Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling.

Lysophosphatidic acid (LPA) is a growth factor for many cells including prostate and ovarian cancer-derived cell lines. LPA stimulates H2O2 production which is required for growth. However, there are significant gaps in our understanding of the spatial and temporal regulation of H2O2-dependent signaling and the way in which signals are transmitted following receptor activation. Herein, we describe the use of two reagents, DCP-Bio1 and DCP-Rho1, to evaluate the localization of active protein oxidation after LPA stimulation by detection of nascent protein sulfenic acids. We found that LPA stimulation causes internalization of LPA receptors into early endosomes that contain NADPH oxidase components and are sites of H2O2 generation. DCP-Rho1 allowed visualization of sulfenic acid formation, indicative of active protein oxidation, which was stimulated by LPA and decreased by an LPA receptor antagonist. Protein oxidation sites colocalized with LPAR1 and the endosomal marker EEA1. Concurrent with the generation of these redox signaling-active endosomes (redoxosomes) is the H2O2- and NADPH oxidase-dependent oxidation of Akt2 and PTP1B detected using DCP-Bio1. These new approaches therefore enable detection of active, H2O2-dependent protein oxidation linked to cell signaling processes. DCP-Rho1 may be a particularly useful protein oxidation imaging agent enabling spatial resolution due to the transient nature of the sulfenic acid intermediate it detects.

Cooperative nuclear localization sequences lend a novel role to the N-terminal region of MSH6.

Human mismatch repair proteins MSH2-MSH6 play an essential role in maintaining genetic stability and preventing disease. While protein functions have been extensively studied, the substantial amino-terminal region (NTR*) of MSH6 that is unique to eukaryotic proteins, has mostly evaded functional characterization. We demonstrate that a cluster of three nuclear localization signals (NLS) in the NTR direct nuclear import. Individual NLSs are capable of partially directing cytoplasmic protein into the nucleus; however only cooperative effects between all three NLSs efficiently transport MSH6 into the nucleus. In striking contrast to yeast and previous assumptions on required heterodimerization, human MSH6 does not determine localization of its heterodimeric partner, MSH2. A cancer-derived mutation localized between two of the three NLS significantly decreases nuclear localization of MSH6, suggesting altered protein localization can contribute to carcinogenesis. These results clarify the pending speculations on the functional role of the NTR in human MSH6 and identify a novel, cooperative nuclear localization signal.

Brainstem nitrergic innervation of the mouse visual thalamus.

Ascending sensory information flowing through the visual thalamus is dynamically regulated by a number of modulatory influences. An important part of this ascending modulation is a cholinergic projection from the parabrachial region of the brainstem (PBR). In addition to containing cholinergic fibers, this projection has been identified in some species as the exclusive extrinsic source of fibers containing the neuronal form of nitric oxide synthase (nNOS). In this study, we examined the nitrergic innervation to the adult mouse visual thalamus. Retrograde tract-tracing with fluorescent microspheres was combined with nNOS and choline acetyltransferase (ChAT) immunocytochemistry, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry to identify the source of nitrergic innervation. Double-labeling revealed that only two regions of the mouse brain contained nitrergic neurons that projected to the visual thalamus: the pedunculopontine tegmentum and, to a lesser extent, the lateral dorsal tegmentum (LDT). Though the LDT projected heavily to the mouse visual thalamus, very few of the retrogradely labeled neurons in that region colocalized NADPH-d. These observations suggest that the parabrachial brainstem region is the primary source of nitrergic fibers in the mouse visual thalamus, similar to that found in cat and monkey. Such similarity suggests that the presence of nNOS in presynaptic terminal fields of the visual thalamus is an important conserved property of thalamic physiology and that the mouse is a valid model for studies of nNOS functions in the brain.

Brain nitric oxide synthase expression in the developing ferret lateral geniculate nucleus: analysis of time course, localization, and synaptic contacts.

Nitric oxide (NO) is a diffusible neurotransmitter that has been implicated in key developmental events, including the refinement of retinogeniculate axons into ON/OFF sublayers in the ferret lateral geniculate nucleus (LGN), and in the formation of eye-specific laminae in other species. To understand the role of NO in the LGN, it is critical to fully characterize the pattern of brain nitric oxide synthase (bNOS) expression within the nucleus, including the phenotype of the neural elements that express it. We have examined the temporal and spatial pattern of bNOS expression in the ferret LGN during the first 6 weeks of postnatal development, and in the adult, by detecting bNOS with a monoclonal antibody as well as beta-nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. We have found that bNOS is expressed in neurons in the A laminae of the LGN as early as postnatal day 7 (P7), a time coincident with eye-specific segregation of retinal axons. This expression continues through P35, with peak somatodendritic expression at P21. Fluorescent double labeling using antibodies to bNOS and glutamic acid decarboxylase indicate that bNOS is expressed in gamma-aminobutyric acid-ergic interneurons within the A laminae. Electron microscopic examination of bNOS-labeled cells showed synaptic contacts from terminals with two distinct morphologic profiles. Expression of bNOS within interneurons that receive contacts from multiple sources indicates that the synaptic circuitry associated with bNOS activation and the potential targets of NO may be more complex than originally thought and supports a potential new role for interneurons as cellular intermediaries in the refinement of pathways in the LGN. Our findings broaden the window of time that bNOS may be active within the developing LGN, suggesting an expanded role for NO during early postnatal development.

Developmental regulation of brain nitric oxide synthase expression in the ferret thalamic reticular nucleus.

We have found that cells in the ferret thalamic reticular nucleus (TRN) express brain nitric oxide synthase (bNOS) in a transient pattern during early postnatal development. Similar to our previous findings in the lateral geniculate nucleus (LGN), bNOS expression in the TRN is first observed at postnatal day 7 (P7) and continues to P35. Quantitative measures show a significant change in the relative numbers of bNOS+ cells from P7-P35, and suggest there is a transition in morphology from a bipolar shape with two primary dendrites, to a more complex, multipolar arrangement. During TRN development, the pattern of bNOS expression shifts from the somatodendritic localization seen during the first postnatal month to expression within axon fibers in the adult. Expression of bNOS within TRN cells demonstrates an additional source of nitric oxide in the developing visual thalamus, perhaps indicating a common function for thalamic nitergic neurons as cellular mediators in the establishment of central topography both in the LGN and the TRN.