Biology subjects

Bugiani, M.

Publications and source records attributed to Bugiani, M..

7 recordsLinked to original sources

bioRxiv2026

Focal and subtle myelin damage in multiple sclerosis-derived post-mortem human brain slice cultures

The mechanisms that drive myelin damage as seen in demyelinating disorders such as multiple sclerosis remain incompletely understood. Much of our current knowledge is derived from animal models, but interspecies differences limit their relevance in the context of human pathology and could explain why various promising preclinical therapies failed during clinical translation. Human post-mortem organotypic brain slice cultures provide a unique platform to study human myelin biology, as they preserve genetic, cytoarchitectural, pathological and species-specific context. Here, we evaluated myelin integrity in a human post-mortem brain organotypic slice culture model and experimentally induce focal myelin damage. Human post-mortem organotypic slices cultures retain key features throughout the culturing period, but exhibit gradual cellular and myelin loss over time. Myelin fibres within the white matter remain detectable and present preserved structural and chemical integrity up to 13 days in vitro, indicated by the conserved paranodal and nodal organization and stable myelin spectroscopic signature. Delivery of lysophosphatidylcholine using cryogel scaffolds enables focal drug administration throughout the full depth of the slice with minimal diffusion into surrounding tissue and induces localized demyelination after lysophosphatidylcholine application. Similar focal application of the selective Nav1.6 stimulator {beta}-mammal scorpion toxin Cn2 induces subtle myelin destabilization. Overall, our results demonstrate the suitability of a human post-mortem brain organotypic slice culture model as an adequate platform for studying myelin damage in a human disease context.

neuroscience↗

bioRxiv2024

Effect of seizures on the severity of myelin vacuolization in a mouse model of megalencephalic leukoencephalopathy with subcortical cysts

ObjectiveMegalencephalic leukoencephalopathy with subcortical cysts (MLC) is a white matter disease characterized by myelin vacuolization and swollen perivascular astrocyte processes. Neuronal activity has been implicated as primary cause of myelin vacuolization and astrocyte swelling. Since acute and excessive increases in neuronal activity occur in MLC during seizures, we investigated whether seizure activity leads to the acute development of myelin vacuoles and swollen astrocyte processes. MethodsGlialcam-null mice (an established MLC mouse model) and wild-type mice received repeated i.p. low dose (5 mg / kg) kainic acid (KA) injections until severe seizures developed. Following a 60-minute period of severe seizure activity, mice were terminated and brains were fixed and processed. Brain tissue was analyzed for myelin vacuolization and astrocyte process thickness using H&E and GFAP stains, respectively. ResultsRepeated low-dose injections of KA resulted in prolonged severe seizure activity in mice of both genotypes. Total amount of seizure activity was comparable between Glialcam-null and wild-type mice. KA-induced severe seizure activity did not significantly increase myelin vacuolization in either Glialcam-null or wild-type mice. The width of perivascular astrocyte processes was also not affected by severe seizure activity. InterpretationWe show that (i) repeatedly injecting a low dose of KA provides the opportunity to regulate seizure development and generate severe seizures in both wild-type and seizure-sensitive Glialcam-null mice, and that (ii) the two major pathological features of MLC, myelin vacuolization and swollen astrocyte endfeet, are not acutely aggravated in response to KA-induced severe seizure activity.

neuroscience↗

bioRxiv2023

Gemcitabine therapeutically disrupts essential SIRT1-mediated p53 repression in Atypical Teratoid/Rhabdoid Tumors

BackgroundAtypical Teratoid/Rhabdoid Tumors (ATRT) are highly malignant embryonal tumors of the central nervous system with a dismal prognosis. Despite recent advances in understanding the molecular characteristics and subclasses of these tumors, effective therapeutic options remain scarce. MethodsIn this study, we developed and validated a novel patient-derived ATRT culture and xenograft model, which we used alongside a panel of other primary ATRT models for large-scale drug discovery assays. The identified hits were mechanistically and therapeutically investigated using an array of molecular assays and two orthotopic xenograft murine models. ResultsWe found that ATRT are selectively sensitive to the nucleoside analogue gemcitabine, with additional efficacy in Sonic Hedgehog (SHH)-subtype ATRT. Gene expression profiles and protein analyses indicated that gemcitabine treatment causes degradation of Sirtuin 1 (SIRT1), resulting in cell death through activation of NF-kB and p53. Furthermore, we discovered that gemcitabine-induced loss of SIRT1 results in a nucleus-to-cytoplasm translocation of the SHH signaling activator GLI2, explaining the additional gemcitabine sensitivity in SHH-subtype ATRT. Treatment of SHH-subgroup ATRT xenograft-bearing mice with gemcitabine resulted in a >30% increase in median survival (p<0.005, log-rank test) and yielded long-term survivors in two independent patient-derived xenograft models. ConclusionsThese findings demonstrate that ATRT are highly sensitive to gemcitabine treatment, and we propose that gemcitabine may form part of a future multimodal treatment strategy for ATRT. Key points- ATRT are specifically sensitive to gemcitabine treatment - SIRT1 may serve as a novel therapeutic target in ATRT - Gemcitabine should be considered for clinical use in ATRT patients Importance of the studyAtypical Teratoid/Rhabdoid Tumors (ATRT) are highly malignant pediatric brain tumors with a 5-year survival of merely 30%, for which effective treatment options are limited. In this study, we propose a potential novel treatment strategy for ATRT patients. We show that ATRT are highly sensitive to the chemotherapeutic gemcitabine, that takes advantage of ATRT-specific SIRT1 overexpression and disrupts p53 suppression and hedgehog signaling. Importantly, we show that gemcitabine significantly prolongs survival of ATRT patient-derived xenograft models, prolonging survival by over 30%. This effect was achieved using gemcitabine concentrations that are achievable in human brain and well-tolerated in pediatric patients. As such, gemcitabine could be readily incorporated into clinical treatment protocols and expand the still very limited therapeutic options for ATRT-patients.

cancer biology↗

bioRxiv2022

Cortical interneuron development is affected in leukodystrophy 4H

4H leukodystrophy is a rare genetic disorder classically characterized by hypomyelination, hypodontia and hypogonadotropic hypogonadism. With the discovery that 4H is caused by mutations that affect RNA polymerase III, mainly involved in the transcription of small non-coding RNAs, also patients with atypical presentations with mainly a neuronal phenotype were identified. Pathomechanisms of 4H brain abnormalities are still unknown and research is hampered by a lack of preclinical models. We aimed to identify cells and pathways that are affected by 4H mutations using induced pluripotent stem cell models. RNA sequencing analysis on induced pluripotent stem cell-derived cerebellar cells revealed several differentially expressed genes between 4H patients and control samples, including reduced ARX expression. As ARX is involved in early brain and interneuron development, we studied and confirmed interneuron changes in primary tissue of 4H patients. Subsequently, we studied interneuron changes in more depth and analyzed induced pluripotent stem cell-derived cortical neuron cultures for changes in neuronal morphology, synaptic balance, network activity and myelination. We showed a decreased percentage of GABAergic synapses in 4H, which correlated to increased neuronal network activity. Treatment of cultures with GABA antagonists led to a significant increase in neuronal network activity in control cells but not in 4H cells, also pointing to lack of inhibitory activity in 4H. Myelination and oligodendrocyte maturation in cultures with 4H neurons was normal, and treatment with sonic hedgehog agonist SAG did not improve 4H related neuronal phenotypes. qPCR analysis revealed increased expression of parvalbumin interneuron marker ERBB4, suggesting that the development rather than generation of interneurons may be affected in 4H. Together, these results indicate that interneurons are involved, possibly parvalbumin interneurons, in disease mechanisms of 4H leukodystrophy.

neuroscience↗

bioRxiv2021

Lipid and nucleocapsid N-protein accumulation in COVID-19 patient lung and infected cells

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the corona virus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS- CoV-2 infected Vero cells, processed identically. Correlative light and electron microscopy on semi- thick cryo-sections, demonstrated induction of electron-lucent, lipid filled compartments after SARS- CoV-2 infection in both lung and cell cultures. In lung tissue, the non-structural protein 4 and the stable nucleocapsid N-protein, were detected on these novel lipid filled compartments. The induction of such lipid filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19, may explain the extensive inflammatory response and provide a new hallmark for SARS- Cov-2 infection at the final, fatal stage of infection.

microbiology↗

bioRxiv2021

SARS-CoV-2 causes brain inflammation and induces Lewy body formation in macaques

SARS-CoV-2 may cause acute respiratory disease, but the infection can also initiate neurological symptoms. Here we show that SARS-CoV-2 infection causes brain inflammation in the macaque model. An increased metabolic activity in the pituitary gland of two macaques was observed by longitudinal positron emission tomography-computed tomography (PET-CT). Post-mortem analysis demonstrated infiltration of T-cells and activated microglia in the brain, and viral RNA was detected in brain tissues from one animal. We observed Lewy bodies in brains of all rhesus macaques. These data emphasize the virus capability to induce neuropathology in this nonhuman primate model for SARS-CoV-2 infection. As in humans, Lewy body formation is an indication for the development of Parkinsons disease, this data represents a warning for potential long-term neurological effects after SARS-CoV-2 infection. TeaserSARS-CoV-2 causes brain inflammation and Lewy bodies, a hallmark for Parkinson, after an asymptomatic infection in macaques.

neuroscience↗

bioRxiv2018

Integrated Stress Response Deregulation underlies Vanishing White Matter and is a target for therapy

Vanishing white matter (VWM) is a fatal, stress-sensitive leukodystrophy that mainly affects children and is currently without treatment. VWM is caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B) that is crucial for initiation of mRNA translation and its regulation under stress conditions. Mice with bi-allelic missense mutations in eIF2B recapitulate human VWM. VWM pathomechanisms are unclear. Using polysomal profiling to screen for mRNAs with altered translation we observed most prominent changes in expression of integrated stress response (ISR) mRNAs in brains of mutant compared to wild-type mice; expression levels correlated with disease severity. We substantiated these findings in VWM patients brains. ISRIB, an ISR inhibitor, normalized expression of mRNA markers, ameliorated white matter pathology and improved motor skills in VWM mice, thus showing that the ISR is central in VWM pathomechanisms and a viable target for therapy.\n\nOne Sentence SummaryISRIB ameliorates ISR deregulation and clinical signs in VWM mice

neuroscience↗