Priprava in določitev fenotipa miši s pogojno izbitim genom Gdi1 v astrocitih

RAB GDP dissociation inhibitor-1 gene (GDI1) encodes for αGDI, a protein controlling the cycling of small GTPases orchestrating intracellular vesicular trafficking. Mutations in human GDI1 are responsible for X-linked intellectual disability (XLID). Adult mice lacking Gdi1 gene (Gdi1 KO), a model of XLID, showed working and associative memory deficits. The cognitive phenotype was explained by a large decrease in the reserve pool of synaptic vesicles (SVs) at the hippocampal synapses, resulting in a slow SV recovery after SV depletion. Thus, cognitive deficits may reflect the temporary depletion of the immediately available SVs. Because Gdi1 is ubiquitously expressed in neurons and in astrocytes, GDI1 mutations could affect trafficking of both cell types impairing astrocyte-neuron cross talk, leading to cognitive defects. To dissect the role of αGDI in neurons and in astrocytes, two conditional models in which Gdi1 is deleted respectively in neurons (CamkII-Cre+-Gdi1flox/Y) or in astrocytes (Glast-CreERT2+-Gdi1flox/Y) were generated. The CamkII-Cre+-Gdi1flox/Y mice phenocopied the Gdi1 KO mouseinstead, Glast-CreERT2+-Gdi1flox/Y mice have a selective impairment in working memory which was rescued by inhibiting glycolysis by 2-deoxy-D-glucose administration. Proteomic analysis showed significant changes in astrocyte-resident glucose handling enzymes. Imaging with [18F]-fluoro-2-deoxy-D-glucose revealed an increased D-glucose uptake in Gdi1-null brain, consistent with the facilitated D-glucose utilisation. These results support a new astrocyte-based mechanism in XLID, opening a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.Gen za inhibitor disociacije GDP-1 (GDI1) RAB kodira beljakovino αGDI, ki nadzoruje kroženje majhnih GTPaz, ki uravnavajo znotrajcelični transport mešičkov. Mutacije v človeškem GDI1 so odgovorne za od X-kromosoma odvisno nespecifično umsko manjzmožnost (XLID). Odrasle miši brez gena Gdi1 (Gdi1-null), model XLID, imajo oslabljeno tvorbo delovnega in asociativnega spomina. Kognitivni fenotip je bil pojasnjen z zmanjšanjem rezervnega bazena sinaptičnih mešičkov (SV) v hipokampalnih sinapsah, zaradi česar se SV po izpraznitvi prepočasi nadomeščajo. Pomanjkanje takoj razpoložljivih SV se lahko odraža kot kognitivni primanjkljaj. Zaradi izražanja Gdi1 tako v nevronih kot astrocitih, bi lahko mutacije v Gdi1 vplivale na transport mešičkov v obeh tipih celic, kar lahko ovira navzkrižno komunikacijo med astrociti in nevroni in s tem povzroča kognitivne okvare. Da bi bolje razumeli vlogo αGDI v nevronih in astrocitih, sta bila ustvarjena dva pogojna modela miši, v katerih je Gdi1 izbrisan v nevronih (CamkII-Cre+-Gdi1flox/Y) ali v astrocitih (GLAST-CreERT2+-Gdi1flox/Y). Miši CamkII-Cre+-Gdi1flox/Y so imele enak fenotip kot miši Gdi1-null, medtem ko so imele miši GLAST-CreERT2+-Gdi1flox/Y selektivno okvaro delovnega spomina, ki je bil obnovljen z inhibicijo glikolize z intraperitonealnim vnosom 2-deoksi-D-glukoze. Proteomska analiza astrocitov je pokazala spremembe v encimih, ki uravnavajo presnovo glukoze. Slikanje s [18F]-fluoro-2-deoksi-D-glukozo je razkrilo zvečan privzem D-glukoze v možganih miši Gdi1-null. Rezultati razkrivajo nov mehanizem pri XLID, ki temelji na astrocitih in uravnavanju aerobne glikolize. Predstavljajo novo terapevtsko priložnost, kar je relevantno za spremembo klinične prakse

The intellectual disability protein RAB39B selectively regulates GluA2 trafficking to determine synaptic AMPAR composition

All rights reserved. RAB39B is a member of the RAB family of small GTPases that controls intracellular vesicular trafficking in a compartment-specific manner. Mutations in the RAB39B gene cause intellectual disability comorbid with autism spectrum disorder and epilepsy, but the impact of RAB39B loss of function on synaptic activity is largely unexplained. Here we show that protein interacting with C-kinase 1 (PICK1) is a downstream effector of GTP-bound RAB39B and that RAB39B-PICK1 controls trafficking from the endoplasmic reticulum to the Golgi and, hence, surface expression of GluA2, a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). The role of AMPARs in synaptic transmission varies depending on the combination of subunits (GluA1, GluA2 and GluA3) they incorporate. RAB39B downregulation in mouse hippocampal neurons skews AMPAR composition towards non GluA2-containing Ca2+ -permeable forms and thereby alters synaptic activity, specifically in hippocampal neurons. We posit that the resulting alteration in synaptic function underlies cognitive dysfunction in RAB39B-related disorders.This work was supported by Comitato Telethon Fondazione ONLUS, Italy (TCP04015), Jerome Lejeune Foundation, France (776) and F. Hoffmann La Roche post-doc program, Switzerland (RPF 138)Peer Reviewe

Loss of HER2 and decreased T-DM1 efficacy in HER2 positive advanced breast cancer treated with dual HER2 blockade: the SePHER Study

HER2-targeting agents have dramatically changed the therapeutic landscape of HER2+ advanced breast cancer (ABC). Within a short time frame, the rapid introduction of new therapeutics has led to the approval of pertuzumab combined with trastuzumab and a taxane in first-line, and trastuzumab emtansine (T-DM1) in second-line. Thereby, evidence of T-DM1 efficacy following trastuzumab/pertuzumab combination is limited, with data from some retrospective reports suggesting lower activity. The purpose of the present study is to investigate T-DM1 efficacy in pertuzumab-pretreated and pertuzumab na\uefve HER2 positive ABC patients. We also aimed to provide evidence on the exposure to different drugs sequences including pertuzumab and T-DM1 in HER2 positive cell lines. Methods: The biology of HER2 was investigated in vitro through sequential exposure of resistant HER2 + breast cancer cell lines to trastuzumab, pertuzumab, and their combination. In vitro experiments were paralleled by the analysis of data from 555 HER2 + ABC patients treated with T-DM1 and evaluation of T-DM1 efficacy in the 371 patients who received it in second line. Survival estimates were graphically displayed in Kaplan Meier curves, compared by log rank test and, when possibile, confirmed in multivariate models. Results: We herein show evidence of lower activity of T-DM1 in two HER2+ breast cancer cell lines resistant to trastuzumab+pertuzumab, as compared to trastuzumab-resistant cells. Lower T-DM1 efficacy was associated with a marked reduction of HER2 expression on the cell membrane and its nuclear translocation. HER2 downregulation at the membrane level was confirmed in biopsies of four trastuzumab/pertuzumab-pretreated patients. Among the 371 patients treated with second-line T-DM1, median overall survival (mOS) from diagnosis of advanced disease and median progression-free survival to second-line treatment (mPFS2) were 52 and 6 months in 177 patients who received trastuzumab/pertuzumab in first-line, and 74 and 10 months in 194 pertuzumab-na\uefve patients (p = 0.0006 and 0.03 for OS and PFS2, respectively). Conclusions: Our data support the hypothesis that the addition of pertuzumab to trastuzumab reduces the amount of available plasma membrane HER2 receptor, limiting the binding of T-DM1 in cancer cells. This may help interpret the less favorable outcomes of second-line T-DM1 in trastuzumab/pertuzumab pre-treated patients compared to their pertuzumab-na\uefve counterpart

The prognostic relevance of HER2-positivity gain in metastatic breast cancer in the ChangeHER trial

Breast cancer (BC) heterogeneity is composite in nature, with a wide variety of factors concurring to define several pathological entities, which differ by clinical presentation, pathologic features, therapy administered, and inherent outcomes1. Additional sources of breast cancer heterogeneity may raise during the disease course. In BC patients whose disease was initially diagnosed in the early stage and subsequently progressed with metastatic involvement of one single or multiple site/s, the molecular characteristics of metastatic lesions do not necessary mimic those of the disease initially diagnosed. A well-depicted molecular landscape is crucial for subtype definition, prognostic evaluation and appropriate therapeutic decisions. Accordingly, current guidelines suggest repeating the immunohistochemical (IHC) assessment in patients with metastatic spread and at least one secondary lesion amenable to biopsy2. Discordance in human epidermal growth factor receptor 2 (HER2) status between the tumor and metastatic lesions is widely acknowledged, and not yet completely unraveled in their biologic meaning and prognostic relevance3\u201311. The overexpression of HER2 or amplification of the related gene is extensively recognized as a feature associated with more aggressive biological behavior12,13. However, the extent to which changes in HER2 status may affect patients\u2019 prognosis is still a matter of debate14. We herein propose an observational study of HER2-positive metastatic breast cancer (mBC) patients treated with the anti-HER2 targeted agents pertuzumab and/or trastuzumab emtansine (T-DM1). Our research question is whether relevant differences exist in long-term outcomes of patients with concordant HER2 status between the primary tumor and its secondary lesion/s compared to patients whose disease revealed HER2-positivity gain at the IHC assessment of metastatic lesions. In our historical cohorts, we also sought to identify factors associated with HER2-positivity gain at the IHC reassessment, for which an impact on prognosis may be foreseen

Outcome and Morphofunctional Changes on Cardiac Magnetic Resonance in Patients With Acute Myocarditis Following mRNA COVID-19 Vaccination

Messenger RNA (mRNA) COVID-19 vaccination has been associated with a higher-than expected occurrence of acute myocarditis, although the benefits of the vaccine greatly outweigh the risk of myocarditis. Even if the short-term prognosis of mRNA vaccine–related myocarditis has been reported to be favorable, scarce information is available on midterm prognosis. The current series included 7 to 9 patients with baseline and follow-up cardiac magnetic resonance imaging (CMRI). Questions on acute myocarditis following mRNA COVID-19 vaccination addressed by this study are the risk of adverse events after discharge and the extent of residual myocardial dysfunction and scar formation