Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative diseases with age as the greatest risk factor. As the general population experiences extended life span, preparation for the prevention and treatment of these and other age-associated neurological diseases are warranted. Since epidemiological studies suggested that non-steroidal anti-inflammatory drug (NSAID) use decreased risk for AD and PD, increasing attention has been devoted to understanding the costs and benefits of the innate neuroinflammatory response to functional recovery following pathology onset. This review will provide a general overview on the role of neuroinflammation in these neurodegenerative diseases and an update on NSAID treatment in recent experimental animal models, epidemiological analyses, and clinical trials.

No abstract

We recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCa-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZinteracting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKCa knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

Abbreviations: CtBP2: C-terminal binding protein 2; GCL: ganglion cell layer; GPR179: G protein-coupled receptor 179; INL: inner nuclear layer; IPL: inner plexiform layer; LRR: leucinerich repeat; OPL: outer plexiform layer; PKCα: protein kinase C-alpha; RBC: rod bipolar cell; TPBG: trophoblast glycoprotein; TRPM1: transient receptor potential cation channel subfamily M member 1 Abstract We recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCa-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZinteracting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKCa knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.SignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.HighlightsPKCα is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.Proteins showing differential phosphorylation between phorbol ester-treated wild type and PKCα-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).The PKCα-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKCα-dependent modulation of RBC physiology.

Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs. We now examine the effects on RBC physiology and retinal processing of TPBG genetic deletion in mice using immunofluorescence and electrophysiological techniques. The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG deletion was observed on RBC morphology, the localization of RBC signal transduction proteins, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Time-resolved capacitance measurements indicated that TPBG deletion reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents. Thus, TPBG is required for efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology.