α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α.

α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism but also by their secretion propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the nerve growth factor-differentiated catecholaminergic PC12 neuronal cell line, which was conferred α-synucleinopathy by inducible expression of αSyn and tubulin polymerization-promoting protein p25α.

Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis.

Background: Secretion of proteopathic α-synuclein (α-SNC) species from neurons is a suspected driving force in the propagation of Parkinson's disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of α-SNC secretion in differentiated PC12 or SH-SY5Y nerve cells. Here we have examined the regulation of exophagy associated with different forms of nerve cell stress relevant to PD.

Macropinocytosis is the entry mechanism of amphotropic murine leukemia virus.

Unlabelled: The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV is internalized not by caveolae or other pinocytic mechanisms but by macropinocytosis. Thus, A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, and NIH 3T3 cells knocked down for caveolin expression, was unaffected.

Tubulin polymerization-promoting protein (TPPP/p25α) promotes unconventional secretion of α-synuclein through exophagy by impairing autophagosome-lysosome fusion.

Aggregation of α-synuclein can be promoted by the tubulin polymerization-promoting protein/p25α, which we have used here as a tool to study the role of autophagy in the clearance of α-synuclein. In NGF-differentiated PC12 catecholaminergic nerve cells, we show that de novo expressed p25α co-localizes with α-synuclein and causes its aggregation and distribution into autophagosomes. However, p25α also lowered the mobility of autophagosomes and hindered the final maturation of autophagosomes by preventing their fusion with lysosomes for the final degradation of α-synuclein.

Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia.

Background: Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton.

Results: Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity.

Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis.

The p21-activated kinase-1 (PAK1) is best known for its role in the regulation of cytoskeletal and transcriptional signaling pathways. We show here in the microglia cell line Ra2 that PAK1 regulates NADPH oxidase (NOX-2) activity in a stimulus-specific manner. Thus, conditional expression of PAK1 dominant-positive mutants enhanced, whereas dominant-negative mutants inhibited, NADPH oxidase-mediated superoxide generation following formyl-methionyl-leucylphenylalanine or phorbol 12-myristate 13-acetate stimulation.