Design and validation of a reporter mouse to study the dynamic regulation of TFEB and TFE3 activity through in vivo imaging techniques.

TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor's validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism.Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

Canine glioblastoma-derived extracellular vesicles as precise carriers for glioblastoma imaging: Targeting across the blood-brain barrier.

The treatment of glioblastoma (GBM) faces significant challenges due to the difficulty of delivering drugs through the blood-brain barrier (BBB). Extracellular vesicles (EVs) have emerged as potential carriers for targeted drug delivery to brain tumors. However, their use and distribution in the presence of an intact BBB and their ability to target GBM tissue are still under investigation. This study explored the use of EVs for GBM targeting across the BBB. Canine plasma EVs from healthy dogs and dogs with glioma were isolated, characterized, and loaded with diagnostic agents. Biodistribution studies were conducted in healthy murine models and a novel intranasal model that preserved BBB integrity while initiating early-stage GBM growth. This model assessed EVs' potential for delivering the contrast agent gadoteric acid to intracranial tumors. Imaging techniques, such as bioluminescence and MRI, confirmed EVs' targeting and delivery capabilities thus revealing a selective accumulation of canine glioma-derived EVs in brain tissue under physiological conditions. In the model of brain tumor, MRI experiments demonstrated the ability of EVs to accumulate gadoteric acid within GBM to enhance contrast of the tumoral mass, even when BBB integrity is maintained. This study underscores the potential of EVs derived from glioma for the targeted delivery of drugs to glioblastoma. EVs from dogs with glioma showed capacity to traverse the BBB and selectively accumulate within the brain tumor. Overall, this research represents a foundation for the application of autologous EVs to precision glioblastoma treatment, addressing the challenge of BBB penetration and targeting specificity in brain cancer therapy.

Sex-Specific Microglial Responses to Glucocerebrosidase Inhibition: Relevance to GBA1-Linked Parkinson's Disease.

Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson's Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5-2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1.

Increased Sensitivity of Computed Tomography Scan for Neoplastic Tissues Using the Extracellular Vesicle Formulation of the Contrast Agent Iohexol.

Computed tomography (CT) is a diagnostic medical imaging modality commonly used to detect disease and injury. Contrast agents containing iodine, such as iohexol, are frequently used in CT examinations to more clearly differentiate anatomic structures and to detect and characterize abnormalities, including tumors. However, these contrast agents do not have a specific tropism for cancer cells, so the ability to detect tumors is severely limited by the degree of vascularization of the tumor itself. Identifying delivery systems allowing enrichment of contrast agents at the tumor site would increase the sensitivity of detection of tumors and metastases, potentially in organs that are normally inaccessible to contrast agents, such as the CNS. Recent work from our laboratory has identified cancer patient-derived extracellular vesicles (PDEVs) as effective delivery vehicles for targeting diagnostic drugs to patients' tumors. Based on this premise, we explored the possibility of introducing iohexol into PDEVs for targeted delivery to neoplastic tissue. Here, we provide preclinical proof-of-principle for the tumor-targeting ability of iohexol-loaded PDEVs, which resulted in an impressive accumulation of the contrast agent selectively into the neoplastic tissue, significantly improving the ability of the contrast agent to delineate tumor boundaries.

DNA aptamers masking angiotensin converting enzyme 2 as an innovative way to treat SARS-CoV-2 pandemic.

All the different coronavirus SARS-CoV-2 variants isolated so far share the same mechanism of infection mediated by the interaction of their spike (S) glycoprotein with specific residues on their cellular receptor: the angiotensin converting enzyme 2 (ACE2). Therefore, the steric hindrance on this cellular receptor created by a bulk macromolecule may represent an effective strategy for the prevention of the viral spreading and the onset of severe forms of Corona Virus disease 19 (COVID-19). Here, we applied a systematic evolution of ligands by exponential enrichment (SELEX) procedure to identify two single strand DNA molecules (aptamers) binding specifically to the region surrounding the K353, the key residue in human ACE2 interacting with the N501 amino acid of the SARS-CoV-2 S. 3D docking in silico experiments and biochemical assays demonstrated that these aptamers bind to this region, efficiently prevent the SARS-CoV-2 S/human ACE2 interaction and the viral infection in the nanomolar range, regardless of the viral variant, thus suggesting the possible clinical development of these aptamers as SARS-CoV-2 infection inhibitors. Our approach brings a significant innovation to the therapeutic paradigm of the SARS-CoV-2 pandemic by protecting the target cell instead of focusing on the virus; this is particularly attractive in light of the increasing number of viral mutants that may potentially escape the currently developed immune-mediated neutralization strategies.

ERα-independent NRF2-mediated immunoregulatory activity of tamoxifen.

Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in estrogen receptor-alpha (ERα)-dependent breast cancers and off-target indications such as infections, although the immune activity of TAM and its active metabolite, 4-OH tamoxifen (4HT), is poorly characterized. Here, we aimed at investigating the endocrine and immune activity of these SERMs in macrophages. Using primary cultures of female mouse macrophages, we analyzed the expression of immune mediators and activation of effector functions in competition experiments with SERMs and 17ß-estradiol (E2) or the bacterial endotoxin LPS. We observed that 4HT and TAM induce estrogen antagonist effects when used at nanomolar concentrations, while pharmacological concentrations that are reached by TAM in clinical settings regulate the expression of VEGFα and other immune activation genes by ERα- and G protein-coupled receptor 1 (GPER1)-independent mechanisms that involve NRF2 through PI3K/Akt-dependent mechanisms. Importantly, we observed that SERMs potentiate cell phagocytosis and modify the effects of LPS on the expression of inflammatory cytokines, such as TNFα and IL1ß, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1ß secretion through caspase-1 activation. Altogether, our data unravel a novel molecular mechanism and immune functions for TAM and 4HT, sustaining their repurposing in infective and other estrogen receptors-unrelated pathologies.

Inhibition of microglial ß-glucocerebrosidase hampers the microglia-mediated antioxidant and protective response in neurons.

BACKGROUND: Homozygotic mutations in the GBA gene cause Gaucher's disease; moreover, both patients and heterozygotic carriers have been associated with 20- to 30-fold increased risk of developing Parkinson's disease. In homozygosis, these mutations impair the activity of ß-glucocerebrosidase, the enzyme encoded by GBA, and generate a lysosomal disorder in macrophages, which changes morphology towards an engorged phenotype, considered the hallmark of Gaucher's disease. Notwithstanding the key role of macrophages in this disease, most of the effects in the brain have been attributed to the ß-glucocerebrosidase deficit in neurons, while a microglial phenotype for these mutations has never been reported. METHODS: We applied the bioluminescence imaging technology, immunohistochemistry and gene expression analysis to investigate the consequences of microglial ß-glucocerebrosidase inhibition in the brain of reporter mice, in primary neuron/microglia cocultures and in cell lines. The use of primary cells from reporter mice allowed for the first time, to discriminate in cocultures neuronal from microglial responses consequent to the ß-glucocerebrosidase inhibition; results were finally confirmed by pharmacological depletion of microglia from the brain of mice. RESULTS: Our data demonstrate the existence of a novel neuroprotective mechanism mediated by a direct microglia-to-neuron contact supported by functional actin structures. This cellular contact stimulates the nuclear factor erythroid 2-related factor 2 activity in neurons, a key signal involved in drug detoxification, redox balance, metabolism, autophagy, lysosomal biogenesis, mitochondrial dysfunctions, and neuroinflammation. The central role played by microglia in this neuronal response in vivo was proven by depletion of the lineage in the brain of reporter mice. Pharmacological inhibition of microglial ß-glucocerebrosidase was proven to induce morphological changes, to turn on an anti-inflammatory/repairing pathway, and to hinder the microglia ability to activate the nuclear factor erythroid 2-related factor 2 response, thus increasing the neuronal susceptibility to neurotoxins. CONCLUSION: This mechanism provides a possible explanation for the increased risk of neurodegeneration observed in carriers of GBA mutations and suggest novel therapeutic strategies designed to revert the microglial phenotype associated with ß-glucocerebrosidase inhibition, aimed at resetting the protective microglia-to-neuron communication.

Cancer-derived EVs show tropism for tissues at early stage of neoplastic transformation.

From the past decade, extracellular vesicles (EVs) have attracted considerable attention as tools for the selective delivery of anti-neoplastic drugs to cancer tissues. Compared to other nanoparticles, EVs display interesting unique features including immune compatibility, low toxicity and the ability to encapsulate a large variety of small- and macro-molecules. However, in virtually all studies, investigations on EVs have been focused on fully transformed cancers: the possibility to apply EV technology also to early-stage tumors has never been explored. Methods: Herein, we studied the ability of cancer-derived EVs to recognize and deliver their cargo also to incipient cancers. To this purpose, EV biodistribution was studied in MMTV-NeuT genetically modified mice during early mammary transformation, in fully developed breast tumors and in the normal gland of wild type syngeneic mice. EVs were loaded with indocyanine green (ICG), a near-infrared (NIR) dye together with oncolytic viruses and i.v. injected in mice. The nanoparticle biodistribution was assayed by in vivo and ex vivo optical imaging (detecting the ICG) and semiquantitative real-time PCR (measuring the adenoviral genome) in different tissues. Results: Our results demonstrate the ability of cancer-derived EVs to recognize early-stage neoplastic tissues opening the possibility to selectively deliver theranostics also for tumor prevention. Conclusions: Taken together our study demonstrates the ability of EVs to recognize and deliver diagnostic and therapeutic agents not only to fully transformed tissues but also to early stage tumors. These findings pave the way for the synthesis of "universal" EVs-based formulation for targeted cancer therapy.

Transplantation of autologous extracellular vesicles for cancer-specific targeting.

Nano- and microsized extracellular vesicles (EVs) are naturally occurring cargo-bearing packages of regulatory macromolecules, and recent studies are increasingly showing that EVs are responsible for physiological intercellular communication. Nanoparticles encapsulating anti-tumor theranostics represent an attractive "exosome-interfering" strategy for cancer therapy. Methods: Herein, by labeling plasma-derived EVs with indocyanine green (ICG) and following their biodistribution by in vivo and ex vivo imaging, we demonstrate the existence of nanoparticles with a highly selective cancer tropism in the blood of colorectal cancer (CRC) patients but not in that of healthy volunteers. Results: In CRC patient-derived xenograft (PDX) mouse models, we show that transplanted EVs recognize tumors from the cognate nanoparticle-generating individual, suggesting the theranostic potential of autologous EVs encapsulating tumor-interfering molecules. In large canine breeds bearing spontaneous malignant skin and breast tumors, the same autologous EV transplantation protocol shows comparable safety and efficacy profiles. Conclusions: Our data show the existence of an untapped resource of intercellular communication present in the blood of cancer patients, which represents an efficient and highly biocompatible way to deliver molecules directly to the tumor with great precision. The novel EV-interfering approach proposed by our study may become a new research direction in the complex interplay of modern personalized cancer therapy.

Reciprocal interference between the NRF2 and LPS signaling pathways on the immune-metabolic phenotype of peritoneal macrophages.

The metabolic and immune adaptation to extracellular signals allows macrophages to carry out specialized functions involved in immune protection and tissue homeostasis. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that coordinates cell redox and metabolic responses to stressors. However, the individual and concomitant activation of NRF2 and inflammatory pathways have been poorly investigated in isolated macrophages. We here took advantage of reporter mice for the transcriptional activities of NRF2 and nuclear factor-kB (NFκB), a key transcription factor in inflammation, and observe a persisting reciprocal interference in the response of peritoneal macrophages to the respective activators, tert-Butylhydroquinone (tBHQ) and lipopolysaccharide (LPS). When analyzed separately by gene expression studies, these pathways trigger macrophage-specific metabolic and proliferative target genes that are associated with tBHQ-induced pentose phosphate pathway (PPP) with no proliferative response, and with opposite effects observed with LPS. Importantly, the simultaneous administration of tBHQ + LPS alters the effects of each individual pathway in a target gene-specific manner. In fact, this co-treatment potentiates the effects of tBHQ on the antioxidant enzyme, HMOX1, and the antibacterial enzyme, IRG1, respectively; moreover, the combined treatment reduces tBHQ activity on the glycolytic enzymes, TALDO1 and TKT, and decreases LPS effects on the metabolic enzyme IDH1, the proliferation-related proteins KI67 and PPAT, and the inflammatory cytokines IL-1ß, IL-6, and TNFα. Altogether, our results show that the activation of NRF2 redirects the metabolic, immune, and proliferative response of peritoneal macrophages to inflammatory signals, with relevant consequences for the pharmacological treatment of diseases that are associated with unopposed inflammatory responses.

The Synaptonuclear Messenger RNF10 Acts as an Architect of Neuronal Morphology.

The Ring Finger Protein 10 [RNF10] is a novel synapse-to-nucleus signaling protein that specifically links activation of synaptic NMDA receptors to modulation of gene expression. RNF10 dissociation from the GluN2A subunit of the NMDA receptor represents the first step of its synaptonuclear transport and it is followed by an importin-dependent translocation into the nucleus. Here, we have identified protein kinase C [PKC]-dependent phosphorylation of RNF10 Ser31 as a key step for RNF10 detachment from NMDA receptor and its subsequent trafficking to the nucleus. We show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture in hippocampal neurons.

In vivo imaging of early signs of dopaminergic neuronal death in an animal model of Parkinson's disease.

The Parkinson's disease (PD) evolves over an extended period of time with the onset occurring long before clinical signs begin to manifest. Characterization of the molecular events underlying the PD onset is instrumental for the development of diagnostic markers and preventive treatments, progress in this field is hindered by technical limitations. We applied an imaging approach to demonstrate the activation of Nrf2 transcription factor as a hallmark of neurodegeneration in neurotoxin-driven models of PD. In dopaminergic SK-N-BE neuroblastoma cells, Nrf2 activation was detected in cells committed to die as proven by time lapse microscopy; in the substantia nigra pars compacta area of the mouse brain, the Nrf2 activation preceded dopaminergic neurodegeneration as demonstrated by in vivo and ex vivo optical imaging, a finding confirmed by co-localization experiments carried out by immunohistochemistry. Collectively, our results identify the Nrf2 signaling as an early marker of neurodegeneration, anticipating dopaminergic neurodegeneration and motor deficits.

Global profiling of TSEC proliferative potential by the use of a reporter mouse for proliferation.

INTRODUCTION: "Tissue-selective estrogen complex" or TSEC is a novel concept of estrogen replacement therapy for the postmenopause based on the combined use of estrogens and selective estrogen receptor modulators (SERMs). The aim of this study was to exploit the potential of a novel transgenic mouse where luciferase expression is associated with cell proliferation (the MITO-luc mouse) to investigate cell proliferation in reproductive and nonreproductive tissues in mice exposed to repetitive treatments with TSEC. MATERIAL AND METHODS: Ovariectomized MITO-Luc mice were subjected to a daily oral treatment with bazedoxifene, conjugated estrogen (CE), TSEC, or raloxifene for 21 days. During the treatment, the proliferative effects of treatments were monitored by bioluminescence-based in vivo imaging. At the end of the treatment, mice were euthanized and cell proliferation assessed in selected tissues by quantitative analysis of luciferase activity and by immunohistochemistry (IHC). RESULTS: In uterus treatment with CE, but not TSEC, induced a large increase in luciferase activity underlying the proliferative effect of the hormone. No accumulation of luciferase was observed in other organs and tissues target of estrogen action. We observed an increase of Ki67 immunoreactivity only in the uterus of mice treated with CE. CONCLUSION: Pairing of an SERM with estrogens results in a complete blockage of CE proliferative effects in uterus and the absence of any undesired proliferative effects in other organs; moreover, the MITO-Luc mouse is an efficacious tool for the global, rapid, and reliable analysis of drug-induced proliferation.

Molecular imaging of cytochrome P450 activity in mice.

Detailed knowledge of drug metabolism is relevant information provided by preclinical drug development research. Oxidative enzymes such as those belonging to P450 family of cytochromes (CYP) play a prominent role in drug metabolism. Here, we propose an innovative method based on bioluminescence in vivo imaging which has the potential to simplify the in vivo measurement of CYP activity also providing a dynamic measure of the effects of a drug on a specific P450 enzyme complex in a living mouse. The method is based on a pro-luciferin which can be converted into the active luciferase substrate by a specific P450 activity. The pro-luciferin is administered to a luciferase reporter mouse which produces luminescent signals in relation to the cytochrome activity present in each tissue. The photon emission generated can be easily localized and quantified by optical imaging. To demonstrate the validity of the system, we pharmacologically induced hepatic Cyp3a in the reporter mouse and proved that pro-luciferin administration generates a Cyp3a selective signal in the chest area that can be efficiently detected by optical imaging. The kind of tool generated has the potential to be exploited for the study of additional CYPs.

Promiscuity, stability and cold adaptation of a newly isolated acylaminoacyl peptidase.

We report on the characterisation of a member of the acylaminoacyl peptidase family, the first isolated from bacteria. The enzyme was obtained from the psychrophilic bacterium Sporosarcina psychrophila and shows the typical features of cold adaptation (low T(m), optimal temperature of 40 °C, poor thermal stability). It was also tested for substrate specificity, effect of metals, temperature dependence and structure stability and revealed promiscuous catalytic activity on at least two chemically distinct substrates, with k(cat)/K(m) values for ester hydrolysis and acylamino acids cleavage of 1.7 × 10(4) s(-1) M(-1) and 6.2 × 10(3) s(-1) M(-1), respectively. Despite some properties cannot be explained with current models, results report on the relevance of structural and catalytic properties for the successful adaptation to cold temperatures.