Chronic Hypergravity Induces a Modification of Histone H3 Lysine 27 Trimethylation at TCRß Locus in Murine Thymocytes.

Gravity changes are major stressors encountered during spaceflight that affect the immune system. We previously evidenced that hypergravity exposure during gestation affects the TCRß repertoire of newborn pups. To identify the mechanisms underlying this observation, we studied post-translational histone modifications. We first showed that among the four studied post-translational histone H3 modifications, only lysine 27 trimethylation (H3K27me3) is downregulated in the thymus of mice exposed to 2× g for 21 days. We then asked whether the TCRß locus chromatin structure is altered by hypergravity exposure. ChIP studies performed on four Vß segments of the murine double-negative SCIET27 thymic cell line, which corresponds to the last maturation stage before V(D)J recombination, revealed increases in H3K27me3 after 2× g exposure. Finally, we evaluated the implication for the EZH2 methyltransferase in the regulation of the H3K27me3 level at these Vß segments by treating SCIET27 cells with the GSK126-specific inhibitor. These experiments showed that the downregulation of H3K27me3 contributes to the regulation of the Vß germline transcript expression that precedes V(D)J recombination. These data show that modifications of H3K27me3 at the TCRß locus likely contribute to an explanation of why the TCR repertoire is affected by gravity changes and imply, for the first time, EZH2 in the regulation of the TCRß locus chromatin structure.

Plasticity of the human IgM repertoire in response to long-term spaceflight.

Immune dysregulation is among the main adverse outcomes of spaceflight. Despite the crucial role of the antibody repertoire in host protection, the effects of spaceflight on the human antibody repertoire are unknown. Consequently, using high-throughput sequencing, we examined the IgM repertoire of five cosmonauts 25 days before launch, after 64 ± 11 and 129 ± 20 days spent on the International Space Station (ISS), and at 1, 7, and 30 days after landing. This is the first study of this kind in humans. Our data revealed that the IgM repertoire of the cosmonauts was different from that of control subjects (n = 4) prior to launch and that two out the five analyzed cosmonauts presented significant changes in their IgM repertoire during the mission. These modifications persisted up to 30 days after landing, likely affected the specificities of IgM binding sites, correlated with changes in the V(D)J recombination process responsible for creating antibody genes, and coincided with a higher stress response. These data confirm that the immune system of approximately half of the astronauts who spent 6 months on the ISS is sensitive to spaceflight conditions, and reveal individual responses indicating that personalized approaches should be implemented during future deep-space exploration missions that will be of unprecedented durations.

Mitochondrial Mechanisms of LRRK2 G2019S Penetrance.

Several mutations in leucine-rich repeat kinase-2 (LRRK2) have been associated with Parkinson's disease (PD). The most common substitution, G2019S, interferes with LRRK2 kinase activity, which is regulated by autophosphorylation. Yet, the penetrance of this gain-of-function mutation is incomplete, and thus far, few factors have been correlated with disease status in carriers. This includes (i) LRRK2 autophosphorylation in urinary exosomes, (ii) serum levels of the antioxidant urate, and (iii) abundance of mitochondrial DNA (mtDNA) transcription-associated 7S DNA. In light of a mechanistic link between LRRK2 kinase activity and mtDNA lesion formation, we previously investigated mtDNA integrity in fibroblasts from manifesting (LRRK2+/PD+) and non-manifesting carriers (LRRK2+/PD-) of the G2019S mutation as well as from aged-matched controls. In our published study, mtDNA major arc deletions correlated with PD status, with manifesting carriers presenting the highest levels. In keeping with these findings, we now further explored mitochondrial features in fibroblasts derived from LRRK2+/PD+ (n = 10), LRRK2+/PD- (n = 21), and control (n = 10) individuals. In agreement with an accumulation of mtDNA major arc deletions, we also detected reduced NADH dehydrogenase activity in the LRRK2+/PD+ group. Moreover, in affected G2019S carriers, we observed elevated mitochondrial mass and mtDNA copy numbers as well as increased expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates antioxidant signaling. Taken together, these results implicate mtDNA dyshomeostasis-possibly as a consequence of impaired mitophagy-in the penetrance of LRRK2-associated PD. Our findings are a step forward in the pursuit of unveiling markers that will allow monitoring of disease progression of LRRK2 mutation carriers.

3D Cultures of Parkinson's Disease-Specific Dopaminergic Neurons for High Content Phenotyping and Drug Testing.

Parkinson's disease (PD)-specific neurons, grown in standard 2D cultures, typically only display weak endophenotypes. The cultivation of PD patient-specific neurons, derived from induced pluripotent stem cells carrying the LRRK2-G2019S mutation, is optimized in 3D microfluidics. The automated image analysis algorithms are implemented to enable pharmacophenomics in disease-relevant conditions. In contrast to 2D cultures, this 3D approach reveals robust endophenotypes. High-content imaging data show decreased dopaminergic differentiation and branching complexity, altered mitochondrial morphology, and increased cell death in LRRK2-G2019S neurons compared to isogenic lines without using stressor agents. Treatment with the LRRK2 inhibitor 2 (Inh2) rescues LRRK2-G2019S-dependent dopaminergic phenotypes. Strikingly, a holistic analysis of all studied features shows that the genetic background of the PD patients, and not the LRRK2-G2019S mutation, constitutes the strongest contribution to the phenotypes. These data support the use of advanced in vitro models for future patient stratification and personalized drug development.