Parkinson's disease and multiple system atrophy patient iPSC-derived oligodendrocytes exhibit alpha-synuclein-induced changes in maturation and immune reactive properties.

SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.

TNF-α and α-synuclein fibrils differently regulate human astrocyte immune reactivity and impair mitochondrial respiration.

Here, we examine the cellular changes triggered by tumor necrosis factor alpha (TNF-α) and different alpha-synuclein (αSYN) species in astrocytes derived from induced pluripotent stem cells. Human astrocytes treated with TNF-α display a strong reactive pro-inflammatory phenotype with upregulation of pro-inflammatory gene networks, activation of the nuclear factor κB (NF-κB) pathway, and release of pro-inflammatory cytokines, whereas those treated with high-molecular-weight αSYN fibrils acquire a reactive antigen (cross)-presenting phenotype with upregulation of major histocompatibility complex (MHC) genes and increased human leukocyte antigen (HLA) molecules at the cell surface. Surprisingly, the cell surface location of MHC proteins is abrogated by larger F110 fibrillar polymorphs, despite the upregulation of MHC genes. Interestingly, TNF-α and αSYN fibrils compete to drive the astrocyte immune reactive response. The astrocyte immune responses are accompanied by an impaired mitochondrial respiration, which is exacerbated in Parkinson's disease (PD) astrocytes. Our data provide evidence for astrocytic involvement in PD pathogenesis and reveal their complex immune reactive responses to exogenous stressors.

Human iPSC-Derived Hippocampal Spheroids: An Innovative Tool for Stratifying Alzheimer Disease Patient-Specific Cellular Phenotypes and Developing Therapies.

The hippocampus is important for memory formation and is severely affected in the brain with Alzheimer disease (AD). Our understanding of early pathogenic processes occurring in hippocampi in AD is limited due to tissue unavailability. Here, we report a chemical approach to rapidly generate free-floating hippocampal spheroids (HSs), from human induced pluripotent stem cells. When used to model AD, both APP and atypical PS1 variant HSs displayed increased Aß42/Aß40 peptide ratios and decreased synaptic protein levels, which are common features of AD. However, the two variants differed in tau hyperphosphorylation, protein aggregation, and protein network alterations. NeuroD1-mediated gene therapy in HSs-derived progenitors resulted in modulation of expression of numerous genes, including those involved in synaptic transmission. Thus, HSs can be harnessed to unravel the mechanisms underlying early pathogenic changes in the hippocampi of AD patients, and provide a robust platform for the development of therapeutic strategies targeting early stage AD.

Cellular alterations identified in pluripotent stem cell-derived midbrain spheroids generated from a female patient with progressive external ophthalmoplegia and parkinsonism who carries a novel variation (p.Q811R) in the POLG1 gene.

Variations in the POLG1 gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma, have recently been associated with Parkinson's disease (PD), especially in patients diagnosed with progressive external ophthalmoplegia (PEO). However, the majority of the studies reporting this association mainly focused on the genetic identification of the variation in POLG1 in PD patient primary cells, and determination of mitochondrial DNA copy number, providing little information about the cellular alterations existing in patient brain cells, in particular dopaminergic neurons. Therefore, through the use of induced pluripotent stem cells (iPSCs), we assessed cellular alterations in novel p.Q811R POLG1 (POLG1Q811R) variant midbrain dopaminergic neuron-containing spheroids (MDNS) from a female patient who developed early-onset PD, and compared them to cultures derived from a healthy control of the same gender. Both POLG1 variant and control MDNS contained functional midbrain regionalized TH/FOXA2-positive dopaminergic neurons, capable of releasing dopamine. Western blot analysis identified the presence of high molecular weight oligomeric alpha-synuclein in POLG1Q811R MDNS compared to control cultures. In order to assess POLG1Q811R-related cellular alterations within the MDNS, we applied mass-spectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1Q811R and control samples. Pro- and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1Q811R MDNS was suggested by the increase in PFKM and LDHA levels and confirmed using functional analysis of glycolytic rate and oxygen consumption levels. Our results validate the use of iPSCs to assess cellular alterations in relation to PD pathogenesis, in a unique PD patient carrying a novel p.Q811R variation in POLG1, and identify several altered pathways that may be relevant to PD pathogenesis.

FGF family members differentially regulate maturation and proliferation of stem cell-derived astrocytes.

The glutamate transporter 1 (GLT1) is upregulated during astrocyte development and maturation in vivo and is vital for astrocyte function. Yet it is expressed at low levels by most cultured astrocytes. We previously showed that maturation of human and mouse stem cell-derived astrocytes - including functional glutamate uptake - could be enhanced by fibroblast growth factor (FGF)1 or FGF2. Here, we examined the specificity and mechanism of action of FGF2 and other FGF family members, as well as neurotrophic and differentiation factors, on mouse embryonic stem cell-derived astrocytes. We found that some FGFs - including FGF2, strongly increased GLT1 expression and enhanced astrocyte proliferation, while others (FGF16 and FGF18) mainly affected maturation. Interestingly, BMP4 increased astrocytic GFAP expression, and BMP4-treated astrocytes failed to promote the survival of motor neurons in vitro. Whole transcriptome analysis showed that FGF2 treatment regulated multiple genes linked to cell division, and that the mRNA encoding GLT1 was one of the most strongly upregulated of all astrocyte canonical markers. Since GLT1 is expressed at reduced levels in many neurodegenerative diseases, activation of this pathway is of potential therapeutic interest. Furthermore, treatment with FGFs provides a robust means for expansion of functionally mature stem cell-derived astrocytes for preclinical investigation.

Generation of an induced pluripotent stem cell line (CSC-46) from a patient with Parkinson's disease carrying a novel p.R301C mutation in the GBA gene.

Mutations in the glucocerebrosidase (GBA) gene have been associated with the development of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line was generated from a 60-year old patient diagnosed with PD and carrying a new mutation variant p.R301C in GBA. Using non-integrating Sendai virus-based technology, we utilized OCT3/4, SOX2, c-MYC and KLF4 transcription factors to reprogram skin fibroblasts into iPSCs. The generated iPSC line retained the mutation, displayed expression of common pluripotency markers, differentiated into the three germ layers, and exhibited normal karyotype. The iPSC line can be further used for studying PD pathogenesis.

Generation of a human induced pluripotent stem cell line (CSC-42) from a patient with sporadic form of Parkinson's disease.

Skin fibroblasts were collected from a 44-year-old patient with sporadic case of Parkinson's disease (PD). The non-integrating Sendai virus vector encoding OCT3/4, SOX2, c-MYC and KLF4 was used to reprogram fibroblasts into induced pluripotent stem cells (iPSCs). Generated iPSCs had normal karyotypes, expressed common stem cell markers, and were capable of differentiating into all three germ layers. Generated line could be used for PD modeling to understand the mechanisms that influence the disorder.

Generation of a human induced pluripotent stem cell line (CSC-40) from a Parkinson's disease patient with a PINK1 p.Q456X mutation.

Parkinson's disease (PD) is a neurodegenerative disease with unknown etiology. Here we show the generation of an induced pluripotent stem cell (iPSC) line, named CSC-40, from dermal fibroblasts obtained from a 59-year-old male patient with a homozygous p.Q456X mutation in the PTEN-induced putative kinase 1 (PINK/PARK6) gene and a confirmed diagnosis of PD, which could be used to model familial PD. A non-integrating Sendai virus-based delivery of the reprogramming factors OCT3/4, SOX2, c-MYC and KLF4 was employed. The CSC-40 cell line showed normal karyotyping and fingerprinting following transduction as well as sustained expression of several pluripotency markers and the ability to differentiate into all three germ layers.