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Summary Chronic lymphocytic leukaemia (CLL) is associated with alterations in T cell number, subset distribution and function. Among these changes, an increase in CD4+ T cells was reported. CD4+ T cells are a heterogeneous population and distinct subsets have been described to exert pro‐ and anti‐tumour functions. In CLL, controversial reports describing the dominance of IFNγ‐expressing Th1 T cells or of IL‐4‐producing Th2 T cells exist. Our study shows that blood of CLL patients is enriched in Th1 T cells producing high amounts of IFNγ. Moreover, we observed that their frequency remains relatively stable in CLL patients over a time course of five years. Furthermore, we provide evidence for an accumulation of Th1 T cells in the Eµ‐TCL1 mouse model of CLL. As TBET (encoded by Tbx21) is a crucial transcription factor for Th1 polarization, we generated Tbx21−/− bone marrow chimaeric mice which showed a lower number of IFNγ‐producing Th1 T cells, and used them for adoptive transfer of Eµ‐TCL1 leukaemia. Disease development in these mice was, however, comparable to that in wild‐type controls, excluding a major role for TBET‐expressing Th1 cells in Eµ‐TCL1 leukaemia. Collectively, our data highlight that Th1 T cells accumulate in CLL but reducing their number has no impact on disease development., publishedVersion

Classification and scoring of phospho-α-synuclein and PK-PET Blot. (A) Classification of phospho-α-synuclein staining into four different scores. Representative pictures for scoring. Score 0: no p-α-synuclein staining, score 1: little staining in brainstem and DpMe, score 2: strong staining in brainstem and DpMe, score 3: strong p-α-synuclein staining in brainstem, DpMe, and cerebellum indicating severe disease progression. (B) Scoring to classify PK-PET Blot. Score 0: no PK resistant aggregates, score 1: light aggregates in brainstem and Deep Mesencepahlic nucleus (DpMe), score 2: clear PK resistant aggregates in brainstem and DpMe, score 3: dominant aggregates in brainstem and DpMe. Score 4: Prominent aggregates in brainstem, DpMe and cerebellum. (PDF 125 kb)

Mating scheme for the generation of mouse cohorts. (A) Breeding scheme for generating 3rd generation telomerase knockout and α-synuclein transgenic mice (αSYNtg/tg G3Terc-/-) and corresponding control cohorts. Heterozygous α-synuclein (αSYN) mice were crossed with heterozygous telomerase knockout mice (Terc) to generate double transgenic αSYNtg/tg G1Terc-/- and single transgenic αSYNtg/tg, G1Terc-/- and Terc+/+ mice (G1 = first generation of telomerase knockout). These double transgenic αSYNtg/tg G1Terc-/- mice were crossed with each other to produce αSYNtg/tg G2Terc-/- mice and finally αSYNtg/tg G3Terc-/- mice. (B) Telomere length was measured in neurons of the brainstem using qFISH telomere staining double-stained with Cy5-Neuron N dye. The graph represents telomere length in brainstem in 75 weeks old mice (n = 5 mice per group). Analyzed were αSYNtg/tg G3Terc-/- in comparison to αSYNtg/tg mice (P = 0.0012) and G3Terc-/- in comparison to Terc+/+ mice (P = 0.0079) as well as αSYNtg/tg G3Terc-/- compared with G3Terc-/- (P = 0.03). (PDF 29 kb)

Exploratory behavior and string agility. Exploratory behavior of the mice has been analysed using the Open Field System. Several parameters, e.g. the activity, duration and the totally travelled distance were measured in three different zones of the open field: Border, intermediate and center of the open field. 7 to 10 mice of the indicated genotypes were recorded each for 10 min. (A) Activity in the open field and (B) duration of stay in the three indicated zones. αSYNtg/tg G3Terc-/- mice did not show a difference in comparison to αSYNtg/tg mice and the two control groups. Although a trend can be seen in duration, αSYNtg/tg and αSYNtg/tg G3Terc-/- stay even shorter in the intermediate and the center zone and prefer the border zone in comparison to Terc+/+ or G3Terc-/- mice. (C) String agility test shows no difference between the different groups. (D) Activated astrocytes were measured using GFAP immunohistochemistry. The histogram depicts GFAP positive astrocytes of the brainstem (n = 4–6 mice). αSYNtg/tg mice show a significant increase in astrocyte activation (62.35 ± 5.656) in comparison to αSYNtg/tg G3Terc-/- mice (39 ± 2.383, P = 0.0159). Furthermore, αSYNtg/tg mice with phenotype (85 weeks) show a significant activation (62.35 ± 5.65) if compared with non-phenotypic mice (75 weeks, 33.37 ± 4.086, P = 0.0095). Terc+/+ (14.3 ± 7.6) and G3Terc-/- mice (10.07 ± 7.10) show low activation levels. (PDF 33 kb)

Primer sequences which were used for RT-PCR. (DOC 40 kb)

Schematic representation of the parameter analyzed in 3D-reconstructed microglia. Automated analysis of each structural characteristic was performed using Imaris Bitplane software. (PDF 50 kb)

Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutations in DNA repair factors lead to accelerated and premature aging. However, the opposite, namely if improved DNA repair will also increase the life or health span is less clear, and only few studies have tested if overexpression of DNA repair factors modulates life and health span in cells or organisms. Recently, we identified and characterized SNEVhPrp19/hPso4, a protein that plays a role in DNA repair and pre-mRNA splicing, and observed a doubling of the replicative life span upon ectopic overexpression, accompanied by lower basal DNA damage and apoptosis levels as well as an increased resistance to oxidative stress. Here we find that SNEVhPrp19/hPso4 is phosphorylated at S149 in an ataxia telangiectasia mutated protein (ATM)-dependent manner in response to oxidative stress and DNA double strand break inducing agents. By overexpressing wild-type SNEVhPrp19/hPso4 and a phosphorylation-deficient point-mutant, we found that S149 phosphorylation is necessary for mediating the resistance to apoptosis upon oxidative stress and is partially necessary for elongating the cellular life span. Therefore, ATM dependent phosphorylation of SNEVhPrp19/hPso4 upon DNA damage or oxidative stress might represent a novel axis capable of modulating cellular life span.

OBJECTIVE: Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC-1α in mitochondria, no studies have addressed whether PGC-1α directly influences oligomerization of α-syn or whether α-syn oligomers impact PGC-1α expression. MATERIALS AND METHODS: We tested whether pharmacological or genetic activation of PGC-1α or PGC-11α knockdown could modulate the oligomerization of α-syn in vitro by using an α-syn -fragment complementation assay. RESULTS: In this study, we found that both PGC-1α reference gene (RG-PGC-1α) and the central nervous system (CNS)-specific PGC-1α (CNS-PGC-1α) are downregulated in human PD brain, in A30P α-syn transgenic animals, and in a cell culture model for α-syn oligomerization. Importantly, downregulation of both RG-PGC-1α and CNS-PGC-1α in cell culture or neurons from RG-PGC-1α-deficient mice leads to a strong induction of α-syn oligomerization and toxicity. In contrast, pharmacological activation or genetic overexpression of RG-PGC-1α reduced α-syn oligomerization and rescued α-syn-mediated toxicity. INTERPRETATION: Based on our results, we propose that PGC-1α downregulation and α-syn oligomerization form a vicious circle, thereby influencing and/or potentiating each other. Our data indicate that restoration of PGC-1α is a promising approach for development of effective drugs for the treatment of PD and related synucleinopathies. ispartof: Annals of Neurology vol:77 issue:1 pages:15-32 ispartof: location:United States status: published

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC-1α in mitochondria, no studies have addressed whether PGC-1α directly influences oligomerization of α-syn or whether α-syn oligomers impact PGC-1α expression.We tested whether pharmacological or genetic activation of PGC-1α or PGC-11α knockdown could modulate the oligomerization of α-syn in vitro by using an α-syn -fragment complementation assay.In this study, we found that both PGC-1α reference gene (RG-PGC-1α) and the central nervous system (CNS)-specific PGC-1α (CNS-PGC-1α) are downregulated in human PD brain, in A30P α-syn transgenic animals, and in a cell culture model for α-syn oligomerization. Importantly, downregulation of both RG-PGC-1α and CNS-PGC-1α in cell culture or neurons from RG-PGC-1α-deficient mice leads to a strong induction of α-syn oligomerization and toxicity. In contrast, pharmacological activation or genetic overexpression of RG-PGC-1α reduced α-syn oligomerization and rescued α-syn-mediated toxicity.Based on our results, we propose that PGC-1α downregulation and α-syn oligomerization form a vicious circle, thereby influencing and/or potentiating each other. Our data indicate that restoration of PGC-1α is a promising approach for development of effective drugs for the treatment of PD and related synucleinopathies.