Biology subjects

Rychkov, G.

Publications and source records attributed to Rychkov, G..

4 recordsLinked to original sources

bioRxiv2025

Splanchnic and pelvic spinal afferent pathways relay sensory information from the mouse colorectum into distinct brainstem circuits.

This study aimed to identify where the sensory information relayed by the two spinal afferent pathways innervating the distal colon and rectum (colorectum), the splanchnic and pelvic spinal afferent pathways, integrates within the brainstem. Localised injections of transneuronal viral tracer (herpes simplex virus H129 strain expressing EGFP (H129-EGFP)) into the distal colon was used to assess the brainstem structures receiving ascending input from the colorectum. H129-EGFP+ cells were distributed in structures involved in ascending sensory relay, descending pain modulation and autonomic regulation in the medulla from 96 hours and in pontine and caudal midbrain 120 hours after inoculation. In a separate cohort of mice, in vivo noxious colorectal distension (CRD) followed by brainstem immunolabelling for phosphorylated MAP kinase ERK 1/2 (pERK) showed that many of the structures in which H129-EGFP+ labelling was observed were relevant to colorectal sensory processing. Surgical removal of dorsal root ganglia (DRG) containing cell bodies of splanchnic colorectal afferent neurons, significantly reduced CRD evoked neuronal activation within the caudal ventrolateral medulla, rostral ventromedial medulla and the lateral parabrachial nuclei. Whilst, removal of DRG containing cell bodies of pelvic colorectal afferent neurons significantly reduced CRD evoked neuronal activation within the rostral ventromedial medulla, lateral parabrachial nuclei, the locus coeruleus, Barringtons nucleus and periaqueductal gray. Collectively, this study showed that the two spinal afferent pathways innervating the colorectum differentially shape colorectal processing within the brainstem and provides new insight into their unique roles to mediating visceromotor responses and defecation associated with colorectal nociception.

neuroscience↗

bioRxiv2023

Modelling human KCNT1-epilepsy in Drosophila: a seizure phenotype and drug responses

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are resistant to current treatments. In vitro studies have shown that KCNT1-epilepsy mutations are gain of function, significantly increasing K+ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which was sensitive to drugs currently used to treat patients with KCNT1-epilepsy. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila can be used to model human KCNT1-epilepsy and potentially used as a tool to assess new treatments for KCNT1 epilepsy.

neuroscience↗

bioRxiv2021

Ubr1-induced selective endo-phagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress

The defence mechanisms against endo-lysosomal homeostasis stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) ubiquitin ligase that counteracts proteostasis stress by enhancing cargo selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations increased intracellular Ca2+ and caused endosomal compartment stress by fusion and enlargement. Endosomal protein QC pathway using ubiquitin QC ligases CHIP and Ubr1 with ESCRT-machinery was able to target only a fraction of MLC1-mutants for lysosomal degradation. As a consequence of the endosomal stress, we found an alternative QC route dependent on Ubr1, SQSTM1/p62 and arginylation to bypass MLC1-mutants to endosomal autophagy (endo-phagy). Significantly, this unfolded a general biological endo-lysosomal QC pathway for arginylated Ubr1-SQSTM1/p62 autophagy targets during Ca2+-assault. Conversely, the loss of Ubr1 with the absence of arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1-dependent endo-phagy/autophagy in constitutive and provoked endo-lysosomal proteostasis stress, and link Ubr1 to Ca2+-homeostasis and proteins implicated in various diseases including cancers and brain disorders.

biochemistry↗

bioRxiv2020

Orai1- and Orai2-, but not Orai3-mediated ICRAC is regulated by intracellular pH

Three Orai (Orai1, Orai2 and Orai3) and two STIM (STIM1 and STIM2; stromal interaction molecule) mammalian protein homologues constitute major components of the store-operated Ca2+ entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+ dependent inactivation, inhibition/potentiation by 2-APB and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2- and Orai3-mediated CRAC current (ICRAC) by intracellular pH. Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1 we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in intracellular pH (pHi). Orai1-mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+ dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi, whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1-Orai3 chimeras suggests that pHi dependence of Orai1 resides in both, the N-terminus and intracellular loop 2, and may also involve pH-dependent interactions with STIM1.

physiology↗