Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity.

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.

Longitudinal alterations in brain perfusion and vascular reactivity in the zQ175DN mouse model of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is marked by a CAG-repeat expansion in the huntingtin gene that causes neuronal dysfunction and loss, affecting mainly the striatum and the cortex. Alterations in the neurovascular coupling system have been shown to lead to dysregulated energy supply to brain regions in several neurological diseases, including HD, which could potentially trigger the process of neurodegeneration. In particular, it has been observed in cross-sectional human HD studies that vascular alterations are associated to impaired cerebral blood flow (CBF). To assess whether whole-brain changes in CBF are present and follow a pattern of progression, we investigated both resting-state brain perfusion and vascular reactivity longitudinally in the zQ175DN mouse model of HD. METHODS: Using pseudo-continuous arterial spin labelling (pCASL) MRI in the zQ175DN model of HD and age-matched wild-type (WT) mice, we assessed whole-brain, resting-state perfusion at 3, 6 and 9 and 13 months of age, and assessed hypercapnia-induced cerebrovascular reactivity (CVR), at 4.5, 6, 9 and 15 months of age. RESULTS: We found increased perfusion in cortical regions of zQ175DN HET mice at 3 months of age, and a reduction of this anomaly at 6 and 9 months, ages at which behavioural deficits have been reported. On the other hand, under hypercapnia, CBF was reduced in zQ175DN HET mice as compared to the WT: for multiple brain regions at 6 months of age, for only somatosensory and retrosplenial cortices at 9 months of age, and brain-wide by 15 months. CVR impairments in cortical regions, the thalamus and globus pallidus were observed in zQ175DN HET mice at 9 months, with whole brain reactivity diminished at 15 months of age. Interestingly, blood vessel density was increased in the motor cortex at 3 months, while average vessel length was reduced in the lateral portion of the caudate putamen at 6 months of age. CONCLUSION: Our findings reveal early cortical resting-state hyperperfusion and impaired CVR at ages that present motor anomalies in this HD model, suggesting that further characterization of brain perfusion alterations in animal models is warranted as a potential therapeutic target in HD.

Transcriptomic and proteomic profiling of bi-partite and tri-partite murine iPSC-derived neurospheroids under steady-state and inflammatory condition.

induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX3CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1ß + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment.

Integrated staging of morphofunctional connectivity in neuronal cultures.

Neurological disorders are defined by synaptic dysfunction. We present a workflow to quantify morphological and functional aspects of synaptic connectivity in neuronal cultures and obtain an integrated readout. We describe steps for measuring synchronous calcium bursting in GCaMP6f-transduced neurons and labeling mature synapses using a proximity ligation assay. The integration of functional and morphological information from the same cultures provides a rich fingerprint of synaptic connectivity, deployable in different experimental conditions. For complete details on the use and execution of this protocol, please refer to Verstraelen et al. and Verschuuren et al.1,2.

Non-viral delivery of RNA for therapeutic T cell engineering.

Adoptive T cell transfer has shown great success in treating blood cancers, resulting in a growing number of FDA-approved therapies using chimeric antigen receptor (CAR)-engineered T cells. However, the effectiveness of this treatment for solid tumors is still not satisfactory, emphasizing the need for improved T cell engineering strategies and combination approaches. Currently, CAR T cells are mainly manufactured using gammaretroviral and lentiviral vectors due to their high transduction efficiency. However, there are concerns about their safety, the high cost of producing them in compliance with current Good Manufacturing Practices (cGMP), regulatory obstacles, and limited cargo capacity, which limit the broader use of engineered T cell therapies. To overcome these limitations, researchers have explored non-viral approaches, such as membrane permeabilization and carrier-mediated methods, as more versatile and sustainable alternatives for next-generation T cell engineering. Non-viral delivery methods can be designed to transport a wide range of molecules, including RNA, which allows for more controlled and safe modulation of T cell phenotype and function. In this review, we provide an overview of non-viral RNA delivery in adoptive T cell therapy. We first define the different types of RNA therapeutics, highlighting recent advancements in manufacturing for their therapeutic use. We then discuss the challenges associated with achieving effective RNA delivery in T cells. Next, we provide an overview of current and emerging technologies for delivering RNA into T cells. Finally, we discuss ongoing preclinical and clinical studies involving RNA-modified T cells.

HPV-specific antibodies in female genital tract secretions captured via first-void urine retain their neutralizing capacity.

Human papillomavirus (HPV) vaccines, primarily relying on neutralizing antibodies, have proven highly effective. Recently, HPV-specific antibodies have been detected in the female genital tract secretions captured by first-void urine (FVU), offering a minimally invasive diagnostic approach. In this study, we investigated whether HPV16-specific antibodies present in FVU samples retain their neutralizing capacity by using pseudovirion-based neutralization assays. Paired FVU and serum samples (vaccinated n = 25, unvaccinated n = 25, aged 18-25) were analyzed using two orthogonal pseudovirion-based neutralization assays, one using fluorescence microscopy and the other using luminescence-based spectrophotometry. Results were compared with HPV16-specific IgG concentrations and correlations between neutralizing antibodies in FVU and serum were explored. The study demonstrated the presence of neutralizing antibodies in FVU using both pseudovirion-based neutralization assays, with the luminescence-based assay showing higher sensitivity for FVU samples, while the fluorescence microscopy-based assay exhibited better specificity for serum and overall higher reproducibility. High Spearman correlation values were calculated between HPV16-IgG and HPV16-neutralizing antibodies for both protocols (rs: 0.54-0.94, p < .001). Significant Spearman correlations between FVU and serum concentrations were also established for all assays (rs: 0.44-0.91, p < .01). This study demonstrates the continued neutralizing ability of antibodies captured with FVU, supporting the hypothesis that HPV vaccination may reduce autoinoculation and transmission risk to the sexual partner. Although further protocol optimizations are warranted, these findings provide a foundation for future research and larger cohort studies that could have implications for the optimal design, evaluation, and implementation of HPV vaccination programs.

Serum Amyloid A3 Fuels a Feed-Forward Inflammatory Response to the Bacterial Amyloid Curli in the Enteric Nervous System.

BACKGROUND & AIMS: Mounting evidence suggests the gastrointestinal microbiome is a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastrointestinal tract and its enteric nervous system. METHODS: To examine the early response, we challenged primary murine myenteric networks with curli, the prototypical bacterial amyloid, and performed shotgun RNA sequencing and multiplex enzyme-linked immunosorbent assay. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. RESULTS: Curli induced a proinflammatory response, with strong up-regulation of Saa3 and the secretion of several cytokines. This proinflammatory state was induced primarily in enteric glia, was accompanied by increased levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant Serum Amyloid A3 (SAA3) was sufficient to recapitulate this specific proinflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Similar to curli, recombinant SAA3 caused a strong up-regulation of Saa3 transcripts, illustrating its self-amplifying potential . Since colonization of curli-producing Salmonella and dextran sulfate sodium-induced colitis triggered a significant increase in Saa3 transcripts as well, we assume SAA3plays a central role in enteric dysfunction. Inhibition of dual leucine zipper kinase, an upstream regulator of the c-Jun N-terminal kinase pathway responsible for SAA3 production, attenuated curli- and recombinant SAA3-induced Saa3 up-regulation, DNA damage, and replication in enteric glia. CONCLUSIONS: Our results position SAA3 as an important mediator of gastrointestinal vulnerability to bacterial-derived amyloids and demonstrate the potential of dual leucine zipper kinase inhibition to dampen enteric pathology.

S100A8-enriched microglia populate the brain of tau-seeded and accelerated aging mice.

Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.