". The advantages and disadvantages of each option will be discussed.