Glioblastoma multiforme (GBM) is characterized by a remarkable cellular and molecular heterogeneity that make the behavior of this tumor highly variable and resistant to therapy. In addition, the ...most serious clinical complication of GBM and other brain tumors is the development of vasogenic edema which dramatically increase the intracranial pressure. In the present study we evaluate the expression, supramolecular organization and spatial distribution of AQP4 and AQP4ex, the new readthrough isoform of AQP4, in relationship with the degree of vasogenic brain edema and tumor progression. To this purpose, tissue samples from regions of tumor core, peritumoral and non-infiltrated tissues of each GBM patient (
n
= 31) were analyzed. Immunofluorescence experiments revealed that the expression of AQP4ex was almost absent in tumoral regions while the canonical AQP4 isoforms appear mostly delocalized. In peritumoral tissues, AQP4 expression was found altered in those perivascular astrocyte processes where AQP4ex appeared reduced and partially delocalized. Protein expression levels measured by immunoblot showed that global AQP4 was reduced mainly in the tumor core. Notably, the relative amount of AQP4ex was more severely reduced starting from the peritumoral region. BN-PAGE experiments showed that the supramolecular organization of AQP4 is only partially affected in GBM. Edema assessment by magnetic resonance imaging revealed that the level of AQP4ex downregulation correlated with edema severity. Finally, the degree of BBB alteration, measured with sodium fluorescein content in GBM biopsies, correlated with the edema index and AQP4ex downregulation. Altogether these data suggest that the AQP4ex isoform is critical in the triggering event of progressive downregulation and mislocalization of AQP4 in GBM, which may affect the integrity of the BBB and contributes to accumulation of edema in the peritumoral tissue. Thus, AQP4ex could be considered as a potential early biomarker of GBM progression.
Translational readthrough (TRT) of aquaporin-4 (AQP4) has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation potential of AQP4. The TRT isoform ...of AQP4, named AQP4ex, is central for both AQP4 polarization and water channel activity in the central nervous system (CNS). Here we evaluate the relevance of the TRT mechanism by analyzing whether AQP4ex is also expressed in peripheral tissues and whether the expression of AQP4ex is necessary for its polarized expression as it occurs in perivascular astrocyte processes. To this purpose, AQP4ex null mice were used, and analysis was performed by immunolocalization and immunoblot. The results demonstrate that AQP4ex is expressed in kidney, stomach, trachea and skeletal muscle with the same localization pattern as the canonical AQP4 isoforms. AQP4ex protein levels vary from 6% to about 13% of the total AQP4 protein levels in peripheral tissues. Immunogold electron microscopy experiments demonstrated the localization of AQP4ex at the astrocytic endfeet, and experiments conducted on AQP4ex null mice CNS confirmed that the expression of AQP4ex is necessary for anchoring of the perivascular AQP4. Without the readthrough isoform, AQP4 assemblies are mis-localized, being uniformly distributed on the astrocyte processes facing the neuropile. No alteration of AQP4 polarization was found in AQP4ex null kidney, stomach, trachea or skeletal muscle, suggesting that AQP4ex does not have a role for proper membrane localization of AQP4 in peripheral tissues. We conclude that a dual role for AQP4ex is limited to the CNS.
Human dermal fibroblasts from a Leigh Syndrome (LS) patient harboring the heterozygous NDUFS1 R557X/D618N compound mutation were reprogrammed to generate integration-free induced pluripotent stem ...cells (iPSCs). The full characterization of IUFi002-A-iPSCs demonstrated that the line is free of exogenous reprogramming genes and maintains the genomic integrity. IUFi002-A-iPSCs' pluripotency was confirmed by the expression of pluripotency markers and embryoid body-based differentiation into cell types representative of each of the three germ layers. The generated iPSC line provides a powerful tool to investigate LS and analyze the molecular mechanisms underlying NDUFS1 mutations-induced pathology.
AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular ...membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine
and serine
, located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser
is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser
was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (
-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser
in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of
-AQP4ex in human brain water homeostasis.
Translational readthrough (TRT) of aquaporin-4 (AQP4) has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation potential of AQP4. The TRT isoform ...of AQP4, named AQP4ex, is central for both AQP4 polarization and water channel activity in the central nervous system (CNS). Here we evaluate the relevance of the TRT mechanism by analyzing whether AQP4ex is also expressed in peripheral tissues and whether the expression of AQP4ex is necessary for its polarized expression as it occurs in perivascular astrocyte processes. To this purpose, AQP4ex null mice were used, and analysis was performed by immunolocalization and immunoblot. The results demonstrate that AQP4ex is expressed in kidney, stomach, trachea and skeletal muscle with the same localization pattern as the canonical AQP4 isoforms. AQP4ex protein levels vary from 6% to about 13% of the total AQP4 protein levels in peripheral tissues. Immunogold electron microscopy experiments demonstrated the localization of AQP4ex at the astrocytic endfeet, and experiments conducted on AQP4ex null mice CNS confirmed that the expression of AQP4ex is necessary for anchoring of the perivascular AQP4. Without the readthrough isoform, AQP4 assemblies are mis-localized, being uniformly distributed on the astrocyte processes facing the neuropile. No alteration of AQP4 polarization was found in AQP4ex null kidney, stomach, trachea or skeletal muscle, suggesting that AQP4ex does not have a role for proper membrane localization of AQP4 in peripheral tissues. We conclude that a dual role for AQP4ex is limited to the CNS.
It is becoming increasingly clear that the communities of microorganisms that populate the surfaces exposed to the external environment, termed microbiota, are key players in the regulation of ...pathogen-host cross talk affecting the onset as well as the outcome of infectious diseases. We have performed a multicenter, prospective, observational study in which nasal and oropharyngeal swabs were collected for microbiota predicting the risk of invasive fungal infections (IFIs) in patients with hematological malignancies. Here, we demonstrate that the nasal and oropharyngeal microbiota are different, although similar characteristics differentiate high-risk from low-risk samples at both sites. Indeed, similar to previously published results on the oropharyngeal microbiota, high-risk samples in the nose were characterized by low diversity, a loss of beneficial bacteria, and an expansion of potentially pathogenic taxa, in the presence of reduced levels of tryptophan (Trp). At variance with oropharyngeal samples, however, low Trp levels were associated with defective host-derived kynurenine production, suggesting reduced tolerance mechanisms at the nasal mucosal surface. This was accompanied by reduced levels of the chemokine interleukin-8 (IL-8), likely associated with a reduced recruitment of neutrophils and impaired fungal clearance. Thus, the nasal and pharyngeal microbiomes of hematological patients provide complementary information that could improve predictive tools for the risk of IFI in hematological patients.
The multi‐site ubiquitination of Tau protein found in Alzheimer's disease filaments hints at the failed attempt of neurons to remove early toxic species. The ubiquitin‐dependent degradation of Tau is ...regulated in vivo by the E3 ligase CHIP, a quality controller of the cell proteome dedicated to target misfolded proteins for degradation. In our study, by using site‐resolved NMR, biochemical and computational methods, we elucidate the structural determinants underlying the molecular recognition between the ligase and its intrinsically disordered substrate. We reveal a multi‐domain dynamic interaction that explains how CHIP can direct ubiquitination of Tau at multiple sites even in the absence of chaperones, including its typical partner Hsp70/Hsc70. Our findings thus provide mechanistic insight into the chaperone‐independent engagement of a disordered protein by its E3 ligase.
An NMR‐based structural investigation of the Tau–CHIP complex revealed a multi‐domain, dynamic interaction that explains the origin of the multiple‐site ubiquitination of Tau operated by CHIP, as well as the ability of this ligase to modify Tau in the absence of chaperones.
Introduction
In 2020, the Italian Medicines Agency (AIFA) approved the reimbursement of calcitonin gene-related peptide (CGRP) pathway monoclonal antibodies (mAbs), including fremanezumab, in ...patients with a Migraine Disability Assessment Scale (MIDAS) score ≥ 11, with prescription renewals for up to 12 months in patients with ≥ 50% reduction in MIDAS score at Months 3 and 6. In this sub-analysis of the Pan-European Real Life (PEARL) study, we provide real-world data on fremanezumab use in Italian routine clinical practice (EUPAS35111).
Methods
This first interim analysis for Italy was conducted when 300 enrolled adult patients with episodic or chronic migraine (EM, CM) completed 6 months of treatment with fremanezumab. The primary endpoint is the proportion of patients achieving ≥ 50% reduction in monthly migraine days (MMD) across the 6 months post-fremanezumab initiation. Secondary endpoints include: proportion of patients achieving ≥ 50% reduction in MIDAS score at Months 3 and 6, and mean change from baseline across Months 1–6 in MMD and headache-related disability. Safety was assessed through adverse events (AEs) reported.
Results
Of 354 patients enrolled at Italian centers, 318 (EM, 35.5%, CM, 64.5%) were included in the effectiveness analysis. Of patients with available data, 109 (61.2%) achieved the primary endpoint. 61.0% and 65.1% achieved ≥ 50% reduction in MMDs at Months 3 and 6, respectively; 79.9% and 81.0% experienced ≥ 50% reduction in MIDAS at the same timepoints.
Conclusion
Fremanezumab was effective and well-tolerated over the first 6 months of treatment, with approximately 80% of patients meeting Italian criteria for treatment continuation at Months 3 and 6.
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