The netrin-1 receptor DCC promotes the survival of a subpopulation of midbrain dopaminergic neurons: Relevance for ageing and Parkinson's disease.

Mechanisms that determine the survival of midbrain dopaminergic (mDA) neurons in the adult central nervous system (CNS) are not fully understood. Netrins are a family of secreted proteins that are essential for normal neural development. In the mature CNS, mDA neurons express particularly high levels of netrin-1 and its receptor Deleted in Colorectal Cancer (DCC). Recent findings indicate that overexpressing netrin-1 protects mDA neurons in animal models of Parkinson's disease (PD), with a proposed pro-apoptotic dependence function for DCC that triggers cell death in the absence of a ligand. Here, we sought to determine if DCC expression influences mDA neuron survival in young adult and ageing mice. To circumvent the perinatal lethality of DCC null mice, we selectively deleted DCC from mDA neurons utilizing DATcre /loxP gene-targeting and examined neuronal survival in adult and aged animals. Reduced numbers of mDA neurons were detected in the substantia nigra pars compacta (SNc) of young adult DATcre /DCCfl/fl mice, with further reduction in aged DATcre /DCCfl/fl animals. In contrast to young adults, aged mice also exhibited a gene dosage effect, with fewer SNc mDA neurons in DCC heterozygotes (DATcre /DCCfl/wt ). Notably, loss of mDA neurons in the SN was not uniform. Neuronal loss in the SN was limited to ventral tier mDA neurons, while mDA neurons in the dorsal tier of the SN, which resist degeneration in PD, were spared from the effect of DCC deletion in both young and aged mice. In the ventral tegmental area (VTA), young adult mice with conditional deletion of DCC had normal mDA neuronal numbers, while significant loss occurred in aged DATcre /DCCfl/fl and DATcre /DCCfl/wt mice compared to age-matched wild-type mice. Our results indicate that expression of DCC is required for the survival of subpopulations of mDA neurons and may be relevant to the degenerative processes in PD.

Evaluating mixtures of 14 hygroscopic additives to improve antibody microarray performance.

Microarrays allow the miniaturization and multiplexing of biological assays while only requiring minute amounts of samples. As a consequence of the small volumes used for spotting and the assays, evaporation often deteriorates the quality, reproducibility of spots, and the overall assay performance. Glycerol is commonly added to antibody microarray printing buffers to decrease evaporation; however, it often decreases the binding of antibodies to the surface, thereby negatively affecting assay sensitivity. Here, combinations of 14 hygroscopic chemicals were used as additives to printing buffers for contact-printed antibody microarrays on four different surface chemistries. The ability of the additives to suppress evaporation was quantified by measuring the residual buffer volume in open quill pins over time. The seven best additives were then printed either individually or as a 1:1 mixture of two additives, and the homogeneity, intensity, and reproducibility of both the spotted protein and of a fluorescently labeled analyte in an assay were quantified. Among the 28 combinations on the four slides, many were found to outperform glycerol, and the best additive mixtures were further evaluated by changing the ratio of the two additives. We observed that the optimal additive mixture was dependent on the slide chemistry, and that it was possible to increase the binding of antibodies to the surface threefold compared to 50 % glycerol, while decreasing whole-slide coefficient of variation to 5.9 %. For the two best slides, improvements were made for both the limit of detection (1.6× and 5.9×, respectively) and the quantification range (1.2× and 2.1×, respectively). The additive mixtures identified here thus help improve assay reproducibility and performance, and might be beneficial to all types of microarrays that suffer from evaporation of the printing buffers.

Antibody Colocalization Microarray for Cross-Reactivity-Free Multiplexed Protein Analysis.

Measuring many proteins at once is of great importance to the idea of personalized medicine, in order to get a snapshot of a person's health status. We describe the antibody colocalization microarray (ACM), a variant of antibody microarrays which avoids reagent-induced cross-reactivity by printing individual detection antibodies atop their corresponding capture antibodies. We discuss experimental parameters that are critical for the success of ACM experiments, namely, the printing positional accuracy needed for the two printing rounds and the need for protecting dried spots during the second printing round. Using small sample volumes (less than 30 µL) and small quantities of reagents, up to 108 different targets can be measured in hundreds of samples with great specificity and sensitivity.