CD4 T cells and toxicity from immune checkpoint blockade.

Immune-related toxicities, otherwise known as immune-related adverse events (irAEs), occur in a substantial fraction of cancer patients treated with immune checkpoint inhibitors (ICIs). Ranging from asymptomatic to life-threatening, ICI-induced irAEs can result in hospital admission, high-dose corticosteroid treatment, ICI discontinuation, and in some cases, death. A deeper understanding of the factors underpinning severe irAE development will be essential for improved irAE prediction and prevention, toward maximizing the benefits and safety profiles of ICIs. In recent work, we applied mass cytometry, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing, and bulk T-cell receptor (TCR) sequencing to identify pretreatment determinants of severe irAE development in patients with advanced melanoma. Across 71 patients separated into three cohorts, we found that two baseline features in circulation-elevated activated CD4 effector memory T-cell abundance and TCR diversity-are associated with severe irAE development, independent of the affected organ system within 3 months of ICI treatment initiation. Here, we provide an extended perspective on this work, synthesize and discuss related literature, and summarize practical considerations for clinical translation. Collectively, these findings lay a foundation for data-driven and mechanistic insights into irAE development, with the potential to reduce ICI morbidity and mortality in the future.

N-Terminal Acetylation of α-Synuclein Slows down Its Aggregation Process and Alters the Morphology of the Resulting Aggregates.

Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibril-catalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein exhibit a lower ß-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates.

Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites.

BACKGROUND: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (εPKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three εPKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by εPKC. METHODS: Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family. RESULTS: We identified S279 as a critical εPKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues (T384, T433 and S471) in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes. CONCLUSION: εPKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.

Deciphering the structural basis of eukaryotic protein kinase regulation.

Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified "Shell" that surrounds the R-spine using site-directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.

Effects of N-terminal Acetylation on the Aggregation of Disease-related α-synuclein Variants.

Mutations in the SNCA gene, which encodes the protein α-synuclein, have been linked with early onset Parkinson's disease. The exact nature of this association, however, is still poorly understood. To investigate this problem, we started from the observation that α-synuclein is constitutively N-terminally acetylated, a post-translational modification that alters the charge and structure of α-synuclein molecules and affects their interaction with lipid membranes, as well as their aggregation process. We thus studied five N-terminal acetylated familial variants (A30P, E46K, H50Q, G51D and A53T) of α-synuclein through a wide range of biophysical assays to probe the microscopic steps in their aggregation process and the structures of the resulting aggregates. Our results reveal a great complexity in the combined effects of the disease-related mutations with N-terminal acetylation on the aggregation of α-synuclein, which underscores the great sensitivity to even relatively small perturbations of the behaviour of this protein.

Staged endovascular treatment of a coexisting parasellar aneurysm and endoscopic resection of a pituitary macroadenoma: illustrative case.

BACKGROUND: Intracranial aneurysms and pituitary adenomas are relatively common pathologies that, in rare instances, may concurrently present. Their management poses considerable clinical and technical challenges. OBSERVATIONS: The authors present a case of a 66-year-old female patient with a fusiform aneurysm of the left internal carotid artery associated with a symptomatic pituitary macroadenoma that had been causing visual deficits for the past several years. She underwent successful placement of flow diverter stents across her aneurysm, followed by routine dual antiplatelet therapy to maintain stent patency. She underwent frequent serial radiographic, endocrine, and ophthalmological evaluations during this time to ensure stability of her pituitary adenoma. Following confirmation of aneurysm obliteration and subsequent de-escalation of antiplatelet medications to aspirin monotherapy, her tumor was subsequently resected via an endoscopic endonasal approach in a delayed fashion. LESSONS: The authors review the literature regarding management of these concurrent pathologies and describe the aspects of the case that led them to their chosen treatment strategy. An algorithm is proposed regarding the management of parasellar aneurysms with a concurrent diagnosis of pituitary tumor pathology.

T cell characteristics associated with toxicity to immune checkpoint blockade in patients with melanoma.

Severe immune-related adverse events (irAEs) occur in up to 60% of patients with melanoma treated with immune checkpoint inhibitors (ICIs). However, it is unknown whether a common baseline immunological state precedes irAE development. Here we applied mass cytometry by time of flight, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing and bulk T cell receptor (TCR) sequencing to study peripheral blood samples from patients with melanoma treated with anti-PD-1 monotherapy or anti-PD-1 and anti-CTLA-4 combination ICIs. By analyzing 93 pre- and early on-ICI blood samples and 3 patient cohorts (n = 27, 26 and 18), we found that 2 pretreatment factors in circulation-activated CD4 memory T cell abundance and TCR diversity-are associated with severe irAE development regardless of organ system involvement. We also explored on-treatment changes in TCR clonality among patients receiving combination therapy and linked our findings to the severity and timing of irAE onset. These results demonstrate circulating T cell characteristics associated with ICI-induced toxicity, with implications for improved diagnostics and clinical management.

Human iPSC-Derived Cerebral Organoids Model Cellular Features of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial Glia.

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy, and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study, to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology, we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging, immunostaining, and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells, accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia, a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study, therefore, deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders.