Muscarinic Acetylcholine Receptor Localization on Distinct Excitatory and Inhibitory Neurons Within the ACC and LPFC of the Rhesus Monkey

Acetylcholine (ACh) can act on pre- and post-synaptic muscarinic receptors (mAChR) in the cortex to influence a myriad of cognitive processes. Two functionally-distinct regions of the prefrontal cortex—the lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC)—are differentially innervated by ascending cholinergic pathways yet, the nature and organization of prefrontal-cholinergic circuitry in primates are not well understood. Using multi-channel immunohistochemical labeling and high-resolution microscopy, we found regional and laminar differences in the subcellular localization and the densities of excitatory and inhibitory subpopulations expressing m1 and m2 muscarinic receptors, the two predominant cortical mAChR subtypes, in the supragranular layers of LPFC and ACC in rhesus monkeys (Macaca mulatta). The subset of m1+/m2+ expressing SMI-32+ pyramidal neurons labeled in layer 3 (L3) was denser in LPFC than in ACC, while m1+/m2+ SMI-32+ neurons co-expressing the calcium-binding protein, calbindin (CB) was greater in ACC. Further, we found between-area differences in laminar m1+ dendritic expression, and m2+ presynaptic localization on cortico-cortical (VGLUT1+) and sub-cortical inputs (VGLUT2+), suggesting differential cholinergic modulation of top-down vs. bottom-up inputs in the two areas. While almost all inhibitory interneurons—identified by their expression of parvalbumin (PV+), CB+, and calretinin (CR+)—expressed m1+, the localization of m2+ differed by subtype and area. The ACC exhibited a greater proportion of m2+ inhibitory neurons compared to the LPFC and had a greater density of presynaptic m2+ localized on inhibitory (VGAT+) inputs targeting proximal somatodendritic compartments and axon initial segments of L3 pyramidal neurons. These data suggest a greater capacity for m2+-mediated cholinergic suppression of inhibition in the ACC compared to the LPFC. The anatomical localization of muscarinic receptors on ACC and LPFC micro-circuits shown here contributes to our understanding of diverse cholinergic neuromodulation of functionally-distinct prefrontal areas involved in goal-directed behavior, and how these interactions maybe disrupted in neuropsychiatric and neurological conditions.

An interolog-based barley interactome as an integration framework for immune signaling

The barley MLA nucleotide-binding, leucine-rich-repeat (NLR) receptor and its orthologs confer recognition specificity to many cereal diseases, including powdery mildew, stem and stripe rust, Victoria blight, and rice blast. We used interolog inference to construct a barley protein interactome (HvInt) comprising 66133 edges and 7181 nodes, as a foundation to explore signaling networks associated with MLA. HvInt was compared to the experimentally validated Arabidopsis interactome of 11253 proteins and 73960 interactions, verifying that the two networks share scale-free properties, including a power-law distribution and small-world network. Then, by successive layering of defense-specific 'omics' datasets, HvInt was customized to model cellular response to powdery mildew infection. Integration of HvInt with expression quantitative trait loci (eQTL) enabled us to infer disease modules and responses associated with fungal penetration and haustorial development. Next, using HvInt and an infection-time-course transcriptome, we assembled resistant (R) and susceptible (S) subnetworks. The resulting differentially co-expressed (R-S) interactome is essential to barley immunity, facilitates the flow of signaling pathways and is linked to Mla through trans eQTL associations. Lastly, next-generation, yeast-two-hybrid screens identified fifteen novel MLA interactors, which were incorporated into HvInt, to predict receptor localization, and signaling response. These results link genomic, transcriptomic, and physical interactions during MLA-specified immunity.

The Multiple Roles of Sphingomyelin in Parkinson’s Disease

Advances over the past decade have improved our understanding of the role of sphingolipid in the onset and progression of Parkinson’s disease. Much attention has been paid to ceramide derived molecules, especially glucocerebroside, and little on sphingomyelin, a critical molecule for brain physiopathology. Sphingomyelin has been proposed to be involved in PD due to its presence in the myelin sheath and for its role in nerve impulse transmission, in presynaptic plasticity, and in neurotransmitter receptor localization. The analysis of sphingomyelin-metabolizing enzymes, the development of specific inhibitors, and advanced mass spectrometry have all provided insight into the signaling mechanisms of sphingomyelin and its implications in Parkinson’s disease. This review describes in vitro and in vivo studies with often conflicting results. We focus on the synthesis and degradation enzymes of sphingomyelin, highlighting the genetic risks and the molecular alterations associated with Parkinson’s disease.

Signaling receptor localization maximizes cellular information acquisition in spatially-structured, natural environments

Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions that are sculpted by processes such as fluid flow and substrate adhesion. Nevertheless, traditional approaches to studying cell sensing assume signals are either uniform or monotonic, neglecting spatial structures of natural environments. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. By viewing cell surface receptors as a sensor network, we develop an information theoretic framework for computing the optimal spatial organization of a sensing system for a given spatial signaling environment. Applying the framework to simulated environments, we find that spatial receptor localization maximizes information acquisition in many natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for redistributing signaling receptors during cell navigation and can be implemented in a cell using a feedback scheme. In a simulated tissue environment, dynamic receptor localization boosts navigation efficiency by 30-fold. Broadly, our framework readily adapts to studying how the spatial organization of signaling components other than receptors can be modulated to improve cellular information processing.

Cytokinin Modulates Cellular Trafficking and the Cytoskeleton, Enhancing Defense Responses

The plant hormone cytokinin (CK) plays central roles in plant development and throughout plant life. The perception of CKs initiating their signaling cascade is mediated by histidine kinase receptors (AHKs). Traditionally thought to be perceived mostly at the endoplasmic reticulum (ER) due to receptor localization, CK was recently reported to be perceived at the plasma membrane (PM), with CK and its AHK receptors being trafficked between the PM and the ER. Some of the downstream mechanisms CK employs to regulate developmental processes are unknown. A seminal report in this field demonstrated that CK regulates auxin-mediated lateral root organogenesis by regulating the endocytic recycling of the auxin carrier PIN1, but since then, few works have addressed this issue. Modulation of the cellular cytoskeleton and trafficking could potentially be a mechanism executing responses downstream of CK signaling. We recently reported that CK affects the trafficking of the pattern recognition receptor LeEIX2, influencing the resultant defense output. We have also recently found that CK affects cellular trafficking and the actin cytoskeleton in fungi. In this work, we take an in-depth look at the effects of CK on cellular trafficking and on the actin cytoskeleton in plant cells. We find that CK influences the actin cytoskeleton and endomembrane compartments, both in the context of defense signaling—where CK acts to amplify the signal—as well as in steady state. We show that CK affects the distribution of FLS2, increasing its presence in the plasma membrane. Furthermore, CK enhances the cellular response to flg22, and flg22 sensing activates the CK response. Our results are in agreement with what we previously reported for fungi, suggesting a fundamental role for CK in regulating cellular integrity and trafficking as a mechanism for controlling and executing CK-mediated processes.

Huntingtin and the Synapse

Huntington disease (HD) is a monogenic disease that results in a combination of motor, psychiatric and cognitive symptoms. HD is caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which results in the production of a pathogenic mutant HTT protein (mHTT). Although there is no cure at present for HD, a number of RNA-targeting therapies have recently entered clinical trials which aim to lower mHTT production through the use of antisense oligonucleotides (ASOs) and RNAi. However, many of these treatment strategies are non-selective in that they cannot differentiate between non-pathogenic wild type HTT (wtHTT) and the mHTT variant. As HD patients are already born with decreased levels of wtHTT, these genetic therapies may result in critically low levels of wtHTT. The consequence of wtHTT reduction in the adult brain is currently under debate, and here we argue that wtHTT loss is not well-tolerated at the synaptic level. Synaptic dysfunction is an extremely sensitive measure of subsequent cell death, and is known to precede neurodegeneration in numerous brain diseases including HD. The present review focuses on the prominent role of wtHTT at the synapse and considers the consequences of wtHTT loss on both pre- and postsynaptic function. We discuss how wtHTT is implicated in virtually all major facets of synaptic neurotransmission including anterograde and retrograde transport of proteins to/from terminal buttons and dendrites, neurotransmitter release, endocytic vesicle recycling, and postsynaptic receptor localization and recycling. We conclude that wtHTT presence is essential for proper synaptic function.

SARS-CoV-2, a Threat to Marine Mammals? A Study from Italian Seawaters

Zoonotically transmitted coronaviruses were responsible for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing the dramatic Coronavirus Disease-2019 (CoViD-19) pandemic, which affected public health, the economy, and society on a global scale. The impact of the SARS-CoV-2 pandemic permeated into our environment and wildlife as well; in particular, concern has been raised about the viral occurrence and persistence in aquatic and marine ecosystems. The discharge of untreated wastewaters carrying infectious SARS-CoV-2 into natural water systems that are home to sea mammals may have dramatic consequences on vulnerable species. The efficient transmission of coronaviruses raises questions regarding the contributions of virus-receptor interactions. The main receptor of SARS-CoV-2 is Angiotensin Converting Enzyme-2 (ACE-2), serving as a functional receptor for the viral spike (S) protein. This study aimed, through the comparative analysis of the ACE-2 receptor with the human one, at assessing susceptibility to SARS-CoV-2 for different species of marine mammals living in Italian waters. We also determined, by means of immunohistochemistry, ACE-2 receptor localization in the lung tissue from different cetacean species, in order to provide a preliminary characterization of ACE-2 expression in the marine mammal respiratory tracts. Furthermore, to evaluate if and how Italian wastewater management and coastal exposition to extreme weather events may led to susceptible marine mammal populations being exposed to SARS-CoV-2, geomapping data were carried out and overlapped. The results showed the potential SARS-CoV-2 exposure for marine mammals inhabiting Italian coastal waters, putting them at risk when swimming and feeding in specific risk areas. Thus, we highlighted the potential hazard of the reverse zoonotic transmission of SARS-CoV-2 infection, along with its impact on marine mammals regularly inhabiting the Mediterranean Sea, while also stressing the need for appropriate action in order to prevent further damage to specific vulnerable populations.

Localization of the Folate Transport Systems in the Murine Central Nervous System

Objectives Folates are critical for normal neurodevelopment, and folate transport in the brain is primarily mediated by folate receptor alpha (FRα) at the blood-cerebrospinal fluid barrier (BCSFB). However, studies have reported folate transporter/receptor expression in other brain compartments, which may significantly contribute to overall brain folate uptake. The objective of this study is to characterize the localization of the folate transport systems i.e., reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and FRα in the mouse central nervous system, which will provide insight on novel routes of brain folate transport. In particular, folate transporter/receptor localization is examined at brain barriers [blood-brain barrier (BBB), BCSFB, arachnoid barrier (AB)] and in brain parenchyma (astrocytes, microglia, neurons). Methods The localization of RFC, PCFT and FRα was observed in the brains of C57BL6/N wildtype mice by applying immunohistochemistry (IHC). Mouse brains were isolated, and IHC was performed on frozen coronal sections. Transporter/receptor localization was examined at brain barriers (BBB, BCSFB, AB) and in brain parenchyma (astrocytes, neurons, microglia) using specific antibodies. Standard IHC markers were utilized to identify various brain compartments, with confocal microscopy used for imaging. Results At the mouse BBB and BCSFB, localization of RFC, PCFT and FRα was observed, which is consistent with previous reported data and our own work. At the AB, in astrocytes and neurons localization of RFC and PCFT (but not FRα) was observed. In microglia, no expression of the folate transporters or receptor was detected. Conclusions RFC and PCFT localization at the AB may represent a novel route of folate transport into the CSF, with transporter expression in neurons and astrocytes facilitating folate uptake into brain parenchyma cellular targets. Modulating folate transport at these brain compartments may provide a novel strategy in increasing brain folate uptake in disorders associated with defective FRα and impaired brain folate transport at the BCSFB. Funding Sources This work is supported by the Natural Sciences and Engineering Research Council of Canada (RB). VS is a recipient of several graduate scholarships.