Signal Transduction Protein Array Analysis Links LRRK2 to Ste20 Kinases and PKC Zeta That Modulate Neuronal Plasticity

substrate phosphorylation..Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations

Leucine-Rich Repeat Kinase 2 Influences Fate Decision of Human Monocytes Differentiated from Induced Pluripotent Stem Cells.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson's disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study, we investigated the effect of the LRRK2 (G2019S) mutation in monocytes, using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant, compared to non-mutant isogenic controls, leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines, demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells, compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision, endorsing the involvement of the immune system in the development of PD

LRRK2 and phospho-LRRK2 levels in iPSC-derived monocytes.

<p>(A) <i>LRRK2</i> mRNA expression relative to RNA polymerase II expression was determined using the 2(-Delta Delta C(T)) method. The relative expression is shown for non-mutant control (WT) and LRRK2 (G2019S) knock-in (GS) cells (n = 3). Data was normalized to the non-mutant control in week 12. No differences between the isogenic cell lines were detected (p > 0.05). (B) Densitometric analysis of immunoblots of three independent cultures per genotype reveal similar LRRK2 protein expression levels (p > 0.05). Error bars represent mean +SEM, *<i>p</i><0.05.</p

The Mycoplasma-Derived Macrophage-Activating 2-Kilodalton Lipopeptide Triggers Global Immune Activation on Nasal Mucosa-Associated Lymphoid Tissues

A better knowledge on how immune responses are initiated in mucosal tissues would facilitate the design of new mucosal vaccines, as well as improve our understanding on host defense against infection. We investigated the mechanisms of adjuvanticity of the Mycoplasma-derived macrophage-activating 2-kDa lipopeptide (MALP-2), which binds to the heterodimer formed by the Toll-like receptors 2 and 6 (TLR2 and -6), at the level of the murine nasal mucosa-associated lymphoid tissues (NALT). TLR2 expression analysis demonstrated that several cell types from the nasal cavity were able to overexpress this receptor, either constitutively (such as B cells) or after stimulation (i.e., T cells). MALP-2 stimulated a strong B-cell activation. In addition, the antigen presentation capacity of dendritic cells was improved after in vivo loading with antigen in the presence of MALP-2. We also observed an up-regulated expression of activation markers and adhesion molecules on T cells, suggesting that they have enhanced responsiveness and interaction potential. Quantitative reverse transcription-PCR analysis showed that MALP-2 administration resulted in the stimulation of a proinflammatory cascade. We observed an early up-regulated expression of IP-10, MCP-1, MCP-3, MIP-1α, MIP-2, and CCR-2 which was reversed within 36 h. The obtained results demonstrated that MALP-2 creates a reversible local microenvironment which promotes effective priming of T and B cells in the NALT

Functional analysis of iPSC-derived monocytes.

<p>(A) Release of the pro-inflammatory cytokine IFNγ showed a trend towards an increase after LPS stimulation (100 ng/ml for 6 hours) as measured by ELISA after 11 weeks of differentiation (n = 3). For IL-1β, IL-6 and TNFα the LPS-induced increase reached significance (IL-1β: p < 0.05; IL-6: p < 0.01; TNFα: p < 0.05). No difference was observed upon comparison of LRRK2 (G2019S) mutant (GS) versus non-mutant control (WT) monocytes. (B) <i>LRRK2</i> (G2019S) cells (GS) monocytes showed significantly reduced (p < 0.05) basal migration compared to control (WT) after 11–18 weeks of differentiation. The trend towards reduced migration upon exposure to the chemotactic stimulus 100 μM ATP did not reach significance (p < 0.05; n = 3).</p