Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson’s disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.     

Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy

Alzheimer''s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo.     

Graph Based Study of Allergen Cross-Reactivity of Plant Lipid Transfer Proteins (LTPs) Using Microarray in a Multicenter Study

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.     

Serotypes and Clonal Diversity of Streptococcus pneumoniae Causing Invasive Disease in the Era of PCV13 in Catalonia,Spain

The aim of this study was to study the serotypes and clonal diversity of pneumococci causing invasive pneumococcal disease in Catalonia, Spain, in the era of 13-valent pneumococcal conjugate vaccine (PCV13). In our region, this vaccine is only available in the private market and it is estimated a PCV13 vaccine coverage around 55% in children. A total of 1551 pneumococcal invasive isolates received between 2010 and 2013 in the Molecular Microbiology Department at Hospital Sant Joan de Déu, Barcelona, were included. Fifty-two serotypes and 249 clonal types—defined by MLST—were identified. The most common serotypes were serotype 1 (n = 182; 11.7%), 3 (n = 145; 9.3%), 19A (n = 137; 8.8%) and 7F (n = 122; 7.9%). Serotype 14 was the third most frequent serotype in children < 2 years (15 of 159 isolates). PCV7 serotypes maintained their proportion along the period of study, 16.6% in 2010 to 13.4% in 2013, whereas there was a significant proportional decrease in PCV13 serotypes, 65.3% in 2010 to 48.9% in 2013 (p<0.01). This decrease was mainly attributable to serotypes 19A and 7F. Serotype 12F achieved the third position in 2013 (n = 22, 6.4%). The most frequent clonal types found were ST306 (n = 154, 9.9%), ST191 (n = 111, 7.2%), ST989 (n = 85, 5.5%) and ST180 (n = 80, 5.2%). Despite their decrease, PCV13 serotypes continue to be a major cause of disease in Spain. These results emphasize the need for complete PCV13 vaccination.     

Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer''s disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.     

Virological Efficacy in Cerebrospinal Fluid and Neurocognitive Status in Patients with Long-Term Monotherapy Based on Lopinavir/Ritonavir: An Exploratory Study

Background

Data on suppression of HIV replication in the CNS and on the subsequent risk of neurocognitive impairment using monotherapy with boosted protease inhibitors are limited.

Methods

Ours was an exploratory cross-sectional study in patients on lopinavir/ritonavir-based monotherapy (LPV/r-MT) or standard triple therapy (LPV/r-ART) for at least 96 weeks who maintained a plasma viral load <50 copies/mL. HIV-1 RNA in CSF was determined by HIV-1 SuperLow assay (lower limit of detection, 1 copy/mL). Neurocognitive functioning was assessed using a recommended battery of neuropsychological tests covering 7 areas. Neurocognitive impairment (NCI) was determined and also a global deficit score (GDS) for study comparisons.

Results

Seventeen patients on LPV/r-MT and 17 on LPV/r-ART were included. Fourteen (82.4%) patients on LPV/r-MT and 16 (94.1%) on LPV/r-ART had HIV-1 RNA <1 copy/mL in CSF (p = 0.601). NCI was observed in 7 patients on LPV/r-MT and in 10 on LPV/r-ART (41% vs 59%; p = 0.494). Mean (SD) GDS was 0.22 (0.20) in patients on LPV/r-MT and 0.47 (0.34) in those on LPV/r-ART (p = 0.012).

Conclusions

Suppression of HIV in CSF is similar in individuals with durable plasma HIV-1 RNA suppression who are receiving LPV/r-MT or LPV/r-ART for at least 96 weeks. Findings for HIV-1 replication in CSF and neurocognitive status indicate that this strategy seems to be safe for CNS functioning.     

CD8 T cells expressing NK associated receptors are increased in melanoma patients and display an effector phenotype

CD8⁺ T cells can express NK-associated receptors (NKRs) that may regulate their cytolytic function. We have characterized the expression of several NKRs on peripheral blood CD8⁺ T cells from melanoma patients and compared them to age-matched healthy donors. The analysis performed includes HLA class I specific receptors (KIRs, LILRB1 and CD94/NKG2) and other NK receptors like CD57, CD56 and CD16. Melanoma patients showed a higher variability in the expression of NKRs on circulating CD8⁺ T cells than age-matched healthy donors. NKR expression on CD8⁺ T cells from melanoma patients showed a significant increase of KIR2DL2/L3/S2 (mAb gl183), CD244, CD57, CD56 and CD16. We have also found an increase of CD8⁺ CD28^– CD27^– T cells in melanoma patients. This subset represents terminally differentiated effector cells expressing CD244 and high levels of perforin. The expression of NKRs was also mainly restricted to this T cell subset. Altogether, circulating CD8⁺ T cells from melanoma patients display a distinct phenotype characterized by downregulation of costimulatory molecules and higher expression of NKRs. We suggest that the increased expression of NKRs on T cells may contribute to the final outcome of the immune response against melanoma both stimulating or inhibiting activation and differentiation to effector cells. Blocking inhibitory receptor function and enhancing activating receptors may represent new strategies with therapeutic potential against melanoma.