Biology subjects

Manikandan, S.

Publications and source records attributed to Manikandan, S..

4 recordsLinked to original sources

bioRxiv2024

Dopamine-driven Increase in IL-1β in Myeloid Cells is Mediated by Differential Dopamine Receptor Expression and Exacerbated by HIV

The catecholamine neurotransmitter dopamine is classically known for regulation of central nervous system (CNS) functions such as reward, movement, and cognition. Increasing evidence also indicates that dopamine regulates critical functions in peripheral organs and is an important immunoregulatory factor. We have previously shown that dopamine increases NF-{kappa}B activity, inflammasome activation, and the production of inflammatory cytokines such as IL-1{beta} in human macrophages. As myeloid lineage cells are central to the initiation and resolution of acute inflammatory responses, dopamine-mediated dysregulation of these functions could both impair the innate immune response and exacerbate chronic inflammation. However, the exact pathways by which dopamine drives myeloid inflammation are not well defined, and studies in both rodent and human systems indicate that dopamine can impact the production of inflammatory mediators through both D1-like dopamine receptors (DRD1, DRD5) and D2-like dopamine receptors (DRD2, DRD3, and DRD4). Therefore, we hypothesized that dopamine-mediated production of IL-1{beta} in myeloid cells is regulated by the ratio of different dopamine receptors that are activated. Our data in primary human monocyte-derived macrophages (hMDM) indicate that DRD1 expression is necessary for dopamine-mediated increases in IL-1{beta}, and that changes in the expression of DRD2 and other dopamine receptors can alter the magnitude of the dopamine-mediated increase in IL-1{beta}. Mature hMDM have a high D1-like to D2-like receptor ratio, which is different relative to monocytes and peripheral blood mononuclear cells (PBMCs). We further confirm in human microglia cell lines that a high ratio of D1-like to D2-like receptors promotes dopamine-induced increases in IL-1{beta} gene and protein expression using pharmacological inhibition or overexpression of dopamine receptors. RNA-sequencing of dopamine-treated microglia shows that genes encoding functions in IL-1{beta} signaling pathways, microglia activation, and neurotransmission increased with dopamine treatment. Finally, using HIV as an example of a chronic inflammatory disease that is substantively worsened by comorbid substance use disorders (SUDs) that impact dopaminergic signaling, we show increased effects of dopamine on inflammasome activation and IL-1{beta} in the presence of HIV in both human macrophages and microglia. These data suggest that use of addictive substances and dopamine-modulating therapeutics could dysregulate the innate inflammatory response and exacerbate chronic neuroimmunological conditions like HIV. Thus, a detailed understanding of dopamine-mediated changes in inflammation, in particular pathways regulating IL-1{beta}, will be critical to effectively tailor medication regimens.

neuroscience↗

bioRxiv2024

Sapap3 knockout mice show threat bias under conflict during platform mediated avoidance task: implications for obsessive compulsive disorder

Obsessive compulsive disorder (OCD) typically involves cycling between symptoms of intrusive aversive thoughts (obsessions) and repetitive rituals aimed at avoiding these aversive outcomes (compulsions), which interferes with patients engagement with other important aspects of their lives. This cycling relationship between obsessions and compulsions highlights a potential role of impaired threat processing and avoidance behavior in OCD symptoms. The most effective behavioral therapy for OCD, exposure with response prevention (ERP), aims to break this cycle. However, it can be difficult for patients to access and engage with, suggesting a need for improved understanding of the neural mechanisms of threat processing and avoidance behavior in OCD to guide development of new and more effective treatments. Platform mediated avoidance (PMA) has proven to be a useful translational paradigm for use in rodents to examine avoidance neurobiology and models relevant to OCD, such as ERP and overtraining-induced persistent avoidance. However, to date this protocol has only been used in rats, and studies in transgenic mouse models relevant to OCD may shed further light on neural mechanisms relevant to disturbances in avoidance and threat processing in the disorder. To address this gap, we tested Sapap3 knockout (KO) mice, a leading preclinical model in OCD research, in the PMA task. Using this paradigm, we examined avoidance acquisition, expression, and extinction, as well as reward seeking under motivational conflict in two separate cohorts conditioned using higher (0.4 mA) or lower (0.24 mA) intensity shock. Surprisingly, the most striking difference observed in Sapap3-KOs vs control mice was heightened suppression of lever pressing for rewards during a tone signaling impending threat, suggesting a shift in action selection under motivational conflict (genotype effect avoidance conditioning: 0.24 mA cohort p=0.011, 0.4 mA cohort p=0.07; avoidance extinction: 0.24 mA p=0.057, 0.4 mA p=0.042). Avoidance responding was also acquired more slowly in Sapap3-KOs trained with a low intensity shock (time x genotype interaction p=0.025) and was extinguished more robustly following ERP (genotype effect p=0.043). In contrast, avoidance was similar between Sapap3 KOs and WT littermate controls trained using higher intensity shock (0.4 mA cohort time effect p<0.0001). Expression of the immediate early gene c-Fos associated with reinstatement of avoidance after ERP showed preliminary evidence for decreased activity of medial orbitofrontal cortex (mOFC) in KOs which may contribute to observed differences in PMA performance (KO vs WT mOFC c-Fos t-test p=0.0456). Together these findings suggest that mOFC dysfunction may contribute to increases in the influence of threats vs rewards over action selection in an animal model with relevance to OCD, and that the Sapap3-KO model presents valuable opportunities for deeper mechanistic investigation of avoidance and threat processing relevant to the human disorder.

neuroscience↗

bioRxiv2023

Formation of clathrin-pits and ATP-independent cholesterol-dependent tubules initiates mechano-regulation on de-adhesion.

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis is not well understood. To investigate this, we study the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical-trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether-force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by a saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes as well as transferrin-labelled pits close to the basal plasma membrane also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked but proceeded uncontrolled. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested on depleting both. The data strongly supports pit-formation to be central to the reduction in fluctuations whether in normal or ATP depleted condition. Furthermore, while in normal conditions the contribution of clathrin-mediated endocytosis is clear, under ATP-depleted conditions we propose that cholesterol-dependent pits spontaneously regulate tension. SummaryWe show that during de-adhesion, cell edges retract, creating membrane folds and increasing fluctuations. Cells increase the rate of endocytosis to regulate back their membrane fluctuations. This is achieved by forming invaginations. Dynamin-dependent pathways are majorly involved, while cholesterol-dependent ATP-independent mechanisms also contribute.

biophysics↗

bioRxiv2022

Tuning aromatic contributions by site-specific encoding of fluorinated phenylalanine residues in bacterial and mammalian cells

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a new group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools will enable deconstruction of biological mechanisms which utilize aromatic-pi interactions in structural and cellular contexts. Statement of SignificanceThe aromatic side-chains of phenylalanine, tyrosine, and tryptophan are crucial for protein function and pharmacology due to their hydrophobic and electrostatic contributions to catalytic centers and ligand-binding pockets. However, few experimental approaches can chemically assess the functional roles of aromatics in cellular environments. The accepted computational method for aromatic interrogation is via serial fluorination, which lacks an experimental correlate in bacterial or mammalian cell systems. We have identified a family of synthetases to encode multiple different types of fluorinated phenylalanine residues in E. coli and HEK cells via nonsense suppression. The efficiency of these synthetases is sufficient to support biochemical characterization and structural determination of proteins with site-specific incorporation of unnatural phenylalanine analogs.

biochemistry↗