Neuromelanin activates proinflammatory microglia through a caspase-8-dependent mechanism

Background We have uncovered a caspase-dependent (caspase-8/caspase-3/7) signaling governing microglia activation and associated neurotoxicity. Importantly, a profuse non-nuclear activation of cleaved caspases 8 and 3 was found in reactive microglia in the ventral mesencephalon from subjects with Parkinson’s disease, thus supporting the existence of endogenous factors activating microglia through a caspase-dependent mechanism. One obvious candidate is neuromelanin, which is an efficient proinflammogen in vivo and in vitro and has been shown to have a role in the pathogenesis of Parkinson’s disease. Consequently, the goal of this study is to test whether synthetic neuromelanin activates microglia in a caspase-dependent manner. Results We found an in-vivo upregulation of CD16/32 (M1 marker) in Iba1-immunolabeled microglia in the ventral mesencephalon after neuromelanin injection. In vitro experiments using BV2 cells, a microglia-derived cell line, demonstrated that synthetic neuromelanin induced a significant chemotactic response to BV2 microglial cells, along with typical morphological features of microglia activation, increased oxidative stress and induction of pattern-recognition receptors including Toll-like receptor 2, NOD2, and CD14. Analysis of IETDase (caspase-8) and DEVDase (caspase-3/7) activities in BV2 cells demonstrated a modest but significant increase of both activities in response to neuromelanin treatment, in the absence of cell death. Conclusions Caspase-8 inhibition prevented typical features of microglia activation, including morphological changes, a high rate of oxidative stress and expression of key proinflammatory cytokines and iNO

Accumulation of Progerin Affects the Symmetry of Cell Division and Is Associated with Impaired Wnt Signaling and the Mislocalization of Nuclear Envelope Proteins

Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/beta-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of beta-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS.Peer reviewe

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1.

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans

At a glance:the largest Niemann-Pick type C1 cohort with 602 patients diagnosed over 15 years

Niemann-Pick type C1 disease (NPC1 [OMIM 257220]) is a rare and severe autosomal recessive disorder, characterized by a multitude of neurovisceral clinical manifestations and a fatal outcome with no effective treatment to date. Aiming to gain insights into the genetic aspects of the disease, clinical, genetic, and biomarker PPCS data from 602 patients referred from 47 countries and diagnosed with NPC1 in our laboratory were analyzed. Patients’ clinical data were dissected using Human Phenotype Ontology (HPO) terms, and genotype–phenotype analysis was performed. The median age at diagnosis was 10.6 years (range 0–64.5 years), with 287 unique pathogenic/likely pathogenic (P/LP) variants identified, expanding NPC1 allelic heterogeneity. Importantly, 73 P/LP variants were previously unpublished. The most frequent variants detected were: c.3019C &gt; G, p.(P1007A), c.3104C &gt; T, p.(A1035V), and c.2861C &gt; T, p.(S954L). Loss of function (LoF) variants were significantly associated with earlier age at diagnosis, highly increased biomarker levels, and a visceral phenotype (abnormal abdomen and liver morphology). On the other hand, the variants p.(P1007A) and p.(S954L) were significantly associated with later age at diagnosis (p &lt; 0.001) and mildly elevated biomarker levels (p ≤ 0.002), consistent with the juvenile/adult form of NPC1. In addition, p.(I1061T), p.(S954L), and p.(A1035V) were associated with abnormality of eye movements (vertical supranuclear gaze palsy, p ≤ 0.05). We describe the largest and most heterogenous cohort of NPC1 patients published to date. Our results suggest that besides its utility in variant classification, the biomarker PPCS might serve to indicate disease severity/progression. In addition, we establish new genotype–phenotype relationships for “frequent” NPC1 variants.</p

No signs of increased apoptosis in the vascular smooth muscle cells of the aortic arch.

<p>Immunohistochemical sections from the aortic arches of wild-type (A) and bi-transgenic tetop-LA^G608G+; sm22α-rtTA⁺ (B) mice supplied with doxycycline from the date of birth to postnatal week 12, stained with an anti-Cleaved Caspase 3 antibody (Asp 175) to highlight apoptotic cells. (C) A section of the ovary from a wild-type animal used as a positive control tissue for apoptotic cells. Arrows indicate apoptotic cells. Scale bars: 100 µm.</p

Expression of mouse lamin A/C and sm22α-actin is unaffected in the aortic arch.

<p>Immunofluorescence staining with an anti-human-lamin A/C antibody (N-18), which also binds to progerin of human origin and lamin A/C of mouse origin, and an antibody for vascular smooth muscle cells (1A4) in wild-type (A–C) and bi-transgenic tetop-LA^G608G+; sm22α-rtTA⁺ animals (D–F). Representative images from the sections of aortic arches of mice with the C57BL/6J; FVB/NCrl genetic background supplied with doxycycline from the date of birth until postnatal week 12. Scale bars: 50 µm. C, F: merge of the lamin A/C, sm22α-actin, and DAPI fluorescence signals. (G–J) Histological examination of aortic sections with haematoxylin eosin staining shows normal structure of the aorta. G, I: aortic arch. H, J: thoraic aorta. Scale bars: 100 µm.</p

Genotype frequencies.

<p>D0, mice supplied with doxycycline from the date of birth; E0, mice supplied with doxycycline during embryogenesis and postnatally; +, presence of transgene; −, absence of transgene. The expected frequency for each individual genotype was 25%.</p

Low levels of transgene expression in the aortic arch.

<p>(A–C) RT-PCR using mRNA from the aortic arch showed very weak amplification products for human lamin A, lamin Adel150 and the reverse transactivator after 35 cycles of PCR. (A, C) RT-PCR on samples from bi-transgenic mice encoding both the reverse transactivator (sm22α-rtTA⁺) and the lamin A minigene (tetop-LA^G608G+) that were supplied with doxycycline from the date of birth until postnatal week 4 (Dox D0, lanes 3–4) or 12 (Dox D0, lanes 7–8), or supplied with dox during embryogenesis and postnatally for 4 weeks (Dox E0, lanes 11–12). (A) C57BL/6J genetic background. (C) C57BL/6J; FVB/NCrl mixed genetic background. (B) RT-PCR for human lamin A and lamin Adel150 in samples from transactivator negative control animals (tetop-LA^G608G+; sm22α-rtTA⁻, lanes 1–6) supplied with doxycycline from the date of birth until postnatal week 4 (Dox D0, lanes 1 and 2, C57BL/6J and C57BL/6J; FVB/NCrl genetic background, respectively) or week 12 (Dox D0, lanes 3 and 4, C57BL/6J and C57BL/6J; FVB/NCrl genetic background, respectively), or that were supplied with doxycycline during embryogenesis and postnatally for 4 weeks (Dox E0, lanes 5 and 6, C57BL/6J and C57BL/6J; FVB/NCrl genetic background, respectively). Bt, bi-transgenic. Wt, wild-type. NC, control with no template. DT, sample from a different transactivator strain was used as a control for the PCR assay and showed amplification for human lamin A and lamin Adel150 with cDNA from the bone of bi-transgenic tetop-LA^G608G+; Sp7-tTA⁺ mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104098#pone.0104098-Schmidt1" target="_blank">[18]</a>. Genomic DNA from a tetop-LA^G608G+; sm22α-rtTA⁺ bi-transgenic animal was used as a positive control for the amplification of the reverse transactivator (350 base pair product) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104098#pone.0104098-BernalMizrachi1" target="_blank">[15]</a>. The RT-PCR results for β-actin served as a control. (D) Western blot analysis on protein extracts from pooled aortic regions from bi-transgenic tetop-LA^G608G+; sm22α-rtTA⁺ (lane 2) and tetop-LA^G608G+; NSE-tTA⁺ (lane 3) animals did not show transgenic expression of human lamin A and progerin. A single transgenic tetop-LA^G608G−; sm22α-rtTA⁺ animal carrying only the transactivator was used as a negative control (lane 4). Protein extract from HGPS patient cell line AG11513A was used as positive control (lane 1). (E) Western blot analysis on protein extracts from pooled aortic regions from bi-transgenic tetop-LA^G608G+; sm22α-rtTA⁺ (lanes 2 and 5) and single transgenic animals, tetop-LA^G608G−; NSE-tTA⁺ (lane 3) and tetop-LA^G608G−; sm22α-rtTA⁺ (lane 4). Protein extracts from wild-type tissue, tetop-LA^G608G−; NSE-tTA⁻, was used as a negative control (lane 1). (F–G and J–K) Very few transgene positive cells, <1%, were detected in the aortic arches of bi-transgenic animals at postnatal week 12 using antibodies specific for human lamin A/C and progerin (JoL2) (F–G), and human progerin (13A4) (J–K). (H–I and L–M) Positive staining was obtained using the same antibodies, on sections of the aortic arch from tetop-LA^G608G+; NSE-tTA+ bi-transgenic mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104098#pone.0104098-Chen1" target="_blank">[17]</a>. (N–O and R–S) Very few transgene protein positive cells, <1%, were detected in the aortic arches of adult bi-transgenic animals, not supplied with doxycycline for the last 4 weeks prior to sacrifice, using antibodies for human lamin A/C and progerin (JoL2) (N–O), and human progerin (13A4) (R–S). (P–Q and T–U) Almost no positive staining was obtained using the same antibodies, on sections of the aortic arch from tetop-LA^G608G+; NSE-tTA+ bi-transgenic mice supplied with doxycycline for 3 weeks (indicating a significant down-regulation of the transgenic expression with the doxycycline supplement). G, I, O, Q: merge of the transgenic lamin A and progerin with DAPI fluorescence signals. K, M, S, U: merge of the progerin and DAPI fluorescence signals. Scale bars: 10 µm.</p

TSPYL5 depletion induces specific death of ALT cells through USP7-dependent proteasomal degradation of POT1

A significant fraction (~10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as Alternative Lengthening of Telomeres(ALT). Currently, there are no known molecular, ALT-specific, therapeutic target. We have identified TSPYL5 (Testis Specific Y-encoded-Like Protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1 – a shelterin component – and protects POT1 from proteasomal degradation, exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies, and identify TSPYL5 as a ALT+ cancer-specific therapeutic target