High LRRK2 Levels Fail to Induce or Exacerbate Neuronal Alpha-Synucleinopathy in Mouse Brain

The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson’s disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy

Inhibitory activity of myelin-associated glycoprotein on sensory neurons is largely independent of NgR1 and NgR2 and resides within Ig-Like domains 4 and 5.

Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-like lectin (Siglec) which has been characterized as potent myelin-derived inhibitor of neurite outgrowth. Two members of the Nogo-receptor (NgR) family, NgR1 and NgR2, have been identified as neuronal binding proteins of MAG. In addition, gangliosides have been proposed to bind to and confer the inhibitory activity of MAG on neurons. In this study, we investigated the individual contribution of NgRs and gangliosides to MAG-mediated inhibition of sensory neurons derived from dorsal root ganglia (DRG) of ngr1, ngr2 or ngr1/ngr2 deletion mutants. We found no disinhibition of neurite growth in the absence of either NgR1 or NgR2. Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth. If treated with Vibrio cholerae neuraminidase (VCN), inhibition by MAG is further attenuated but still not annulled. Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG. Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity. These findings provide new insights into the inhibitory function of MAG and suggest similarities but also major differences in MAG inhibition between sensory and central nervous system (CNS) neurons

IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling.

Signaling through innate immune receptors such as the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily proceeds via the assembly of large membrane-proximal complexes or "signalosomes." Although structurally distinct, the IL-17 receptor family triggers cellular responses that are typical of innate immune receptors. The IL-17RA receptor subunit is shared by several members of the IL-17 family. Using a combination of crystallographic, biophysical, and mutational studies, we show that IL-17A, IL-17F, and IL-17A/F induce IL-17RA dimerization. X-ray analysis of the heteromeric IL-17A complex with the extracellular domains of the IL-17RA and IL-17RC receptors reveals that cytokine-induced IL-17RA dimerization leads to the formation of a 2:2:2 hexameric signaling assembly. Furthermore, we demonstrate that the formation of the IL-17 signalosome potentiates IL-17-induced IL-36γ and CXCL1 mRNA expression in human keratinocytes, compared with a dimerization-defective IL-17RA variant

Transgenic expression of beta 1 antibody in brain neurons impairs age-dependent amyloid deposition in APP23 mice

Heterologous expression of the functional amyloid beta (A beta) antibody beta 1 in the central nervous system was engineered to maximize antibody exposure in the brain and assess the effects on A beta production and accumulation in these conditions. A single open reading frame encoding the heavy and light chains of beta 1 linked by the mouth and foot virus peptide 2A was expressed in brain neurons of transgenic mice. Two of the resulting BIN66 transgenic lines were crossed with APP23 mice, which develop severe central amyloidosis. Brain concentrations at steady-state 5 times greater than those found after peripheral beta 1 administration were obtained. Similar brain and plasma beta 1 concentrations indicated robust antibody efflux from the brain. In preplaque mice, beta 1 formed a complex with A beta that caused a modest A beta increase in brain and plasma. At 11 months of age, beta 1 expression reduced amyloid by 97% compared with age-matched APP23 mice. Interference of beta 1 with beta-secretase cleavage of amyloid precursor protein was relatively small. Our data suggest that severely impaired amyloid formation was primarily mediated by a complex of beta 1 with soluble A beta, which might have prevented A beta aggregation or favored transport out of the brain. (C) 2013 Elsevier Inc. All rights reserved

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

Putting the brakes on demyelination: Fingolimod (FTY720) was recently shown to significantly decrease relapse rates in patients with multiple sclerosis. This drug attenuates the trafficking of harmful T-cells entering the brain by regulating sphingosine-1-phosphate (S1P) receptors. We designed, synthesized, evaluated 2H-phthalazin-1-one derivatives (e.g., 1 L) as selective S1P5 receptor agonists; these compounds are highly potent and selective, with good PK properties, and significant activity in oligodendrocytes

ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Using both pharmacological and genetic approaches we report here that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Using mutational analysis, we demonstrate that the main cleavage site of the sheddases is located within the stalk region of TREM2 proximal to the plasma membrane. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimer’s disease. O-glycosylation is detected within the stalk region, but distant to the cleavage site. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered

High LRRK2 transgene levels do not exacerbate α-synuclein-driven phenotypes.

<p>(A) Schematic representation of the four different transgenic lines used to generate double transgenics. (B) 3-Step accelerated rotarod performance of females and males comparing single and double transgenics. The different genotypes and the number of mice per genotype are indicated. p-values were determined by repeated measures ANOVA (group effects for the respective panels: 1: F(1,22) = 0.483, p = 0.494; 2: F(1,26) = 0.000, p = 0.983; 3: F(1,11) = 0.738, p = 0.409; 4: F(1,22) = 2.048, p = 0.166; 5: F(1,16) = 1.255, p = 0.279; 6: F(1,27) = 5.171, p = 0.031). (C) Kaplan-Meier curves showing the time-of-sacrifice when mice had to be killed because of too severe motor deficits (1 = 100% and 0 = 0% of mice alive). The different genotypes, gender, number of mice per genotype and the p-values (nonparametric Kaplan-Meier) are indicated.</p

aSN and phospho-S129-aSN protein levels in spinal cord and forebrain of end-stage disease single and double transgenic mice.

<p>Tris-soluble and -insoluble fractions of spinal cord and forebrain lysates were immunoblotted and stained with antibodies detecting total α-synuclein (aSN) or specifically phosphorylated S129-aSN (paSN). β-actin (βAc) levels were measured as loading control and for normalization. For reference, LRRK2 levels detected via immunoblot are shown comparing single and double-transgenics. Different α-synuclein protein species/forms are marked as follows: mo, monomer; ol, oligomer; tr, truncated. For reference, in the upper panels the performance and specificity of the antibodies are illustrated in the two right lanes comparing WT and KO (aSN knock-out) brain samples and were added to indicate unspecific cross-reactive proteins (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone.0036581.s007" target="_blank">Figure S7</a>). Graphs represent quantifications of monomeric aSN and paSN/aSN, all normalized to βAc. Circles represent individual mice, the means are indicated as horizontal bars and % are normalized to the levels in haSN(A53T) single transgenics. p-values were determined by two-tailed, unequal variances Student’s t-test. Genotypes: aSN = haSN(A53T), aSN/LRRK2 = haSN(A53T)/hLRRK2(G2019S), Ntg = non-transgenic wildtype littermate control and KO = aSN knock-out mice.</p

Motor assessment and aSN/Tau protein characterization in hLRRK2(G2019S) mice.

<p>(A) Motor skill learning of 4-month-old male and 6-month-old female hLRRK2(G2019S) and Ntg controls in the 3-step accelerated rotarod task over four consecutive days. The number of mice per genotype is indicated. Three batches of animals were included in this graph (single transgenic and Ntg animals from experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone-0036581-g003" target="_blank">Figure 3B</a> as well as a separate batch). p-values were determined by repeated measure ANOVA (group effect males: F(1,119) = 9.42, p = 0.003, group effect females: F(1,52) = 3.74, p = 0.059). (B) Novelty-induced horizontal locomotor activity of 7.3- and 28.2-month-old hLRRK2(G2019S) and Ntg mice. Bar graphs show the sum of the distance travelled from 5–30 min and from 35–60 min. The number of mice per genotype is indicated. p-values were determined either by repeated measure ANOVA (group effect males 7.3 M: F(1,16) = 4.044, p = 0.061; group effect males 28.2 M: F(1,16) = 0.093, p = 0.764) or by two-tailed, unequal variances Student’s t-test. (C) Western blotting of forebrain homogenates from 15-month-old hLRRK2(G2019S) (TG) and Ntg male mice. Lower panel: Shown are levels of mouse α-synuclein (aSN) and phospho-α-synuclein Ser129 (paSN) as well as mouse microtubule-associated protein Tau and phospho-Tau Ser202/Thr205 (pTau). β-actin (βAc) was used as loading control and for normalization. Upper panel shows the results of the immunoblot quantifications. Circles represent individual mice, the means (% normalized to Ntg) are indicated as horizontal bars. p-values were determined by two-tailed, unequal variances Student’s t-test. Ntg: non-transgenic wildtype littermate control.</p

Microgliosis in end-stage haSN(A53T) transgenic mouse brain is unaltered by high LRRK2 levels.

<p>DAB-immunohistochemistry for Iba1 shows activated microglia on a representative sagittal brain section of a haSN(A53T) mouse (A and 20×higher magnification from brainstem in B) and a haSN(A53T)/hLRRK2(G2019S) double transgenic mouse (C and 20×higher magnification from brainstem in D). (E) Quantification of the brainstem results. Values represent % of the area in the brainstem that is covered by Iba1-positive microglia. p-value (p = 0.179) was determined by two-tailed, unequal variances Student’s t-test. Dots represent quantifications of single individuals. Control images obtained from a separate experiment but from littermate hLRRK2(G2019S) single transgenic (F and 20×higher magnification from brainstem in G) and from non-transgenic wildtype littermate control (Ntg) (H and 20×higher magnification from brainstem in I) mouse.</p