Abstract Body

Human stem cell models of dementias provide platforms to investigate the cellular pathways involved in disease pathogenesis. We have used human stem cell-derived neurons and microglial cells to study the cell and molecular biology of mutations causal for different forms of neurodegeneration, focusing on mutations causal for monogenic Alzheimer’s disease (PSEN1, APP, SORL1) and frontotemporal dementia (MAPT). In addition to highlighting insights into mechanisms of dementia initiation and progression that have come from these approaches, this talk will also discuss progress in using these systems for whole-genome CRISPR screens for understanding disease pathogenesis and to identify candidate therapeutic pathways for disease modification.

Aims

The sorting receptor SorLA is encoded by the well-established Alzheimer’s disease risk gene SORL1. SorLA is established as a sorting receptor that mediates endosome to Golgi trafficking of the amyloid precursor protein. Loss of SORL1 impedes retrograde trafficking of APP, leads to early endosome traffic jams and increases processing of APP to amyloid beta. We have recently shown that loss of SORL1 leads to early endosome enlargement, an established AD cytopathology. Our objective in this study is to further explore endo-lysosomal trafficking impairments in neurons that lead to neurodegeneration.

Methods

We used isogenic hiPSC-derived neurons deficient in or with enhanced SORL1 expression and examined endocytic cargo trafficking, lysosomal degradation, neuronal functional activity, and transcriptomic responses to loss of SORL1.

Results

We find that loss of SORL1 impairs trafficking of multiple cargo including the neurotrophin receptor TrkB and the Glutamate receptor GLUR1. Functionally, loss of SORL1 impedes lysosomal degradation and impacts neuronal synaptic function. RNA-sequencing analyses shows altered network interaction changes in neurotrophin and synaptic genes, demonstrating that endosomal traffic jams induced by loss of SorLA expression lead to impairment of pathways that are necessary for neuronal health and function. Finally, we demonstrate both genetically and pharmacologically that enhanced SORL1 expression and small molecules that enhance endosomal trafficking promote cargo trafficking and rescue endosome enlargement induced by SORL1 deficiency in neurons.

Conclusions

Collectively our data suggests that SORLA plays multiple roles in neuronal endo-lysosomal trafficking that impact health and function of neurons. Furthermore, strategies enhancing endosomal trafficking should be considered as therapeutic targets for AD.

Aims

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an important model to study Parkinson’s disease (PD). About 100 iPSC used in this study were derived from subjects within the Parkinson’s Progression Marker Initiative (PPMI) and have their genetic background characterized. We included healthy controls, idiopathic Parkinson's Disease (iPD) patients with mutations in LRRK2, GBA and SNCA as well as unaffected mutation carriers. Using single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq), we aim to explore the cell population heterogeneity, compare the transcriptome profiles and define molecular networks that are perturbed in PD, cell-type specific enrichment of common-SNPs and novel associations with brain disorders, providing disease insight, therapeutic targets and relevant readouts for interventional testing.

Methods

Dopaminergic neurons were produced with an automated culture system, harvested on day 65 and diluted to 1000 cells/ul for capture on the 10X Genomics Chromium controller. We used Seurat (v3) within the R environment for filtering, normalization, integration of multiple single-cell libraries, unsupervised clustering, visualization, and differential expression analysis. We performed pseudotime analysis using Monocle (v3).

Results

Cell clustering produced multiple transcriptionally distinct populations. iPSC-derived dopaminergic neurons cluster showed the highest correlation with the human substantia nigra dopaminergic neurons. Comparing control cell lines and cell lines with different mutations allow us to identify differentially expressed genes that can be associated with PD across cell types.

Conclusions

Our results deliver multiple novelties: (A) a first catalogue of the cell type-specific information for patient-specific iPSC-derived dopaminergic neurons; (B) potential novel genetic associations with PD.

Aims

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by protein inclusions mostly composed of aggregated α-synuclein (α-Syn) and by the progressive degeneration of midbrain dopaminergic neurons (mDANs). The relevance of α-Syn aggregation for the preferential loss of mDANs in PD pathology is not completely understood yet. We therefore aimed to elucidate the mechanisms of the brain region-specific neuronal vulnerability in PD.

Methods

We applied human induced pluripotent stem cells (iPSCs) from familial PD cases with a duplication (Dupl) of the α-Syn gene (SNCA) locus to model human PD. Human iPSCs from PD Dupl patients and a control individual were differentiated into mDANs and cortical projection neurons (CPNs) and assessed for neuronal cell death and α-Syn pathology degree.

Results

Elevated α-Syn pathology, as revealed by enhanced α-Syn insolubility and phosphorylation, was determined in PD-derived mDANs compared to PD CPNs. PD-derived mDANs exhibited higher levels of reactive oxygen species and protein nitration levels compared to CPNs, which might underlie elevated α-Syn pathology observed in mDANs. Finally, increased neuronal death was observed in PD-derived mDANs compared to PD CPNs and to control mDANs and CPNs. Inhibition of α-Syn oligomerization by a NPT100-18A compound rescued PD mDAN cell death. Our results reveal, for the first time, a higher α-Syn pathology, oxidative stress level, and neuronal death rate in human PD mDANs compared to PD CPNs from the same patient.

Conclusions

The finding implies the contribution of pathogenic α-Syn, probably induced by oxidative stress, to selective vulnerability of substantia nigra dopaminergic neurons in human PD.

Aims

Parkinson's disease (PD) is pathologically characterized by progressive loss of dopaminergic neurons (DAn) in the substantia nigra (SNc) and accumulation of α-syn. However, neuroinflammation and glial reactivity are increasingly gaining interest as they might sustain or exacerbate DAn degeneration. We recently demonstrated that LRRK2-PD astrocytes accumulate α-syn due to dysfunctional protein degradation pathways. Using a human co-culture system, we showed that α-syn is transferred from astrocytes to surrounding DAn affecting their survival.

Yet, whether astrocytes secrete other neurotoxic factors and actively function as immune mediators inducing neuronal degeneration remains elusive.

Methods

We generated induced pluripotent stem cells (iPSC)-derived astrocytes from familial LRRK2-G2019S PD patients and age/gender-matched controls.

Results

PD-derived astrocytes are robustly reactive without any stimulation, since they display high GFAP levels along with retracted morphology and abnormal cytokine profile, most likely due to inherent alterations leading to increased α-syn accumulation. These astrocytes overexpress inflammasome and Toll-like receptors that may act as chronic inflammation stimulators. RNA-seq transcriptome profiling confirmed inflammation-related changes occurring only in PD astrocytes.

We investigated the mechanisms by which astrocytes induce neuronal cell death and found pro-inflammatory IL-6 released by LRRK2-PD astrocytes as key mediator of neuronal degeneration. Matching these in vitro findings, we detected IL6-expressing astrocytes in postmortem brains. IL-6 via IL-6R leads to downstream activation of STAT pathway in PD neurons. Blockage of IL-6R rescued astrocyte-induced neuronal degeneration.

Conclusions

Our results provide substantial new insights into the contribution of astrocytes to the human pathology of PD and identify a new therapeutic target for halt or rescue PD pathogenesis.

Aims

Sustained gene silencing throughout the nervous system has been achieved using “designer DNA drugs”, a.k.a., antisense oligonucleotides or ASOs. ASOs slow disease progression or produce disease reversal in models of inherited ALS and Huntington’s disease, respectively, with pivotal Phase III trials now in progress, as well as trials for C9orf72-mediated ALS/FTD or to suppress tau in Alzheimer’s or LRRK2 or alpha synuclein in Parkinson’s.

Here we show that ASOs can also be used to produce “identify theft”, direct conversion of astrocytes into new functional nigral or hippocampal neurons in a single step by depleting the RNA binding protein PTB.

Methods

Suppression of PTB using a therapeutically viable approach with injection of an antisense oligonucleotide (ASO) into cerebral spinal fluid of healthy adult mice is shown to convert astrocytes into new neurons, especially in the hippocampus.

Results

ASO-mediated suppression of PTB in the brains of aged mice is shown to generate many new hippocampal neurons that are electrically active, send axons into CA3, and receive both inhibitory and excitatory inputs. Suppression of PTB in mice with chemically induced Parkinson’s disease potently reverses disease through converting astrocytes into new substantia nigral neurons to restore striatal dopamine. Using a therapeutically viable injection into cerebral spinal fluid, PTB targeting ASOs drive astrocyte to nigral neuron conversion that mediates reversal of chemically induced Parkinson’s-like disease.

Conclusions

ASO-mediated suppression of PTB may be a generalizable, therapeutically feasible strategy for treating neurodegenerative disorders, including Parkinson’s, Alzheimer’s, and Huntington’s, by converting astrocytes into neuronal replacements for those lost to disease.

Abstract Body

The innovation of induced pluripotent stem cells (iPSCs) are drawing attention to their application for regenerative medicine. Parkinson’s disease is one of the most promising target diseases 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 iPSCs offers a limitless and more advantageous donor source. Our research aim is to apply the stem cell technology to the clinic in cell therapy for PD.

Our group has successfully established a protocol for donor induction with clinically compatible grade. The non-clinical studies transplanted these donor neurons into PD models of mice, rats, and cynomolgus monkeys.

These studies showed the graft survival with functional recovery and without any tumorization or side effect. Based on these non-clinical results, 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.

Aims

Molecular studies in rodent models suggest a direct contribution of astrocytes to neuroinflammatory and neurodegenerative processes causing Alzheimer’s disease. Yet these models may not fully recapitulate human disease as human and rodent astrocytes differ considerably in morphology, functionality, and gene expression.

Methods

To address this limitation, we developed an approach to study human astroglia within a host wild-type and Alzheimer’s brain environment.

Results

Transplantation of human induced pluripotent stem cell (hiPSC)-derived glia progenitors into neonatal brains of immune-deficient mice results in differentiation into astrocytes that integrate functionally within the mouse host brain and mature in a cell-autonomous way retaining human-specific morphologies, unique features and properties. Importantly, in Alzheimer’s mouse brains, xenografted hiPSC-derived astrocytes exhibit differential responses to amyloid-beta plaques that seem not dependent on the ApoE genetic background.

Conclusions

In sum, hiPSC-derived astroglia transplanted into the neonatal mouse brain assumes human-specific phenotypes in homeostatic and disease conditions. Thus, this chimeric model is a powerful tool to analyze the role of patient-derived and genetically modified astroglia in Alzheimer’s disease.