Optogenetic delivery of trophic signals in a genetic model of Parkinson’s disease

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1^B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.     

Genetic variation at hair length candidate genes in elephants and the extinct woolly mammoth

Background  

Like humans, the living elephants are unusual among mammals in being sparsely covered with hair. Relative to extant elephants, the extinct woolly mammoth, Mammuthus primigenius, had a dense hair cover and extremely long hair, which likely were adaptations to its subarctic habitat. The fibroblast growth factor 5 (FGF5) gene affects hair length in a diverse set of mammalian species. Mutations in FGF5 lead to recessive long hair phenotypes in mice, dogs, and cats; and the gene has been implicated in hair length variation in rabbits. Thus, FGF5 represents a leading candidate gene for the phenotypic differences in hair length notable between extant elephants and the woolly mammoth. We therefore sequenced the three exons (except for the 3' UTR) and a portion of the promoter of FGF5 from the living elephantid species (Asian, African savanna and African forest elephants) and, using protocols for ancient DNA, from a woolly mammoth.     

Differential sensitivity of ERBB2 kinase domain mutations towards lapatinib

Background

Overexpression of the ERBB2 kinase is observed in about one-third of breast cancer patients and the dual ERBB1/ERBB2 kinase inhibitor lapatinib was recently approved for the treatment of advanced ERBB2-positive breast cancer. Mutations in the ERBB2 receptor have recently been reported in breast cancer at diagnosis and also in gastric, colorectal and lung cancer. These mutations may have an impact on the clinical responses achieved with lapatinib in breast cancer and may also have a potential impact on the use of lapatinib in other solid cancers. However, the sensitivity of lapatinib towards clinically observed ERBB2 mutations is not known.

Methodology/Principal Findings

We cloned a panel of 8 clinically observed ERBB2 mutations, established stable cell lines and characterized their sensitivity towards lapatinib and alternative ERBB2 inhibitors. Both lapatinib-sensitive and lapatinib-resistant ERBB2 mutations were observed. Interestingly, we were able to generate lapatinib resistance mutations in wt-ERBB2 cells incubated with lapatinib for prolonged periods of time. This indicates that these resistance mutations may also cause secondary resistance in lapatinib-treated patients. Lapatinib-resistant ERBB2 mutations were found to be highly resistant towards AEE788 treatment but remained sensitive towards the dual irreversible inhibitors CL-387785 and WZ-4002.

Conclusions/Significance

Patients harbouring certain ERBB2 kinase domain mutations at diagnosis may not benefit from lapatinib treatment. Moreover, secondary lapatinib resistance may develop due to kinase domain mutations. Irreversible ERBB2 inhibitors may offer alternative treatment options for breast cancer and other solid tumor patients harbouring lapatinib resistance mutations. In addition, these inhibitors may be of interest in the scenario of secondary lapatinib resistance.     

Functional GPR37 trafficking protects against toxicity induced by 6‐OHDA,MPP+ or rotenone in a catecholaminergic cell line

G protein‐coupled receptor 37 (GPR37) is suggested to be implicated in the pathogenesis of Parkinson's disease and is accumulating in Lewy bodies within afflicted brain regions. Over‐expressed GPR37 is prone to misfolding and aggregation, causing cell death via endoplasmic reticulum stress. Although the cytotoxicity of misfolded GPR37 is well established, effects of the functional receptor on cell viability are still unknown. An N2a cell line stably expressing green fluorescent protein (GFP)‐tagged human GPR37 was created to study its trafficking and effects on cell viability upon challenge with the toxins 1‐methyl‐4‐phenylpyridinium (MPP+), rotenone and 6‐hydroxydopamine (6‐OHDA). Neuronal‐like differentiation into a tyrosine hydroxylase expressing phenotype, using dibutyryl‐cAMP, induced trafficking of GPR37 to the plasma membrane. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cell viability and lactate dehydrogenase (LDH) cell death assays revealed that GPR37 was protective against all three toxins in differentiated cells. In undifferentiated cells, the majority of GPR37 was cytoplasmic and the protective effects were more variable: GPR37 expression protected against rotenone and MPP+ but not against 6‐OHDA in MTT assays, while it protected against 6‐OHDA but not against MPP+ or rotenone in lactate dehydrogenase (LDH) assays. These results suggest that GPR37 functionally trafficked to the plasma membrane protects against toxicity.     

Interactions of the dipeptide paralysin beta-Ala-Tyr and the aminoacid Glu with phospholipid bilayers

Existing evidence points out that the biological activity of beta-Ala-Tyr may in part related to its interactions with the cell membranes. For comparative reasons the effects of Glu were also examined using identical techniques and conditions. In order to examine their thermal and dynamic effects on membrane bilayers a combination of DSC, Raman and solid state NMR spectroscopy on DPPC/water model membranes were applied and the results were compared. DSC data showed that Glu perturbs to a greater degree the model membrane compared to beta-Ala-Tyr. Thus, alteration of the phase transition temperature and half width of the peaks, abolishment of the pretransition and influence on the enthalpy of the phase transition were more pronounced in the Glu loaded bilayers. Raman spectroscopy showed that incorporation of Glu in DPPC/water bilayers increased the order in the bilayers in contrast to the effect of the dipeptide. Several structural and dynamical properties of the DPPC multilamellar bilayers with and without the dipeptide or Glu were compared using high resolution C-13 MAS (Magic Angle Spinning) spectra and spectral simulations of inhomogeneously broadened, stationary P-31 NMR lineshapes measured under CP (Cross-polarization) conditions. These methods revealed that the aminoacid Glu binds in the close realm of the phosphate in the hydrophilic headgroup of DPPC while beta-Ala-Tyr is located more deeply inside the hydrophobic zone of the bilayer. The P-31 NMR simulations indicated restricted fast rotary motion of the phospholipids about their long axes in the organized bilayer structure. Finally, by the applied methodologies it is concluded that the two molecules under study exert dissimilar thermal and dynamic effects on lipid bilayers, the Glu improving significantly the packing of the lipids in contrast to the smaller and opposite effect of the dipeptide.     

Patterns of protein synthesis in the chick blastula: A comparison of the component areas of the epiblast and the primary hypoblast

The patterns of protein synthesis are examined in the hypoblast and in the areas that comprise the epiblast, that is, the area opaca, the marginal zone, and the central area, during the blastula stage which marks the beginning of the interaction between the epiblast and hypoblast for induction of the primitive streak. The results demonstrate that there are distinct qualitative and quantitative differences in protein patterns in individual areas of blastoderm, the differences being most distinct between the hypoblast and any of the component areas of the epiblast. These differences in patterns of proteins suggest that the component areas of the chick blastula have already diverged to different developmental fates before any apparent morphogenetic differentiation, that is, the appearance of the primitive streak.     

Does soil aging affect the N2O mitigation potential of biochar? A combined microcosm and field study

The application of biochar as a soil amendment to improve soil fertility has been suggested as a tool to reduce soil‐borne CO₂ and non‐CO₂ greenhouse gas emissions, especially nitrous oxide (N₂O). Both laboratory and field trials have demonstrated N₂O emission reduction by biochar amendment, but the long‐term effect (>1 year) has been questioned. Here, we present results of a combined microcosm and field study using a powdered beech wood biochar from slow pyrolysis. The field experiment showed that both CO₂ and N₂O emissions were still effectively reduced by biochar in the third year after application. However, biochar did not influence the biomass yield of sunflower for biogas production (Helianthus annuus L.). Biochar reduced bulk density and increased soil aeration and thus reduced the water‐filled pore space (WFPS) in the field, but was also able to suppress N₂O emission in the microcosms experiment conducted at constant WFPS. For both experiments, biochar had limited impact on soil mineral nitrogen speciation, but it reduced the accumulation of nitrite in the microcosms. Extraction of soil DNA and quantification of functional marker genes by quantitative polymerase chain reaction showed that biochar did not alter the abundance of nitrogen‐transforming bacteria and archaea in both field and microcosm experiments. In contradiction to previous experiments, this study demonstrates the long‐term N₂O emission suppression potential of a wood biochar and thus highlights its overall climate change mitigation potential. While a detailed understanding of the underlying mechanisms requires further research, we provide evidence for a range of biochar‐induced changes to the soil environment and their change with time that might explain the often observed N₂O emission suppression.