Contribution of serine racemase/d-serine pathway to neuronal apoptosis

Recent data indicate that age-related N-methyl-d-aspartate receptor (NMDAR) transmission impairment is correlated with the reduction in serine racemase (SR) expression and d-serine content. As apoptosis is associated with several diseases and conditions that generally occur with age, we investigated the modulation of SR/d-serine pathway during neuronal apoptosis and its impact on survival. We found that in cerebellar granule neurons (CGNs), undergoing apoptosis SR/d-serine pathway is crucially regulated. In the early phase of apoptosis, the expression of SR is reduced, both at the protein and RNA level through pathways, upstream of caspase activation, involving ubiquitin proteasome system (UPS) and c-Jun N-terminal kinases (JNKs). Forced expression of SR, together with treatment with NMDA and d-serine, blocks neuronal death, whereas pharmacological inhibition and Sh-RNA-mediated suppression of endogenous SR exacerbate neuronal death. In the late phase of apoptosis, the increased expression of SR contribute to the last, NMDAR-mediated, wave of cell death. These findings are relevant to our understanding of neuronal apoptosis and NMDAR activity regulation, raising further questions as to the role of SR/d-serine in those neuro-pathophysiological processes, such as aging and neurodegenerative diseases characterized by a convergence of apoptotic mechanisms and NMDAR dysfunction.     

Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER

Several regulators of endoplasmic reticulum (ER)-associated degradation (ERAD) have a shorter half-life compared to conventional ER chaperones. At steady state, they are selectively removed from the ER by poorly defined events collectively referred to as ERAD tuning. Here we identify the complex comprising the type-I transmembrane protein SEL1L and the cytosolic protein LC3-I as an ERAD tuning receptor regulating the COPII-independent, vesicle-mediated removal of the lumenal ERAD regulators EDEM1 and OS-9 from the ER. Expression of?folding-defective polypeptides enhances the lumenal content of EDEM1 and OS-9 by inhibiting their SEL1L:LC3-I-mediated segregation. This raises ERAD activity in the absence of UPR-induction. The mouse hepatitis virus (MHV) subverts ERAD tuning for replication. Consistently, SEL1L or LC3 silencing impair the MHV life cycle. Collectively, our data provide new molecular information about the ERAD tuning mechanisms that regulate ERAD in mammalian cells at the post translational level and how these mechanisms are hijacked by a pathogen.     

A role for Atg8-PE deconjugation in autophagosome biogenesis

Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8-PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8-PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8-PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.     

Modulation of urinary peptidome in humans exposed to high altitude hypoxia

The exposure of healthy subjects to high altitude represents a model to explore the pathophysiology of diseases related to tissue hypoxia and to evaluate pharmacological approaches potentially useful as a therapy for chronic diseases related to hypoxia. We explored the urinary peptidome to detect alterations induced by the exposure of subjects to different altitudes (sea level, high altitude = 3500 m, very high altitude = 5400 m) and to pharmacological treatment. Urine samples were collected from 47 subjects, randomly and blindly assigned to placebo (n = 24) or Telmisartan (n = 23). Samples were purified by the use of magnetic beads, then analysed by MALDI-TOF MS. Results showed that the urinary peptidome is not affected by the administration of Telmisartan, neither at the sea level nor at high and very high altitudes. In contrast, the urinary protein profiles are modified when subjects are exposed to high and very high altitudes, and we detected six peptides differentially expressed in hypobaric hypoxia at high or very high altitude compared to the sea level. Two of them were identified as fragments of the glycoprotein uromodulin and of the α1-antitrypsin. This is the first proteomic study showing that hypobaric hypoxia conditions affect the urinary peptidome.     

Distinct localization of T cell Agrin during antigen presentation--evidence for the expression of Agrin receptor(s) in antigen-presenting cells

Agrin is over-expressed by activated and autoimmune T cells, and synergizes with the T cell receptor (TCR) to augment cell activation. In the present study, we show that Agrin accumulates to distinct areas of the plasma membrane and that cell activation causes its redistribution. During antigen presentation, Agrin primarily accumulates to the periphery of the mature immunological synapse, mostly in lamellipodia-like protrusions that wrap around the antigen-presenting cell and, conversely, anti-Agrin sera induced a significant redistribution of TCR at the plasma membrane. We also provide evidence for the expression of Agrin receptors in peripheral blood monocytes, dendritic cells and a fraction of B cells. Interestingly, interferon-α treatment, which induces the expression of Agrin in T cells, also augmented Agrin binding to monocytes. Stimulation of monocytes with recombinant Agrin induced the clustering of surface receptors, including major histocompatibility complex class II, activation of intracellular signalling cascades, as well as enhanced dsRNA-induced expression of pro-inflammatory cytokines interleukin-6 and tumour necrosis factor-α. Collectively, these results confirm the location of Agrin at the immunological synapse between T cells and antigen-presenting cells and justify further characterization of its receptors in the immune system.     

An Optimized Method for Manufacturing a Clinical Scale Dendritic Cell-Based Vaccine for the Treatment of Glioblastoma

Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE₂, IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with “new method” lysate compared to DC pulsed with “classical method” lysate. Our results indicate that immunomagnetic isolation of CD14⁺ monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.