Biology subjects

Ivanova, E.

Publications and source records attributed to Ivanova, E..

At least 19 recordsLinked to original sources

bioRxiv2026

A dynamic molecular glue targets a plastic MDA5 filament interface to stimulate innate immunity

Innate immune signaling is a double-edged sword: uncontrolled activation drives autoinflammatory diseases, while controlled activation enhances antiviral and antitumor immunity. MDA5, a cytosolic dsRNA sensor that activates type I interferon signaling, mediates both immunopathology and protective immunity, yet small-molecule modulators have remained elusive. Here we report MA2, a first-in-class selective MDA5 agonist that acts as a molecular glue. MA2 binds a cryptic inter-protomer pocket within MDA5 filaments that undergoes ATPase-associated conformational remodeling. Rather than rigidly locking the interface, MA2 remains bound while accommodating and reshaping these structural transitions, strengthening MDA5-dsRNA and inter-protomer interactions and attenuating ATP hydrolysis. MA2 does not induce new filament assembly but potentiates pre-existing filaments, enabling context-dependent activation in dsRNA-enriched cells. MA2 enhances antiviral activity and synergizes with chemotherapeutic agents and natural killer cells to promote tumor cell killing. These findings establish dynamic molecular glues as a strategy for targeting conformationally plastic interfaces previously considered undruggable.

immunology↗

bioRxiv2026

Two epidemics, one genotype, different outcomes: evolutionary changes of Avian Influenza H5N1, genotype EA-2024-DI

Since 2020, high pathogenicity avian influenza H5Nx viruses of clade 2.3.4.4b have become enzootic in Europe, causing recurrent epidemic waves characterized by extensive reassortment events. Here, we describe the emergence of a single high-fitness genotype (EA-2024-DI) that has driven two consecutive waves, evolving into distinct sub-lineages. While its circulation is ongoing, during the 2025-2026 wave it caused an unprecedented number of cases in wild birds. Using phylodynamic analyses of a large dataset of genomic sequences, we compared the spatial diffusion and host transmission pattern of the EA-2024-DI sub-lineages across the three most recent epidemic waves (2023-2024, 2024-2025 and 2025-2026). We show that the genotype has persisted over time and has spread primarily through wild Anseriformes, but with a marked change in the transmission patterns between the different waves and a shift in the epicenter from Eastern to Central Europe, the latter having emerged as an important hub for virus diffusion throughout Europe. Our results reveal a recent increase in the frequency of viruses from wild and domestic mammals carrying mutations enhancing virus replication in mammalian hosts, highlighting the importance of proactive monitoring of this group of hosts to better understand its role in the virus ecology and evolution.

evolutionary biology↗

bioRxiv2026

Epigenomic profiling of cerebrospinal fluid cells identifies immune regulatory alterations and implicates protocadherins in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), where DNA methylation may play a role by connecting genetic and environmental risk factors. We performed whole-genome DNA methylation profiling of cerebrospinal fluid (CSF) cells from relapsing-remitting MS patients and matched controls, identifying 2,710 differentially methylated positions (DMPs) and 4,330 regions (DMRs). These changes were enriched in immune signaling, adhesion and migration processes, and were accompanied by corresponding RNA expression changes. MS-associated methylation changes enriched in the cohesin chromatin regulation pathway mapped to enhancers of T helper 17 (Th17) cells, whereas in other T cell types they were mapping to bivalent enhancers and repressed chromatin. Notably, this pathway comprised multiple Protocadherin (PCDH) genes, typically expressed in neuronal cells, that displayed consistent methylation and expression changes in CSF cells. Expression of shared intracellular domain of PCDH{gamma} cluster proteins was confirmed in peripheral blood T cells by flow cytometry as well as expression of PCDH{gamma} cluster genes in memory CD4+ T cell subsets. Moreover, co-expression analysis suggests a role of PCDH genes in aryl hydrocarbon receptor (AHR) signaling. In summary, DNA methylation changes in CSF resident cells reflect dysregulated T cell activation and migration in MS and suggest a novel role of protocadherin molecules in MS pathogenesis.

immunology↗

bioRxiv2026

CRISPR-engineered inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY(R) VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

bioengineering↗

bioRxiv2026

Live-cell imaging enables reporter-free monitoring of the circadian rhythm in individual Synechocystis cells

In vivo monitoring of circadian rhythms depends on reliable and non-invasive detection methods. This is often achieved by expressing reporter genes heterologously under the control of a circadian promoter. The activity or fluorescence of the gene product is then used as a readout. To avoid the need for generation of such reporter strains, we recently established a reporter-free detection method for cyanobacterial batch cultures. To determine whether these rhythms are driven at the level of individual cells or result from population-based effects, such as gating of cell division, we analyzed individual Synechocystis sp. PCC 6803 cells by combining a microfluidic cultivation technique with multipoint time-lapse microscopy imaging at the single-cell resolution. Hundreds of time-lapse image sequences, acquired over a period of up to ten days, were processed using our deep learning cell segmentation workflow. Although the cells had been entrained by a 12-hour light-dark cycle, neither cell size nor cell division displayed circadian rhythms. This indicates the absence of circadian gating of cell division in Synechocystis. Instead, we observed circadian oscillation in the average brightness of the phase contrast of individual Synechocystis cells. To demonstrate how phase-contrast analysis of single cells can be complemented by backscatter analysis of batch cultures, we investigated the wildtype, a deletion mutant known to affect circadian rhythms ({Delta}kaiC3) and complementation strains at both, the single-cell and batch levels. We concluded that phase contrast and backscatter likely measured the same rhythmic changes in the refractive index of the cells. The method presented here will advance circadian research by enabling the analysis of circadian rhythms in individual cells without the need for expression of reporter molecules.

microbiology↗

bioRxiv2026

Choosing the best motion artefact correction: simplified and advanced how-to guides using QT-NIRS

SignificanceSelecting the appropriate motion artefact (MA) correction method for functional near-infrared spectroscopy (fNIRS) data is quite challenging, particularly in light of the need for standardised practice, replication, and transparency in the field. A clear framework for making measurable and replicable decisions is therefore essential. AimThis paper proposes a guide based on an open-source data quality assessment tool (QT-NIRS) that enables a transparent and evidence-driven choice of MA correction method. ApproachWe present the guide in two approaches: a simplified version that is easy to run for beginners, and an advanced version providing more informative output at the cost of additional computations and minor changes to the original QT-NIRS code. Due to its high flexibility and within-subject nature, the method is applicable across samples with varied characteristics. ResultsWe applied the guide to two challenging datasets from 60 British preschoolers (mean age = 3.94 years, SD = 0.49) and 39 South African school children (mean age = 12.00 years, SD = 0.51). Both simplified and advanced approaches supported similar MA correction methods. ConclusionsWhile both approaches can be used interchangeably, we recommend the advanced approach when possible due to its more informative and straightforward output, and advise caution when using the simplified version.

neuroscience↗

bioRxiv2026

Counting apples - How does the prefrontal region support early numerical understanding in preschool children?

Because early maths skills strongly predict later outcomes, it is crucial to understand the mechanisms that shape early learning in children. The recent years have seen an increase in studying the neural correlates that support the acquisition of maths skills. However, existing work in early childhood has primarily focused on core number-processing regions in the parietal regions, with comparatively little attention to the supportive role of prefrontal regions. In this study, we examined the engagement of the prefrontal regions when matching numbers and objects. Children (N=60, 25 girls, aged 2.74-5.18 years) matched auditory small (1-3) and large (5-7) numbers, as well as objects (fruits) to corresponding visual pictures while their frontoparietal brain responses were recorded using functional near-infrared spectroscopy (fNIRS). Importantly, matching large numbers was substantially more difficult than matching small numbers or objects. The analysis revealed that children had increased activation in the right middle frontal gyrus when matching large numbers, compared to small numbers. However, there was no difference in the prefrontal region between matching small numbers and objects. The connectivity analysis further revealed increased frontoparietal connectivity when matching small numbers, but not large numbers or objects. Our findings suggest that prefrontal involvement during early numerical knowledge acquisition relies primarily on domain-general mechanisms, with number-specific responses likely to emerge later in development.

neuroscience↗

bioRxiv2026

Terminally Differentiated Influenza-Specific Effector Memory B Cells Circulate after Live Attenuated Influenza Vaccination

Live attenuated influenza vaccination (LAIV) is the only FDA approved mucosal vaccine. Easily assayed, circulating correlates of protection after LAIV are lacking. Using fluorochrome labeled hemagglutinin (HA) antigen, we previously identified a subset of HA-specific (HA+) IgDneg memory B cells that circulate after inactivated intramuscular vaccination (IIV), express the master transcriptional regulator, T-bet, as well as effector memory genes and predict durable antibody (Ab) responses to IIV. Here we profile the circulating HA+ IgDneg memory B cell response in a cohort of immunized patients who seroconvert after LAIV to identify which, if any, circulating HA+ B cells predict antibody responses after LAIV. Although we report LAIV elicits circulating T-bet+ HA+ IgDneg B cells that are phenotypically similar to those we described after IIV, we find no correlation between the magnitude of these cells and systemic HA-IgG responses after LAIV. Supervised and unsupervised analyses demonstrate that unlike IIV, LAIV preferentially elicits circulating HA+ IgDneg B cells that co-express TBX21 and the terminal effector cell gene, Zeb2. Consistent with their terminal differentiation status, LAIV-elicited T-bet+ cells cannot be recalled as short-lived antibody-secreting cells (ASCs) after systemic or mucosal antigen re-challenge. We conclude that the transcriptional profiles and functions of HA+ IgDneg B cells vary by influenza vaccine platform.

immunology↗

bioRxiv2024

Detection of titanium nanoparticles in human, animal and infant formula milk

The sustainability of mammals on Earth relies on milk. During lactation, maternal exposure to pollutants like metal nanoparticles (NPs) can affect offspring development and survival. Despite being banned from food applications in Europe due suspected toxicity, titanium dioxide (TiO2) NPs are still massively manufactured for countless other uses. While contamination of ecosystems is well documented, contamination of mammals remains underexplored. Here, we used synchrotron X-ray fluorescence and single particle inductively coupled plasma mass spectrometry to analyse human, animal, and infant formula milk. Titanium containing micro- and nano-particles were detected in all samples, regardless of the species, location, and processing. We identified varying concentrations, sizes, and combinations of rutile and anatase TiO2, ilmenite FeTiO3 and possibly titanite CaTiSiO5 or pseudobrookite Fe2TiO5. These findings suggest that milk serves as a carrier for titanium-containing nanomaterials to expose newborns on a daily basis until weaning.

physiology↗

bioRxiv2024

During haptic communication, the central nervous system compensates distinctly for delay and noise

Connected humans have been previously shown to exploit the exchange of haptic forces and tactile information to improve their performance in joint action tasks. As human interactions are increasingly mediated through robots and networks it is important to understand the impact that network features such as lag and noise may have on human behaviour. In this paper, we investigated the interaction with a human-like robot controller that provides similar haptic communication behaviour as human-human interaction and examined the influence and compensation mechanisms for delay and noise on haptic communication. The results of our experiments show that participants can distinguish between noise and delay, and make use of compensation mechanisms to preserve performance in both cases. However, while noise is compensated for by increasing co-contraction, delay compensation could not be explained by this strategy. Instead, computational modelling suggested that a feed-forward prediction mechanism is used to compensate for the temporal delay and yield an efficient haptic communication. Author summaryIncreasingly humans are making use of networks and robots to coordinate haptic interactions through teleoperation. However, with networks comes delays and noise that can change both the force that is transmitted and how we perceive that force. The haptic communication involved in joint actions, such as moving a piano or performing a pair spin, has been shown to improve performance, but how does delay affect this behaviour? We tested how participants tracked a moving target with their right hand when connected to a human-like robotic partner, when perturbed by delay or noise. Through a comparison between noise and delay perturbation in experimental performance and in simulation with a computational model, we found that participants could from small values of perturbation identify if the perturbation was from delay or noise and that they adopted different compensation strategies in each case.

neuroscience↗

bioRxiv2023

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.

cancer biology↗

bioRxiv2022

FOXA2 controls the antioxidant response in FH-deficient cells independent of NRF2

Hereditary Leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The activation of the anti-oxidant response is key for cellular survival in FH-deficient cells, yet the extent to which chromatin remodelling shapes the anti-oxidant response is currently unknown. Here, we explored the global effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the highly remodelled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor which directly regulates anti-oxidant response genes and subsequent metabolic rewiring. Moreover, we also find that FOXA2 regulates anti-oxidant genes independent of the canonical anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides new insights into the molecular mechanisms behind cell responses to fumarate accumulation, and potentially provides new avenues for therapeutic intervention for HLRCC.

cancer biology↗

bioRxiv2021

KEAP1 mutation in lung adenocarcinoma promotes immune evasion and immunotherapy resistance

Lung cancer treatment has benefited greatly from the development of effective immune-based therapies. However, these strategies still fail in a large subset of patients. Tumor-intrinsic mutations can drive immune evasion via recruiting immunosuppressive populations or suppressing anti-tumor immune responses. KEAP1 is one of the most frequently mutated genes in lung adenocarcinoma patients and is associated with poor prognosis and inferior response to all therapies, including checkpoint blockade. Here, we established a novel antigenic lung cancer model and showed that Keap1-mutant tumors promote dramatic remodeling of the tumor immune microenvironment. Combining single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. Importantly, analysis of KEAP1 mutant patient tumors revealed analogous decrease in dendritic cell and T cell infiltration. Our study provides new insight into the role of KEAP1 mutations in promoting immune evasion and suggests a path to novel immune-based therapeutic strategies for KEAP1 mutant lung cancer. Statement of significanceThis study establishes that tumor-intrinsic KEAP1 mutations contribute to immune evasion through suppression of dendritic cell and T cell responses, explaining the observed resistance to immunotherapy of KEAP1 mutant tumors. These results highlight the importance of stratifying patients based on KEAP1 status and paves the way for novel therapeutic strategies.

cancer biology↗

bioRxiv2021

Retinoschisin deficiency induces persistent aberrant waves of activity affecting neuroglial signaling in the retina

Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early onset inherited condition caused by mutations in the RS1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of mice lacking RS1, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 13. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Muller glia, spanning all retinal layers and disrupting light-induced signaling. This study highlights a critical role for RS1 in early retinal development with a potential to disrupt circuit formation to central targets. Additionally, it confers a functional role to RS1 beyond the scope of an adhesion molecule and identifies an early onset for dysfunction, a potential temporal target for therapeutic intervention and diagnosis. Significance Statement/ HighlightsO_LIPhotoreceptor inner segments express Rs1 at P5, after which RS1 protein is detected in the inner segments by P9 and throughout the retina at later ages, with structural abnormalities observed by optical coherence tomography at P13 in Rs1 mutant mouse models. C_LIO_LIAberrant glutamate-driven wavelets identified by GCaMP6f-based analyses are a novel pathophysiological feature of RS1 deficient mice that emerge after maximal RS1 expression. C_LIO_LIMuller glia display abnormal radial glutamate-driven coordinated and sporadic bursts of activity in RS1-deficient mice. C_LIO_LIThese data identify a novel pathophysiological feature of RS1-deficient mice and define a window where treatments might be most effective. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/457777v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@3f27eborg.highwire.dtl.DTLVardef@55e5a4org.highwire.dtl.DTLVardef@1577435org.highwire.dtl.DTLVardef@8e55b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

AAV-BR1 targets endothelial cells in the retina to reveal their morphological diversity and to deliver Cx43.

Endothelial cells (ECs) are key players in the development and maintenance of the vascular tree, the establishment of the blood brain barrier and control of blood flow. Disruption in ECs is an early and active component of vascular pathogenesis. However, our ability to selectively target ECs in the CNS for identification and manipulation is limited. Here, in the mouse retina, a tractable model of the CNS, we utilized a recently developed AAV-BR1 system to identify distinct classes of ECs along the vascular tree using a GFP reporter. We then developed an inducible EC-specific ectopic Connexin 43 (Cx43) expression system using AAV-BR1-CAG-DIO-Cx43-P2A-DsRed2 in combination with a mouse line carrying inducible CreERT2 in ECs. We targeted Cx43 because its loss has been implicated in microvascular impariment in numerous diseases such as diabetic retinopathy and vascular edema. GFP-labeled ECs were numerous, evenly distributed along the vascular tree and their morphology was polarized with respect to the direction of blood flow. After tamoxifen induction, ectopic Cx43 was specifically expressed in ECs. Similarly to endogenous Cx43, ectopic Cx43 was localized at the membrane contacts of ECs and it did not affect tight junction proteins. The ability to enhance gap junctions in ECs provides a precise and potentially powerful tool to treat microcirculation deficits, an early pathology in numerous diseases.

neuroscience↗

bioRxiv2020

Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy

Local blood flow control within the CNS is critical to proper function and is dependent on coordination between neurons, glia and blood vessels. Macroglia such as astrocytes and Muller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial involvement, since mice lacking Cx3cr1, exhibited no fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan, abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 weeks due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy. Significance StatementThis work identifies a novel role for microglia, the innate immune cells of the CNS, in the local control of the retinal vasculature and identifies deficits early in diabetes. Microglia contact neurons and vasculature and express several vasoactive agents. Activation of microglial fractalkine-Cx3cr1 signalling leads to capillary constriction and blocking the renin-angiotensin system (RAS) with candesartan abolishes microglial-mediated vasoconstriction in the retina. In early diabetes, reduced retinal blood flow is coincident with capillary constriction, increased microglial-vessel association, loss of microglial-capillary regulation and altered microglial expression of the RAS pathway. While candesartan restores retinal capillary diameter early in diabetes, targeting of microglial-vascular regulation is required to prevent coincident dilation of large retinal vessels and reduced retinal blood flow.

cell biology↗

bioRxiv2020

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. In this study, we investigated the response of oligo-conjugated antibodies towards four variables: Concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations cause unnecessarily high background and that concentrations can be drastically reduced without loss of biological information. Reducing volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background and reduces costs. Background signal can account for a major fraction of the total sequencing and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved. Impact statementOligo-conjugated antibodies are a powerful tool but require thorough optimization to reduce background signal, increase sensitivity, and reduce sequencing costs.

genomics↗

bioRxiv2020

Retina-specific targeting of pericytes reveals structural diversity and enables control of capillary blood flow

Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. Across three lines, under either the PDGFRb or NG2 promoter, the specificity and pattern of Cre activation varied significantly. In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes. Furthermore, in one PDGFRb line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficit, a common pathology in numerous diseases.

neuroscience↗