The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 (SH2B1α-SH2B1δ) are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1-/- [knockout (KO)] mice exhibit decreased neurite complexity and length, and BDNF-induced expression of the synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, SH2B1ß and SH2B1γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ resides primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc and FosL1.

2022 Cannon lecture: an ode to signal transduction: how the growth hormone pathway revealed insight into height, malignancy, and obesity.

Walter Cannon was a highly regarded American neurologist and physiologist with extremely broad interests. In the tradition of Cannon and his broad interests, we discuss our laboratory's multifaceted work in signal transduction over the past 40+ years. We show how our questioning of how growth hormone (GH) in the blood communicates with cells throughout the body to promote body growth and regulate body metabolism led to insight into not only body height but also important regulators of malignancy and body weight. Highlights include finding that 1) A critical initiating step in GH signal transduction is GH activating the GH receptor-associated tyrosine kinase JAK2; 2) GH activation of JAK2 leads to activation of a number of signaling proteins, including STAT transcription factors; 3) JAK2 is autophosphorylated on multiple tyrosines that regulate the activity of JAK2 and recruit signaling proteins to GH/GH receptor/JAK2 complexes; 4) Constitutively activated STAT proteins are associated with cancer; 5) GH activation of JAK2 recruits the adapter protein SH2B1 to GH/GH receptor/JAK2 complexes where it facilitates GH regulation of the actin cytoskeleton and motility; and 6) SH2B1 is recruited to other receptors in the brain, where it enhances satiety, most likely in part by regulating leptin action and neuronal connections of appetite-regulating neurons. These findings have led to increased understanding of how GH functions, as well as therapeutic interventions for certain cancer and obese individuals, thereby reinforcing the great importance of supporting basic research since one never knows ahead of time what important insight it can provide.

Acidification Activates Toxoplasma gondii Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity.

Pathogenic microbes rely on environmental cues to initiate key events during infection such as differentiation, motility, egress and invasion of cells or tissues. Earlier investigations showed that an acidic environment activates motility of the protozoan parasite T. gondii. Conversely, potassium ions, which are abundant in the intracellular milieu that bathes immotile replicating parasites, suppress motility. Since motility is required for efficient parasite cell invasion and egress we sought to better understand its regulation by environmental cues. We found that low pH stimulates motility by triggering Ca2+-dependent secretion of apical micronemes, and that this cue is sufficient to overcome suppression by potassium ions and drive parasite motility, cell invasion and egress. We also discovered that acidification promotes membrane binding and cytolytic activity of perforin-like protein 1 (PLP1), a pore-forming protein required for efficient egress. Agents that neutralize pH reduce the efficiency of PLP1-dependent perforation of host membranes and compromise egress. Finally, although low pH stimulation of microneme secretion promotes cell invasion, it also causes PLP1-dependent damage to host cells, suggesting a mechanism by which neutral extracellular pH subdues PLP1 activity to allow cell invasion without overt damage to the target cell. These findings implicate acidification as a signal to activate microneme secretion and confine cytolytic activity to egress without compromising the viability of the next cell infected.

Cerebral complement C1q activation in chronic Toxoplasma infection.

Exposure to the neurotropic parasite, Toxoplasma gondii, causes significant brain and behavioral anomalies in humans and other mammals. Understanding the cellular mechanisms of T. gondii-generated brain pathologies would aid the advancement of novel strategies to reduce disease. Complement factor C1q is part of a classic immune pathway that functions peripherally to tag and remove infectious agents and cellular debris from circulation. In the developing and adult brain, C1q modifies neuronal architecture through synapse marking and pruning. T. gondii exposure and complement activation have both been implicated in the development of complex brain disorders such as schizophrenia. Thus, it seems logical that mechanistically, the physiological pathways associated with these two factors are connected. We employed a rodent model of chronic infection to investigate the extent to which cyst presence in the brain triggers activation of cerebral C1q. Compared to uninfected mice, cortical C1q was highly expressed at both the RNA and protein levels in infected animals bearing a high cyst burden. In these mice, C1q protein localized to cytoplasm, adjacent to GFAP-labeled astrocytes, near degenerating cysts, and in punctate patterns along processes. In summary, our results demonstrated an upregulation of cerebral C1q in response to latent T. gondii infection. Our data preliminarily suggest that this complement activity may aid in the clearance of this parasite from the CNS and in so doing, have consequences for the connectivity of neighboring cells and synapses.

ER-associated degradation adapter Sel1L is required for CD8+ T cell function and memory formation following acute viral infection.

The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here, we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal bioenergetics and c-Myc expression. Finally, we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together, these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence.

Role of the Beta and Gamma Isoforms of the Adapter Protein SH2B1 in Regulating Energy Balance.

Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperphagia, and insulin resistance-phenotypes mimicked by mice lacking Sh2b1. SH2B1ß and γ isoforms are expressed ubiquitously, whereas SH2B1α and δ isoforms are expressed primarily in the brain. Restoring SH2B1ß driven by the neuron-specific enolase promoter largely reverses the metabolic phenotype of Sh2b1-null mice, suggesting crucial roles for neuronal SH2B1ß in energy balance control. Here we test this hypothesis by using CRISPR/Cas9 gene editing to delete the ß and γ isoforms from the neurons of mice (SH2B1ßγ neuron-specific knockout [NKO] mice) or throughout the body (SH2B1ßγ knockout [KO] mice). While parameters of energy balance were normal in both male and female SH2B1ßγ NKO mice, food intake, body weight, and adiposity were increased in male (but not female) SH2B1ßγ KO mice. Analysis of long-read single-cell RNA seq data from wild-type mouse brain revealed that neurons express almost exclusively the α and δ isoforms, whereas neuroglial cells express almost exclusively the ß and γ isoforms. Our work suggests that neuronal SH2B1ß and γ are not primary regulators of energy balance. Rather, non-neuronal SH2B1ß and γ in combination with neuronal SH2B1α and δ suffice for body weight maintenance. While SH2B1ß/γ and SH2B1α/δ share some functionality, SH2B1ß/γ appears to play a larger role in promoting leanness.

Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem).

We present a short insight into the problem of parasitophorous vacuole (PV) formation as a most peculiar kind of cell vacuolization occurring in the course of intracellular development of coccidian pathogens of the genera Eimeria, Isospora, Toxoplasma, Sarcocystis, Cryptosporidium, Epieimeria, and Karyolysus. The review focuses on the morpho-functional diversity of PVs in these parasites. By the present time, the PVs containing different parasite genera and species have been examined to different extent. The membrane of the PV (PVM) obviously derives from the host cell plasmalemma. But soon after parasite penetration, the morphofunctional organization and biochemical composition of the PVM drastically changes: its proteins are selectively excluded and those of the parasite are incorporated. As the result, the PV becomes not fusigenic for lysosomes or any other vacuoles or vesicles, because host cell surface markers necessary for membrane fusion are eliminated from the PVM during parasite invasion. The pattern of the PVs is parasite specific and demonstrates a broad diversity within the same genera and species and even at different stages of the endogenous development. The PV is far from being an indifferent membrane vesicle containing the parasite. Instead, it represents a dynamic system that reflects the innermost events of host-parasite relationships, thus promoting the accomplishing of the parasite life cycle, which, in its turn, is a necessary prerequisite of the parasite eventual survival as a species.