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Background Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. Methods We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. Results The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. Conclusions Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society

Here, we present the characterization of three iPSC lines derived from dermalfibroblasts of old healthy subjects. Fibroblasts were reprogrammed using Sendai viralvectors encoding OCT4, SOX2, KLF4 and c-MYC. The iPSCs expressed endogenous pluripotency markers, could generate the three germ layers (ectoderm, mesodermand endoderm), maintained a stable karyotype, and were free from Sendai vectors and reprogramming factors. These integration-free iPSCs can serve for establishingcontrol cell cultures in studies searching for phenotypes and mechanisms that could potentially be dysregulated in degenerative diseases., This work was funded by grants from the Spanish Ministerio de Economía y Competitividad (MINECO: SAF2013-47596-R and CIBERNED: CB06/05/0065) to C.V., from CIBERNED CB06/05/0055 to R.M, from CIBERNED CB06/05/0041 to J.K., and from Fundación Ramón Areces (CIVP18A3941) to C.V. and R.M. E.R.-T. was supported by a FPIFellowship from the MINECO.

Genetic polymorphism of apolipoprotein E (APOE) confers differential susceptibility to late-onset Alzheimer's disease (LOAD). The ε3 allele of APOE, the most common isoform, does not represent a risk factor for LOAD. In contrast, the ε4 allele is the strongest genetic risk factor for this disease. Here, we present the characterization of four iPSC lines generated from dermal fibroblasts of diagnosed sporadic AD patients using Sendai viral vectors encoding OCT4, SOX2, KLF4 and c-MYC. The iPSCs expressed endogenous pluripotency markers, could be differentiated into the three germ layers, maintained the original genotypes, and were free from Sendai vectors and reprogramming factors., We thank Mª José Román for technical assistance. This work was funded by grants from the Spanish Ministerio de Economía y Competitividad (MINECO: SAF2016-80419-R and CIBERNED: CB06/ 05/0065, PI2013/01 and PI2015-2/02-4) to C.V.

The L444P mutation in the GBA1 gene which encodes β-glucocerebrosidase-1, is a major risk factor for developing Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We report the generation and characterization of an induced pluripotent stem cell (iPSC) line derived from a female PD patient carrying the L444P/wt mutation. The iPSC line presented a normal morphology, expressed endogenous pluripotency markers, could be differentiated into endodermal, mesodermal and ectodermal cells, was free from Sendai vectors and reprogramming factors, had a normal karyotype and maintained the original GBA1 genotype. Thus, this iPSC line can serve to establish cellular models of PD., We thank Drs. Marta Martínez and Miquel Vila for sharing with us PD5 fibroblasts. We also thank Mª José Román for technical assistance. This work was funded by grants from the Spanish Ministerio de Economía y Competitividad (MINECO: SAF2013-47596-R and CIBERNED CB06/05/0065) to C.V., from CIBERNED CB06/05/0055 to R.M, from CIBERNED CB06/05/0041 to J.K., and from Fundación Ramón Areces (CIVP18A3941) to C.V. and R.M. E.R.-T. was supported by a FPI Fellowship from the MINECO.

The transcription factor Nurr1 is expressed in the mouse olfactory bulb (OB), although it remains unknown whether it influences the generation of dopaminergic neurons (DA) (DA neurons) in cells isolated from this brain region. We found that expressing Nurr1 in proliferating olfactory bulb stem cells (OBSCs) produces a marked inhibition of cell proliferation and the generation of immature neurons immunoreactive for tyrosine hydroxylase (TH) concomitant with marked upregulations of Th, Dat, Gad, and Fgfr2 transcripts. In long-term cultures, these cells develop neurochemical and synaptic markers of mature-like mesencephalic DA neurons, expressing GIRK2, VMAT2, DAT, calretinin, calbindin, synapsin-I, and SV2. Concurring with the increase in both Th and Gad expression, a subpopulation of induced cells was both TH- and GAD-immunoreactive indicating that they are dopaminergic-GABAergic neurons. Indeed, these cells could mature to express VGAT, suggesting they can uptake and store GABA in vesicles. Remarkably, the dopamine D1 receptor agonist SKF-38393 induced c-Fos in TH(+) cells and dopamine release was detected in these cultures under basal and KCl-evoked conditions. By contrast, cotransducing the Neurogenin2 and Nurr1 transcription factors produced a significant decrease in the number of TH-positive neurons. Our results indicate that Nurr1 overexpression in OBSCs induces the formation of two populations of mature dopaminergic neurons with features of the ventral mesencephalon or of the OB, capable of responding to functional dopaminergic stimuli and of releasing dopamine. They also suggest that the accumulation of Fgfr2 by Nurr1 in OBSCs may be involved in the generation of DA neurons.

NURR1 is an essential transcription factor for the differentiation, maturation, and maintenance of midbrain dopaminergic neurons (DA neurons) as it has been demonstrated using knock-out mice. DA neurons of the substantia nigra pars compacta degenerate in Parkinson’s disease (PD) and mutations in the Nurr1 gene have been associated with this human disease. Thus, the study of NURR1 actions in vivo is fundamental to understand the mechanisms of neuron generation and degeneration in the dopaminergic system. Here, we present and discuss findings indicating that NURR1 is a valuable molecular tool for the in vitro generation of DA neurons which could be used for modeling and studying PD in cell culture and in transplantation approaches. Transduction of Nurr1 alone or in combination with other transcription factors such as Foxa2, Ngn2, Ascl1, and Pitx3, induces the generation of DA neurons, which upon transplantation have the capacity to survive and restore motor behavior in animal models of PD. We show that the survival of transplanted neurons is increased when the Nurr1-transduced olfactory bulb stem cells are treated with GDNF. The use of these and other factors with the induced pluripotent stem cell (iPSC)-based technology or the direct reprogramming of astrocytes or fibroblasts into human DA neurons has produced encouraging results for the study of the cellular and molecular mechanisms of neurodegeneration in PD and for the search of new treatments for this disease.

The transcription factor Nurr1 is expressed in the mouse olfactory bulb (OB), although it remains unknown whether it influences the generation of dopaminergic neurons (DA) (DA neurons) in cells isolated from this brain region. We found that expressing Nurr1 in proliferating olfactory bulb stem cells (OBSCs) produces a marked inhibition of cell proliferation and the generation of immature neurons immunoreactive for tyrosine hydroxylase (TH) concomitant with marked upregulations of Th, Dat, Gad, and Fgfr2 transcripts. In long-term cultures, these cells develop neurochemical and synaptic markers of mature-like mesencephalic DA neurons, expressing GIRK2, VMAT2, DAT, calretinin, calbindin, synapsin-I, and SV2. Concurring with the increase in both Th and Gad expression, a subpopulation of induced cells was both TH- and GAD- immunoreactive indicating that they are dopaminergic- GABAergic neurons. Indeed, these cells could mature to express VGAT, suggesting they can uptake and store GABA in vesicles. Remarkably, the dopamine Dl receptor agonist SKF-38393 induced c-Fos in TH(+) cells and dopamine release was detected in these cultures under basal and KCl-evoked conditions. By contrast, cotransducing the Neurogenin2 and Nurr1 transcription factors produced a significant decrease in the number of TH- positive neurons. Our results indicate that Nurr1 overexpression in OBSCs induces the formation of two populations of mature dopaminergic neurons with features of the ventral mesencephalon or of the OB, capable of responding to functional dopaminergic stimuli and of releasing dopamine. They also suggest that the accumulation of Fgfr2 by Nurr1 in OBSCs may be involved in the generation of DA neurons.