Amyloid-like fibrillar aggregates of intracellular proteins are common pathological features of human neurodegenerative diseases. However, the nature of pathogenic aggregates and the biological consequences of their formation remain elusive. Here, we describe (i) a model cellular system in which prefibrillar ␣-synuclein aggregates and fibrillar inclusions are naturally formed in the cytoplasm with distinctive kinetics and (ii) a tight correlation between the presence of prefibrillar aggregates and the Golgi fragmentation. Consistent with the structural abnormality of Golgi apparatus, trafficking and maturation of dopamine transporter through the biosynthetic pathway were impaired in the presence of ␣-synuclein aggregates. Reduction in cell viability was also observed in the prefibrillar aggregate-forming condition and before the inclusion formation. The fibrillar inclusions, on the other hand, showed no correlation with Golgi fragmentation and were preceded by these events. Furthermore, at the early stage of inclusion formation, active lysosomes and mitochondria were enriched in the juxtanuclear area and co-aggregate into a compact inclusion body, suggesting that the fibrillar inclusions might be the consequence of an attempt of the cell to remove abnormal protein aggregates and damaged organelles. These results support the hypothesis that prefibrillar ␣-synuclein aggregates are the pathogenic species and suggest that Golgi fragmentation and subsequent trafficking impairment are the specific consequence of ␣-synuclein aggregation.

Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in Parkinson’s disease (PD). Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD, the mechanism by which α-synuclein aggregates spread remains undefined. Here, we show that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates, co-aggregation with endogenous α-synuclein, and exocytosis of the co-aggregates. Moreover, we found that glucocerebrosidase depletion, which has previously been strongly associated with PD and increased cognitive impairment, promoted propagation of α-synuclein aggregates. These studies define how α-synuclein aggregates spread among neuronal cells and explain how glucocerebrosidase mutations increase the risk of developing PD and other synucleinopathies.

The generation of reactive oxygen species (ROS) in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the Rac protein. We report here that the COOHterminal region of Nox1, a protein related to gp91 phox (Nox2) of phagocytic cells, is constitutively associated with ␤Pix, a guanine nucleotide exchange factor for Rac. Both growth factor-induced ROS production and Rac1 activation were completely blocked in cells depleted of ␤Pix by RNA interference. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Moreover, the depletion of Nox1 by RNA interference inhibited growth factor-induced ROS generation. These results suggest that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, ␤Pix, and Rac1, which then binds to Nox1 to stimulate its NADPH oxidase activity.Reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide (H 2 O 2 ), are produced in mammalian cells in response to the activation of various cell surface receptors and contribute to intracellular signaling and to the regulation of various biological activities, including host defense and metabolic conversion (15,23,35). Receptor-mediated ROS production has been studied extensively in phagocytic cells. The enzyme NADPH oxidase of such cells is composed of at least five protein components, namely two transmembrane flavocytochrome b components (gp91 phox and p22 phox ) and three cytosolic components (p47 phox , p67 phox , and p40 phox ) (2). The exposure of resting phagocytic cells to an appropriate stimulus results in extensive phosphorylation of the cytosolic components of NADPH oxidase and their association with the transmembrane flavocytochrome b components (1,11,33,36). The assembled oxidase complex catalyzes the transfer of an electron to molecular oxygen to yield the superoxide anion, which is then spontaneously or enzymatically converted to H 2 O 2 . The small GTPase Rac is also required for the activation of NADPH oxidase in phagocytic leukocytes (12, 28). Hematopoietic cell-specific Rac2 and the ubiquitously expressed protein Rac1 are the major and minor Rac isoforms, respectively, in human neutrophils (19).Nonphagocytic cells also produce superoxide anions in response to a variety of extracellular stimuli, including plateletderived growth factor (PDGF) and epidermal growth factor (EGF) (3, 5, 35, 38) Several homologs (Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2) of gp91 phox (Nox2) have been identified in various nonphagocytic cells (8,13,21,23,37). Nox proteins contain binding sites for FAD, NADPH, and heme, and their NH 2 -terminal portions contain a cluster of six hydrophobic segments that are predicted to form transmembrane ␣ helices (23). Some of the gp91 phox homologs likely associate with p22 phox to form functional cytochrome b in nonphagocytes, given that the latter protein is widely expressed (8) and that p22 phox antisense RNA was shown to inhibit angiotensin II-induced ROS gener...

A single amyloidogenic protein is implicated in multiple neurological diseases and capable of generating a number of aggregate “strains” with distinct structures. Among the amyloidogenic proteins, α-synuclein generates multiple patterns of proteinopathies in a group of diseases, such as Parkinson disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, the link between specific conformations and distinct pathologies, the key concept of the strain hypothesis, remains elusive. Here we show that in the presence of bacterial endotoxin, lipopolysaccharide (LPS), α-synuclein generated a self-renewable, structurally distinct fibril strain that consistently induced specific patterns of synucleinopathies in mice. These results suggest that amyloid fibrils with self-renewable structures cause distinct types of proteinopathies despite the identical primary structure and that exposure to exogenous pathogens may contribute to the diversity of synucleinopathies.

Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, ␤-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrixassisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of ␤-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified ␤-actin potently inhibited both phosphatidylinositol-4,5-bisphosphateand oleate-dependent PLD2 activities in a concentrationdependent manner (IC 50 ‫؍‬ 5 nM). In a previous paper, we reported that ␣-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 In vitro binding analyses showed that ␤-actin could displace ␣-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of ␤-actin on PLD2. We also observed that ␤-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, ␤-actin and ␣-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions. Mammalian phospholipase D (PLD)1 hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid and choline in response to a variety of signals, which can include hormones, neurotransmitters, and growth factors (1). phosphatidic acid itself has been shown to be an intracellular lipid second messenger and to be involved in multiple physiological events such as the promotion of mitogenesis, stimulation of respiratory bursts, secretory processes, actin cytoskeletal reorganization, and the activation of Raf-1 kinase and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinase isoforms in a large number of cells. These relationships suggest that agonist-induced PLD activation may play roles in multiple signaling events (2-7). The mammalian PLD isozymes identified thus far, PLD1 and PLD2, share a sequence homology of ϳ50%, but they have very different regulatory properties. PLD1 has low basal activity in the presence of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and can be activated by several cytosolic factors including protein kinase C ␣ and small GTP-binding proteins such as Rho A, Rac-1, ARF1, RalA, and CDC42 (8 -15). PLD2 also depends on PIP 2 but has a higher basal activity than PLD1 (16), and it has been proposed that PLD2 may be closely associated with different cellular inhibitors. Alth...