Background:The deviant ATP-binding site in the important drug resistance-linked Pdr subfamily is unique. Results: Mutations in conserved residues exhibit significant ATPase activity, but reduced transport activity. Conclusion: Conserved residues Cys-199, Glu-1013, and Asp-1042 are not directly involved in ATP hydrolysis but are actively involved in the transport cycle. Significance: Our results indicate a new role for deviant ATP-binding sites.

Chemotherapy induced peripheral neuropathy (CIPN) is a common side effect of anti-cancer treatment with microtubule targeted agents (MTAs). The frequency of severe CIPN, which can be dose limiting and even life threatening, varies widely among different MTAs. For example, paclitaxel induces a higher frequency of severe CIPN than does eribulin. Different MTAs also possess distinct mechanisms of microtubule-targeted action. Recently, we demonstrated that paclitaxel and eribulin differentially affect sciatic nerve axons, with paclitaxel inducing more pronounced neurodegenerative effects and eribulin inducing greater microtubule stabilizing biochemical effects. Here, we complement and extend these axonal studies by assessing the effects of paclitaxel and eribulin in the cell bodies of sciatic nerve axons, housed in the dorsal root ganglia (DRG). Importantly, the microtubule network in cell bodies is known to be significantly more dynamic than in axons. Paclitaxel induced ATF3 expression, a marker of neuronal stress/injury. Paclitaxel also increased expression levels of acetylated tubulin and EB1, markers of microtubule stability and growth, respectively. These effects are hypothesized to be detrimental to the dynamic microtubule network within the cell bodies. In contrast, eribulin had no significant effect on any of these parameters in the cell bodies. Taken together, DRG cell bodies and their axons, two distinct neuronal cell compartments, contain functionally distinct microtubule networks that exhibit unique biochemical responses to different MTA treatments. We hypothesize that these distinct mechanistic actions may underlie the variability seen in the initiation, progression, persistence and recovery from CIPN.

Sequence variants modulating gene function or expression affect various heritable traits, including the number of neurons within a population. The present study employed a forward-genetic approach to identify candidate causal genes and their sequence variants controlling the number of one type of retinal neuron, the AII amacrine cell. Data from twenty-six recombinant inbred (RI) strains of mice derived from the parental C57BL/6J (B6/J) and A/J laboratory strains were used to identify genomic loci regulating cell number. Large variation in cell number is present across the RI strains, from a low of ∼57,000 cells to a high of ∼87,000 cells. Quantitative trait locus (QTL) analysis revealed three prospective controlling genomic loci, on Chromosomes (Chrs) 9, 11, and 19, each contributing additive effects that together approach the range of variation observed. Composite interval mapping validated two of these loci, and chromosome substitution strains, in which the A/J genome for Chr 9 or 19 was introgressed on a B6/J genetic background, showed increased numbers of AII amacrine cells as predicted by those two QTL effects. Analysis of the respective genomic loci identified candidate controlling genes defined by their retinal expression, their established biological functions, and by the presence of sequence variants expected to modulate gene function or expression. Two candidate genes, Dtx4 on Chr 19, being a regulator of Notch signaling, and Dixdc1 on Chr 9, a modulator of the WNT-β-catenin signaling pathway, were explored in further detail. Postnatal overexpression of Dtx4 was found to reduce the frequency of amacrine cells, while Dixdc1 knockout retinas contained an excess of AII amacrine cells. Sequence variants in each gene were identified, being the likely sources of variation in gene expression, ultimately contributing to the final number of AII amacrine cells.

Multiple factors regulate the differentiation of neuronal morphology during development, including interactions with afferents, targets, and homotypic neighbors, as well as cell-intrinsic transcriptional regulation. Retinal bipolar cells provide an exemplary model system for studying the control of these processes, as there are 15 transcriptionally and morphologically distinct types, each extending their dendritic and axonal arbors in respective strata within the synaptic layers of the retina. Here we have examined the role of the transcription factor Sox5 in the control of the morphological differentiation of one type of cone bipolar cell (CBC), the Type 7 cell. We confirm selective expression of SOX5 in this single bipolar cell type, emerging at the close of the first post-natal week, prior to morphological differentiation. Conditional knockout mice were generated by crossing a bipolar cell-specific cre-expressing line with mice carrying floxed Sox5 alleles, as well as the Gustducin-gfp reporter which labels Type 7 CBCs. Loss of SOX5 was confirmed in the bipolar cell stratum, in GFP+ Type 7 cells. Such SOX5-deficient Type 7 cells differentiate axonal and dendritic arbors that are each reduced in areal extent. The axonal arbors exhibit sprouting in the inner plexiform layer (IPL), thereby extending their overall radial extent, while the dendritic arbors connect with fewer cone pedicles in the outer plexiform layer, showing an increase in the average number of dendritic contacts at each pedicle. SOX5-deficient Type 7 CBCs should therefore exhibit smaller receptive fields derived from fewer if now hyper-innervated pedicles, transmitting their signals across a broader depth through the IPL.