Molecular phenotypes of mitochondrial dysfunction in clinically non-manifesting heterozygous PRKN variant carriers.

Homozygous or compound heterozygous (biallelic) variants in PRKN are causal for PD with highly penetrant symptom expression, while the much more common heterozygous variants may predispose to PD with highly reduced penetrance, through altered mitochondrial function. In the presence of pathogenic heterozygous variants, it is therefore important to test for mitochondrial alteration in cells derived from variant carriers to establish potential presymptomatic molecular markers. We generated lymphoblasts (LCLs) and human induced pluripotent stem cell (hiPSC)-derived neurons from non-manifesting heterozygous PRKN variant carriers and tested them for mitochondrial functionality. In LCLs, we detected hyperactive mitochondrial respiration, and, although milder compared to a biallelic PRKN-PD patient, hiPSC-derived neurons of non-manifesting heterozygous variant carriers also displayed several phenotypes of altered mitochondrial function. Overall, we identified molecular phenotypes that might be used to monitor heterozygous PRKN variant carriers during the prodromal phase. Such markers might also be useful to identify individuals at greater risk of eventual disease development and for testing potential mitochondrial function-based neuroprotective therapies before neurodegeneration advances.

Generation and characterization of induced pluripotent stem cell (iPSC) lines of two asymptomatic individuals carrying a heterozygous exon 7 deletion in Parkin (PRKN) and two non-carriers from the same family.

Mutations in the Parkin (PRKN) gene are the most frequent known cause of autosomal recessive early-onset Parkinson's disease (PD). Heterozygous mutations might predispose to disease with a highly reduced penetrance. We generated iPSC lines from two individuals carrying a heterozygous deletion of exon 7 in the PRKN gene and two controls from the same family. PBMCs were reprogrammed using non-integrating episomal plasmids. The iPSC lines exhibit expression of pluripotency markers, the potential to differentiate into the three germ layers, and a stable karyotype. These lines will serve to study mechanisms of reduced penetrance in heterozygous PRKN mutation carriers.

Generation of an induced pluripotent stem cell line (EURACi014-A) from a Parkinson's disease patient with an A53T mutation in the SNCA gene by an integration-free reprogramming method.

The SNCA gene encodes the presynaptic α-synuclein (aSyn) protein, and its mutations are associated with autosomal dominant Parkinson's disease (PD). We describe the generation of an induced pluripotent stem cell (iPSC) line of a patient carrying a pathogenic Ala53Thr missense mutation in the SNCA gene. Human dermal fibroblasts were reprogrammed using a non-integrating episomal method. The generated iPSC line (EURACi014-A; iPS-1.1) shows expression of pluripotency markers, the potential to differentiate into all three germ layers, and a stable karyotype. Hence, this line represents a valuable resource for the study and modeling of the processes directly controlled by aSyn.

Generation of hiPSC-Derived Functional Dopaminergic Neurons in Alginate-Based 3D Culture.

Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, overcoming the hurdle of restricted accessibility to disease-affected tissue for mechanistic studies on Parkinson's disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. In this work we established a new in vitro cell culture system based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimization of hydrogel matrix concentrations and composition allowed a high viability of embedded hiPSCs. Neural differentiation competence and efficiency of DA neuronal generation were increased in the 3D cultures compared to a conventional 2D culture methodology. Additionally, electrophysiological parameters and metabolic switching profile confirmed increased functionality and an anticipated metabolic resetting of neurons grown in alginate scaffolds with respect to their 2D counterpart neurons. We also report long-term maintenance of neuronal cultures and preservation of the mature functional properties. Furthermore, our findings indicate that our 3D model system can recapitulate mitochondrial superoxide production as an important mitochondrial phenotype observed in neurons derived from PD patients, and that this phenotype might be detectable earlier during neuronal differentiation. Taken together, these results indicate that our alginate-based 3D culture system offers an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons from hiPSCs.

Interaction of Alpha-Synuclein With Lipids: Mitochondrial Cardiolipin as a Critical Player in the Pathogenesis of Parkinson's Disease.

Alpha-Synuclein (α-Syn) is a central protein in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders including Parkinson's disease (PD). Although its role in neurotransmission is well established, the precise role of this protein in disease pathogenesis is still not fully understood. It is, however, widely regarded to be associated with the misfolding and accumulation of toxic intracellular aggregates. In fact, α-Syn is the most abundant protein component of Lewy bodies and Lewy neurites, which are also characterized by a high lipid content. Lipids, the main constituents of cellular membranes, have been implicated in many aspects of PD-related processes. α-Syn interacts with membrane phospholipids and free fatty acids via its N-terminal domain, and altered lipid-protein complexes might enhance both its binding to synaptic and mitochondrial membranes and its oligomerization. Several studies have highlighted a specific interaction of α-Syn with the phospholipid cardiolipin (CL), a major constituent of mitochondrial membranes. By interacting with CL, α-Syn is able to disrupt mitochondrial membrane integrity, leading to mitochondrial dysfunction. Additionally, externalized CL is able to facilitate the refolding of toxic α-Syn species at the outer mitochondrial membrane. In this review, we discuss how α-Syn/lipid interactions, in particular the α-Syn/CL interaction at the mitochondrial membrane, may affect α-Syn aggregation and mitochondrial dysfunction and may thus represent an important mechanism in the pathogenesis of PD.

Acid bone lysate activates TGFß signalling in human oral fibroblasts.

Demineralized bone matrix is a widely used allograft from which not only the inorganic mineral but also embedded growth factors are removed by hydrochloric acid (HCl). The cellular response to the growth factors released during the preparation of demineralized bone matrix, however, has not been studied. Here we investigated the in vitro impact of acid bone lysate (ABL) prepared from porcine cortical bone chips on oral fibroblasts. Proteomic analysis of ABL revealed a large spectrum of bone-derived proteins including TGF-ß1. Whole genome microarrays and RT-PCR together with the pharmacologic blocking of TGF-ß receptor type I kinase with SB431542 showed that ABL activates the TGF-ß target genes interleukin 11, proteoglycan 4, and NADPH oxidase 4. Interleukin 11 expression was confirmed at the protein level by ELISA. Immunofluorescence and Western blot showed the nuclear localization of Smad2/3 and increased phosphorylation of Smad3 with ABL, respectively. This effect was independent of whether ABL was prepared from mandible, calvaria or tibia. These results demonstrate that TGF-ß is a major growth factor that is removed upon the preparation of demineralized bone matrix.