α-Synuclein (αSyn) is the major gene linked to sporadic Parkinson's disease (PD), whereas the G209A (p.A53T) αSyn mutation causes a familial form of PD characterized by early onset and a generally severe phenotype, including nonmotor manifestations. Here we generated de novo induced pluripotent stem cells (iPSCs) from patients harboring the p.A53T mutation and developed a robust model that captures PD pathogenic processes under basal conditions. iPSC-derived mutant neurons displayed novel disease-relevant phenotypes, including protein aggregation, compromised neuritic outgrowth, and contorted or fragmented axons with swollen varicosities containing αSyn and Tau. The identified neuropathological features closely resembled those in brains of p.A53T patients. Small molecules targeting αSyn reverted the degenerative phenotype under both basal and induced stress conditions, indicating a treatment strategy for PD and other synucleinopathies. Furthermore, mutant neurons showed disrupted synaptic connectivity and widespread transcriptional alterations in genes involved in synaptic signaling, a number of which have been previously linked to mental disorders, raising intriguing implications for potentially converging disease mechanisms.

SummaryRecent studies demonstrate that astroglia from non-neurogenic brain regions can be reprogrammed into functional neurons through forced expression of neurogenic factors. Here we explored the effect of CEND1 and NEUROG2 on reprogramming of mouse cortical astrocytes and embryonic fibroblasts. Forced expression of CEND1, NEUROG2, or both resulted in acquisition of induced neuronal cells expressing subtype-specific markers, while long-term live-cell imaging highlighted the existence of two different modes of neuronal trans-differentiation. Of note, a subpopulation of CEND1 and NEUROG2 double-transduced astrocytes formed spheres exhibiting neural stem cell properties. mRNA and protein expression studies revealed a reciprocal feedback loop existing between the two molecules, while knockdown of endogenous CEND1 demonstrated that it is a key mediator of NEUROG2-driven neuronal reprogramming. Our data suggest that common reprogramming mechanisms exist driving the conversion of lineage-distant somatic cell types to neurons and reveal a critical role for CEND1 in NEUROG2-driven astrocytic reprogramming.

In experimental models, both in vivo and cellular, over-expression of Parkinson’s linked mutant leucine-rich repeat kinase 2 (LRRK2) is sufficient to induce neuronal death. While several cell death associated proteins have been linked to LRRK2, either as protein interactors or as putative substrates, characterization of the neuronal death cascade remains elusive. In this study, we have mapped for the first time the domain within LRRK2 that mediates the interaction with FADD, thereby activating the molecular machinery of the extrinsic death pathway. Using homology modeling and molecular docking approaches, we have identified a critical motif within the N-terminal armadillo repeat region of LRRK2. Moreover, we show that co-expression of fragments of LRRK2 that contain the FADD binding motif, or deletion of this motif itself, blocks the interaction with FADD, and is neuroprotective. We further demonstrate that downstream of FADD, the mitochondrial proteins Bid and Bax are recruited to the death cascade and are necessary for neuronal death. Our work identifies multiple novel points within neuronal death signaling pathways that could potentially be targeted by candidate therapeutic strategies and highlight how the extrinsic pathway can be activated intracellularly in a pathogenic context.

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Parkinson’s disease (PD) is a common progressive neurodegenerative disorder characterized by loss of striatal-projecting dopaminergic neurons of the ventral forebrain, resulting in motor and cognitive deficits. Despite extensive efforts in understanding PD pathogenesis, no disease-modifying drugs exist. Recent advances in cell reprogramming technologies have facilitated the generation of patient-derived models for sporadic or familial PD and the identification of early, potentially triggering, pathological phenotypes while they provide amenable systems for drug discovery. Emerging developments highlight the enhanced potential of using more sophisticated cellular systems, including neuronal and glial co-cultures as well as three-dimensional systems that better simulate the human pathophysiology. In combination with high-throughput high-content screening technologies, these approaches open new perspectives for the identification of disease-modifying compounds. In this review, we discuss current advances and the challenges ahead in the use of patient-derived induced pluripotent stem cells for drug discovery in PD. We address new concepts implicating non-neuronal cells in disease pathogenesis and highlight the necessity for functional assays, such as calcium imaging and multi-electrode array recordings, to predict drug efficacy. Finally, we argue that artificial intelligence technologies will be pivotal for analysis of the large and complex data sets obtained, becoming game-changers in the process of drug discovery.

Combining high throughput screening approaches with induced pluripotent stem cell (iPSC)-based disease modeling represents a promising unbiased strategy to identify therapies for neurodegenerative disorders. Here we applied high content imaging on iPSC-derived neurons from patients with familial Parkinson’s disease bearing the G209A (p.A53T) α-synuclein (αSyn) mutation and launched a screening campaign on a small kinase inhibitor library. We thus identified the multi-kinase inhibitor BX795 that at a single dose effectively restores disease-associated neurodegenerative phenotypes. Proteomics profiling mapped the molecular pathways underlying the protective effects of BX795, comprising a cohort of 118 protein-mediators of the core biological processes of RNA metabolism, protein synthesis, modification and clearance, and stress response, all linked to the mTORC1 signaling hub. In agreement, expression of human p.A53T-αSyn in neuronal cells affected key components of the mTORC1 pathway resulting in aberrant protein synthesis that was restored in the presence of BX795 with concurrent facilitation of autophagy. Taken together, we have identified a promising small molecule with neuroprotective actions as candidate therapeutic for PD and other protein conformational disorders.

Combining high throughput screening approaches with induced pluripotent stem cell (iPSC)based disease models represents a promising unbiased strategy to identify therapies for neurodegenerative disorders. Here we applied high content imaging on iPSC-derived neurons from patients with familial Parkinson's disease bearing the G209A (p.A53T) α-synuclein (αSyn) mutation and launched a screening campaign on a small kinase inhibitor library. We thus identified the multi-kinase inhibitor BX795 that at a single dose effectively restores diseaseassociated neurodegenerative phenotypes. Proteomics profiling mapped the molecular pathways underlying the neuroprotective effects of BX795 that comprised a cohort of 118 protein-mediators of the core biological processes of RNA metabolism, protein synthesis, modification and clearance, and stress response, all linked to the mTORC1 signaling hub. In agreement, expression of human p.A53T-αSyn in neuron-like cells affected key components of the mTORC1 pathway resulting in aberrant protein synthesis that was restored in the presence of BX795 with concurrent facilitation of autophagy. Taken together, we have developed an adaptable platform based on p.A53T iPSC-derived neurons for drug screening and identified a promising small molecule with potent neuroprotective actions as candidate therapeutic for PD and other protein conformational disorders.3 Key words α-synuclein, p.A53T α-synuclein mutation, induced pluripotent stem cell derived neurons, drug screening, mTOR signaling, mRNA metabolism, proteostasis AcknowledgementsWe thank Drs. Tamotsu Yoshimori and Terje Johansen for providing GFP-LC3 and mChery-GFP-p62 plasmids, respectively.

Microalgae as unicellular eukaryotic organisms demonstrate several advantages for biotechnological and biological applications. Natural derived microalgae products demand has increased in food, cosmetic and nutraceutical applications lately. The natural antioxidants have been used for attenuation of mitochondrial cell damage caused by oxidative stress. This study evaluates the in vitro protective effect of Chlorella vulgaris bioactive extracts against oxidative stress in human mesenchymal stromal/stem cells (MSCs). The classical solid-liquid and the supercritical extraction, using biomass of commercially available and laboratory cultivated C. vulgaris, are employed. Oxidative stress induced by 300 μM H2O2 reduces cell viability of MSCs. The addition of C. vulgaris extracts, with increased protein content compared to carbohydrates, to H2O2 treated MSCs counteracted the oxidative stress, reducing reactive oxygen species levels without affecting MSC proliferation. The supercritical extraction was the most efficient extraction method for carotenoids resulting in enhanced antioxidant activity. Pre-treatment of MSCs with C. vulgaris extracts mitigates the oxidative damage ensued by H2O2. Initial proteomic analysis of secretome from licensed (TNFα-activated) MSCs treated with algal extracts reveals a signature of differentially regulated proteins that fall into clinically relevant pathways such as inflammatory signaling. The enhanced antioxidative and possibly anti-inflammatory capacity could be explored in the context of future cell therapies.