Several neurological diseases, includingThe importance of ␣-synuclein to the pathogenesis of Parkinson disease (PD) 4 and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1)(2)(3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4 -6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7-9), which increase levels of ␣-synuclein with no alteration in sequence, also cause PD or DLB.␣-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11,12). Phosphorylation at tyrosine residues has been observed by some investigators (13,14) but not by others (10 -12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress ␣-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of ␣-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate ␣-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an ␣-synuclein kinase. The ability of PLK2 to directly phosphorylate ␣-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates ␣-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brai...

The developing brain is uniquely susceptible to the neurotoxic hazard posed by mercurials. Host differences in maturation, metabolism, nutrition, sex, and autoimmunity influence outcomes. How population-based variability affects the safety of the ethylmercury-containing vaccine preservative, thimerosal, is unknown. Reported increases in the prevalence of autism, a highly heritable neuropsychiatric condition, are intensifying public focus on environmental exposures such as thimerosal. Immune profiles and family history in autism are frequently consistent with autoimmunity. We hypothesized that autoimmune propensity influences outcomes in mice following thimerosal challenges that mimic routine childhood immunizations. Autoimmune disease-sensitive SJL/J mice showed growth delay; reduced locomotion; exaggerated response to novelty; and densely packed, hyperchromic hippocampal neurons with altered glutamate receptors and transporters. Strains resistant to autoimmunity, C57BL/ 6J and BALB/cJ, were not susceptible. These findings implicate genetic influences and provide a model for investigating thimerosal-related neurotoxicity.

BackgroundCheckpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME).MethodsWe developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI.ResultsUsing a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs.ConclusionTaken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer.Statement of significanceCPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.

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Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.