Cranial sensory neuron development in the absence of brain-derived neurotrophic factor in BDNF/Bax double null mice

AbstractTo investigate the role of brain-derived neurotrophic factor (BDNF) in differentiation of cranial sensory neurons in vivo, we analyzed development of nodose (NG), petrosal (PG), and vestibular (VG) ganglion cells in genetically engineered mice carrying null mutations in the genes encoding BDNF and the proapoptotic Bcl-2 homolog Bax. In bax −/− mutants, ganglion cell numbers were increased significantly compared to wild-type animals, indicating that naturally occurring cell death in these ganglia is regulated by Bax signaling. Analysis of bdnf −/− bax −/− mutants revealed that, although the Bax null mutation completely rescued cell loss in the absence of BDNF, it did not rescue the lethality of the BDNF null phenotype. Moreover, despite rescue of BDNF-dependent neurons by the bax null mutation, sensory target innervation was abnormal in double null mutants. Vagal sensory innervation to baroreceptor regions of the cardiac outflow tract was completely absent, and the density of vestibular sensory innervation to the cristae organs was markedly decreased, compared to wild-type controls. Moreover, vestibular afferents failed to selectively innervate their hair cell targets within the cristae organs in the double mutants. These innervation failures occurred despite successful navigation of sensory fibers to the peripheral field, demonstrating that BDNF is required locally for afferent ingrowth into target tissues. In addition, the bax null mutation failed to rescue expression of the dopaminergic phenotype in a subset of NG and PG neurons. These data demonstrate that BDNF signaling is required not only to support survival of cranial sensory neurons, but also to regulate local growth of afferent fibers into target tissues and, in some cells, transmitter phenotypic expression is required

Large Multidomain Protein NMR: HIV-1 Reverse Transcriptase Precursor in Solution

NMR studies of large proteins, over 100 kDa, in solution are technically challenging and, therefore, of considerable interest in the biophysics field. The challenge arises because the molecular tumbling of a protein in solution considerably slows as molecular mass increases, reducing the ability to detect resonances. In fact, the typical 1H-13C or 1H-15N correlation spectrum of a large protein, using a 13C- or 15N-uniformly labeled protein, shows severe line-broadening and signal overlap. Selective isotope labeling of methyl groups is a useful strategy to reduce these issues, however, the reduction in the number of signals that goes hand-in-hand with such a strategy is, in turn, disadvantageous for characterizing the overall features of the protein. When domain motion exists in large proteins, the domain motion differently affects backbone amide signals and methyl groups. Thus, the use of multiple NMR probes, such as 1H, 19F, 13C, and 15N, is ideal to gain overall structural or dynamical information for large proteins. We discuss the utility of observing different NMR nuclei when characterizing a large protein, namely, the 66 kDa multi-domain HIV-1 reverse transcriptase that forms a homodimer in solution. Importantly, we present a biophysical approach, complemented by biochemical assays, to understand not only the homodimer, p66/p66, but also the conformational changes that contribute to its maturation to a heterodimer, p66/p51, upon HIV-1 protease cleavage