Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic causes of Parkinson disease (PD) and genome-wide association studies identify LRRK2 sequence variants as risk factors for sporadic PD. Intact kinase function appears critical for the toxicity of LRRK2 PD mutants, yet our understanding of how LRRK2 causes neurodegeneration remains limited. We find that most LRRK2 PD mutants abnormally enhance LRRK2 oligomerization, causing it to form filamentous structures in transfections of cell lines or primary neuronal cultures. Strikingly, ultrastructural analyses, including immuno-electron microscopy and electron microscopic tomography, demonstrate that these filaments consist of LRRK2 recruited onto part of the cellular microtubule network in a well-ordered, periodic fashion. Like LRRK2-related neurodegeneration, microtubule association requires intact kinase function and the WD40 domain, potentially linking microtubule binding and neurodegeneration. Our observations identify a novel effect of LRRK2 PD mutations and highlight a potential role for microtubules in the pathogenesis of LRRK2-related neurodegeneration.

PKCθ plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-κB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCθ to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCθ, we addressed here the question of whether the translocation and activation of PKCθ in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCγ1-deficient T cell line, or a dominant negative PLCγ1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCθ are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCα. Membrane or lipid raft recruitment of PKCθ (but not PKCα) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCθ, but did not associate with it. These results provide evidence that a nonconventional PI3-K– and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCθ in T cells.

Modifications to the gene encoding human alpha-synuclein have been linked to development of Parkinson’s disease. The highly conserved structure of alpha-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human alpha-synuclein including new genetic tags developed for correlated LM and EM (the tetracysteine-biarsenical labeling system or the new fluorescent protein for EM, MiniSOG), we determined the distribution of alpha-synuclein when over-expressed in primary neurons at supramolecular and cellular scales, in three dimensions (3D). We observed specific association of alpha-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, alpha-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice over-expressing human alpha-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. 3D electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulo-vesicular structures similar to what observed in vitro. We propose that alpha-synuclein over-expression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that alpha- synuclein is involved in processes associated with the sorting, channeling, packaging and transport of synaptic material destined for degradation.

Protein kinase C-h (PKCh) plays an important role in T-cell activation via stimulation of AP-1 and NF-jB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCh-interacting kinase. SPAK interacted with PKCh (but not with PKCa) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCh, but not by PKCa. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCh-deficient T cells. Transfected SPAK synergized with constitutively active PKCh to activate AP-1, but not NF-jB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKChinteracting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAKspecific RNAi or a dominant-negative SPAK mutant inhibited PKCh-and TCR/CD28-induced AP-1, but not NF-jB, activation. These results define SPAK as a substrate and target of PKCh in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-jB) activation.

CD8+ T cells are crucial for host defense against invading pathogens and malignancies. However, relatively little is known about intracellular signaling events that control the genetic program of their activation and differentiation. Using CD8+ T cells from TCR-transgenic mice crossed to protein kinase C-θ (PKCθ)-deficient mice, we report that PKCθ is not required for Ag-induced CD8+ T cell proliferation, but is important for T cell survival and differentiation into functional, cytokine-producing CTLs. Ag-stimulated PKCθ−/− T cells underwent accelerated apoptosis associated with deregulated expression of Bcl-2 family proteins and displayed reduced activation of ERKs and JNKs. Some defects in the function of PKCθ−/− T cells (poor survival and reduced Bcl-2 and Bcl-xL expression, CTL activity, and IFN-γ expression) were partially or fully restored by coculture with wild-type T cells or by addition of exogenous IL-2, whereas others (increased BimEL expression and TNF-α production) were not. These findings indicate that PKCθ, although not essential for initial Ag-induced proliferation, nevertheless plays an important role in promoting and extending T cell survival, thereby enabling the complete genetic program of effector CD8+ differentiation. The requirement for PKCθ in different types of T cell-dependent responses may, therefore, depend on the overall strength of signaling by the TCR and costimulatory receptors and may reflect, in addition to its previously established role in activation, an important, hitherto unappreciated, role in T cell survival.