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Biology subjects

Kramer, P.

Publications and source records attributed to Kramer, P..

7 recordsLinked to original sources

Seizures in a Dravet Syndrome mouse model inhibit the locus coeruleus but locally evoke supraphysiologic norepinephrine release

Locus coeruleus (LC) neurons are the primary source of norepinephrine / noradrenaline (NE) released to most brain structures, including seizure-prone neocortical and limbic regions. LC-NE networks regulate numerous physiological processes, including arousal, attention, and memory. Previous evidence suggests that LC-NE neurons may be involved in epilepsy and that their activation may have therapeutic effects on seizures, but the endogenous activity of the LC-NE network during seizures remains largely unclear. We combined electrocorticography recordings and in vivo imaging to measure somatic activity of LC-NE neurons as well as downstream NE release during hyperthermia-induced seizures in a well-characterized pre-clinical model of Dravet Syndrome. We found that population LC-NE somatic activity was inhibited during seizures, whereas downstream NE release was dramatically elevated. To reconcile these discordant findings, we established that activation of downstream regions is capable of locally evoking NE release, in part via activation of glutamate receptors, consistent with the Glutamate Amplifies Noradrenergic Effects (GANE) model. Finally, we confirmed that ictal NE release occurred across additional downstream brain regions, epilepsy models, and seizure induction methods. Thus, disruption of NE signaling is likely a widespread ictal signature, with implications for impaired consciousness during and after seizures.

neuroscience↗

Tex15 is required for vomeronasal sensory neuron diversity and male pheromone detection

The mouse vomeronasal organ (VNO) detects pheromones, which provide vital information needed to find a mate, detect predators, or alert to the presence of an intruder. Within the VNO, pheromones are sensed by two families of G-protein Coupled Receptors (GPCRs) the type 1 and type 2 vomeronasal receptors (V1Rs and V2Rs). However, little is known about the regulatory mechanisms that control the expression of V1R and V2R genes by vomeronasal sensory neurons (VSNs). Here we identify a transcriptional repressor, Testis-expressed gene 15 (Tex15), that regulates vomeronasal receptor gene choice and is required for the proper functioning of the vomeronasal system in regulating male behavior. We find that Tex15 is transiently expressed by VSN precursors prior to strong VR gene expression. The absence of Tex15 results in a pervasive change in the expression of V1R and V2R genes, which results in a less diverse repertoire of VR expressing cells and includes a dramatic reduction in the expression of specific receptors that have been tied to intermale aggression. Accordingly, Tex15 knockout mice exhibit lowered activation in the Accessory Olfactory Bulb (AOB) after exposure to male odorants, and a loss of stereotyped aggression between male mice. Taken together, these results show that Tex15 plays a critical role in pheromone sensing by ensuring that VSNs express a diverse set of receptor proteins.

molecular biology↗

Generation of apical-out nasal organoids to facilitate viral infection and drug screening

Advanced culture systems such as organoids can serve as powerful platforms to study epithelial physiology, as they recapitulate the organisation and many key functions of the tissue of origin. The nasal epithelium is the first respiratory epithelium that is exposed to inhaled airborne pathogens. As a result, it is crucial to model host-pathogen interactions occurring in this tissue. To facilitate the efficient modelling of these interactions, we have developed a method to generate de novo apical-out nasal organoids from nasal epithelial cell aggregates. Optimisation of this method revealed a stark tissue-specific effect of the culture temperature, as apical-out nasal organoids were generated in much higher efficiency at 32.5 {degrees}C, compared to more widely used temperatures of 37{degrees}C. These organoids are composed of ciliated, basal and goblet cells and are produced in a completely standardised and scalable manner, devoid of any extracellular matrix hydrogel. Moreover, they displayed high homogeneity in size and cellular composition, as well as susceptibility to viral infections and capability to model antiviral drug responses. Here, we describe a method for the efficient and reproducible generation of apical-out nasal organoids with high potential to be utilised in host-pathogen interaction studies and personalised medicine from easy-to-access nasal swabs.

cell biology↗

Effects of Unexpected Underfoot Perturbations During Turning on Measures of Mediolateral Stability and Corresponding Recovery Strategies

Humans regularly walk across uneven terrain, which demands the use of reactive control strategies to maintain forward progress and stability. While reactive control during walking has been well described during straight gait, it is unclear how reactive control differs during turning gait. Because turning is asymmetrical, perturbations to the inside and outside limbs may elicit different reactive adjustments. This study investigates how unexpected underfoot perturbations alter stability measures during turning and how individuals alter their foot placement to maintain stability after such perturbations. Seven healthy adults completed walking trials around a circular track while wearing mechanized shoes that pseudo-randomly delivered underfoot perturbations to either the inside or outside limb. We calculated mediolateral margin of stability corrected for centripetal acceleration (ML MoSC), step width, and step length from kinematic data. Linear mixed effects models compared the effects of perturbation type (inversion vs eversion), perturbation limb (inside vs outside), and their interaction for each outcome measure. ML MoSC was affected by both perturbation type and perturbation limb, with larger changes observed during eversion, and outside, perturbations. Changes to step width and step time during two recovery steps after each perturbation were primarily influenced by the perturbation limb - outside perturbations elicited consistent changes during two recovery steps compared to one altered step after inside perturbations. Perturbations to the outside limb during turning disrupt gait longer than perturbations to the inside limb. This difference across perturbation limb may indicate that outside steps are more important to maintaining and recovering stability than inside ones during turning.

bioengineering↗

Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells

The fetal liver is a hematopoietic organ, hosting a diverse and evolving progenitor population. While human liver organoids derived from pluripotent stem cells (PSCs) mimic aspects of embryonic and fetal development, they typically lack the complex hematopoietic niche and the interaction between hepatic and hematopoietic development. We describe the generation of human Fetal Liver-like Organoids (FLOs), that model human hepato-hematopoietic interactions previously characterized in mouse models. Developing FLOs first integrate a yolk sac-like hemogenic endothelium into hepatic endoderm and mesoderm specification. As the hepatic and hematopoietic lineages differentiate, the FLO culture model establishes an autonomous niche capable of driving subsequent progenitor differentiation without exogenous factors. Consistent with yolk sac-derived waves, hematopoietic progenitor cells (HPCs) within FLOs exhibit multipotency with a preference for myeloid lineage commitment, while retaining fetal B and T cell differentiation potential. We reconstruct in FLOs the embryonic monocyte-to-macrophage and granulocyte immune trajectories within the FLO microenvironment and assess their functional responses in the liver niche. In vivo, FLOs demonstrate a liver engraftment bias of hematopoietic cells, recapitulating a key phenomenon of human hematopoietic ontogeny. Our findings highlight the intrinsic capacity of liver organoids to support hematopoietic development, establishing FLOs as a platform for modeling and manipulating human blood-liver niche interactions during critical stages of development and disease.

developmental biology↗

The dynamic response of quorum-sensing to density is robust to signal supplementation and signal synthase knockouts

Quorum sensing (QS) is a widespread mechanism of environment sensing and behavioral coordination in bacteria. At its core, QS is based on the production, sensing and response to small signaling molecules. Previous work with Pseudomonas aeruginosa shows that QS can be used to achieve quantitative resolution and deliver a dosed response to the bacterias density environment, implying a sophisticated mechanism of control. To shed light on how the mechanistic signal components contribute to graded responses to density, we assess the impact of genetic (AHL signal synthase deletion) and/or signal supplementation (exogenous AHL and PQS addition) perturbations on lasB reaction-norms to changes in density. Our approach condenses data from 2,000 timeseries (over 74,000 individual observations) into a comprehensive view of QS-controlled gene expression across variation in genetic, environmental, and signal determinants of lasB expression. We first confirm that deleting either ({Delta}lasI, {Delta}rhlI) or both ({Delta}lasIrhlI) signal synthase genes attenuates QS response. In the {Delta}rhlI background we show persistent yet attenuated density-dependent lasB expression due to native 3-oxo-C12 signaling. We then test if density-independent quantities of signal (3-oxo-C12, C4, PQS or combined) added to the WT either flatten or increase the reaction norm and find that the WT response is robust to all tested concentrations of signal, alone or in combination. We then move to progressively supplementing the genetic knockouts and find that cognate signal supplementation ({Delta}lasI+3-oxo-C12, {Delta}rhlI+C4) is sufficient to restore lasB expression and as well as reactivity to density encoded by the intact signal synthase. We also find that dual supplementation of the double synthase knockout restores expression but does not flatten the reaction norm. Despite adding a density-independent amount of AHL, the double signal synthase can still quantitively sense density. Our results show that a positive reaction norm to density is robust to multiple combinations of gene deletion and density-independent signal supplementation and that while density-independent signal supplementation can increase mean expression, the WT QS still retains the ability to quantitatively respond to density. Our work develops a modular approach to query the robustness and mechanistic bases of the central environmental sensing phenotype of quorum sensing.

microbiology↗

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion

A rare loss-of-function variant p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8) enriched in Western Finland protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, especially compared with individuals matched for the genotype of a common T2D risk variant in SLC30A8, p.Arg325. In genome-edited human IPS-derived {beta}-like cells, we establish that the p.Arg138* variant results in reduced SLC30A8 expression due to haploinsufficiency. In human {beta}-cells loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function, which was also seen in isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aiming at maintaining insulin secretion capacity in T2D.

genetics↗