Ischemic brain injury occurs in many clinical settings, including stroke, cardiac arrest, hypovolemic shock, cardiac surgery, cerebral edema, and cerebral vasospasm. Decades of work have revealed many important mechanisms related to ischemic brain injury. However, there remain significant gaps in the scientific knowledge to reconcile many ischemic brain injury events. Brain ischemia leads to protein misfolding and aggregation, and damages almost all types of subcellular organelles including mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, etc. Irreparably damaged organelles and insoluble protein aggregates are normally removed by autophagy. The build-up of common autophagic components, such as LC3, p62, and ubiquitinated proteins, are generally observed in brain tissue samples in animal models of both global and focal brain ischemia, but the interpretation of the role of these autophagy-related changes in ischemic brain injury in the literature has been controversial. Many pathological events or mechanisms underlying dysfunctional autophagy after brain ischemia remain unknown. This review aims to provide an update of the current knowledge and future research directions regarding the critical role of dysfunctional autophagy in ischemic brain injury.

Phosphoinositides are essential signaling molecules. The PI5P4K family of phosphoinositide kinases and their substrates and products, PI5P and PI4,5P 2 , respectively, are emerging as intracellular metabolic and stress sensors. We performed an unbiased screen to investigate the signals that these kinases relay and the specific upstream regulators controlling this signaling node. We found that the core Hippo pathway kinases MST1/2 phosphorylated PI5P4Ks and inhibited their signaling in vitro and in cells. We further showed that PI5P4K activity regulated several Hippo- and YAP-related phenotypes, specifically decreasing the interaction between the key Hippo proteins MOB1 and LATS and stimulating the YAP-mediated genetic program governing epithelial-to-mesenchymal transition. Mechanistically, we showed that PI5P interacted with MOB1 and enhanced its interaction with LATS, thereby providing a signaling connection between the Hippo pathway and PI5P4Ks. These findings reveal how these two important evolutionarily conserved signaling pathways are integrated to regulate metazoan development and human disease.

The molecular heterogeneity of Tau is a critical contributor to Alzheimer’s disease (AD) progression and pathology. Currently, very little is known about the prevalence or impact of the diverse collection of Tau proteoforms. Additionally, individual proteoforms may have different interactions or frequencies within the broader proteome. Here, we detected and analyzed Tau proteoforms in the context of the broader proteome in model systems using a novel single-molecule proteomic analysis platform with future applicability in AD samples. We used a single-molecule proteomic analysis platform that leverages a set of target isoform specific and target PTM specific affinity reagents combined with novel instrumentation, single-molecule biochemistry, and machine learning bioinformatics to enable deep proteoform and broad proteome analysis. In this study, we first evaluated the proteoform distribution of Tau using commercially available antibody reagents. Proteoform heterogeneity was compared (specifically splicing and phosphorylation variants) in both healthy and diseased samples. Then, total protein counts were assessed across the proteome using Protein Identification by Short-epitope Mapping (PrISM), which leverages proprietary multi-affinity probes designed to recognize short epitopes and a machine learning algorithm that decodes binding of hundreds of multi-affinity probes into protein quantifications. The approach was evaluated by first measuring defined mixtures of recombinant Tau proteins. Next, we examined Tau enriched from induced pluripotent stem cell (iPSC)-derived neurons and tau-expressing cell lines. The platform revealed the molecular heterogeneity of Tau proteoforms missed by bulk measurements and peptide-centric proteomics approaches. The proteoform data was then supplemented with broader proteome analysis in order to identify proteome-wide changes – differences in proteins and pathways due to or in combination with specific Tau proteoforms and disease. Understanding the biology of complex diseases like AD and other Tauopathies depends on understanding both the specific Tau proteoforms that exist at baseline and in disease as well as the pathways and processes impacted in the proteome. Single-molecule tools that can analyze proteoforms in depth and the proteome broadly and link molecular signatures to disease states are essential. These tools will enable improved biomarkers, improved understanding of disease, and improved therapies for Alzheimer’s disease in the future.

Millions of traumatic brain injuries (TBIs) occur annually. TBIs commonly result from falls, traffic accidents, and sports-related injuries, all of which involve rotational acceleration/deceleration of the brain. During these injuries, the brain endures a multitude of primary insults including compression of brain tissue, damaged vasculature, and diffuse axonal injury. All of these deleterious effects can contribute to secondary brain ischemia, cellular death, and neuroinflammation that progress for weeks, months, and lifetime after injury. While the linear effects of head trauma have been extensively modeled, less is known about how rotational injuries mediate neuronal damage following injury. Here, we developed a new model of repetitive rotational head trauma in rodents and demonstrated acute and prolonged pathological, behavioral, and electrophysiological effects of rotational TBI (rTBI). We identify aberrant Cyclin-dependent kinase 5 (Cdk5) activity as a principal mediator of rTBI. We utilized Cdk5-enriched phosphoproteomics to uncover potential downstream mediators of rTBI and show pharmacological inhibition of Cdk5 reduces the cognitive and pathological consequences of injury. These studies contribute meaningfully to our understanding of the mechanisms of rTBI and how they may be effectively treated.

INTRODUCTION:A wide array of post-translational modifications of the tau protein occurs in Alzheimer's disease (AD) and they are critical to pathogenesis and biomarker development. Several promising tau markers, pT181, pT217, and pT231, rely on increased phosphorylation within a common molecular motif threonine-proline-proline (TPP). METHODS:We validated new and existing antibodies against pT217, pT231, pT175, and pT181, then combined immunohistochemistry (IHC) and immunoassays (ELISA) to broadly examine the phosphorylation of the tau TPP motif in AD brains. RESULTS:The tau burden, as examined by IHC and ELISA, correlates to Braak stages across all TPP sites. Moreover, we observed regional variability across four TPP motif phosphorylation sites in multiple brains of sporadic AD patients. DISCUSSION:We conclude that there is an elevation of TPP tau phosphorylation in AD brains as disease advances. The regional variability of pTPP tau suggests that examining different phosphorylation sites is essential for a comprehensive assessment of tau pathology.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

ABSTRACT Millions of traumatic brain injuries (TBIs) occur annually. TBIs commonly result from falls, traffic accidents, and sports-related injuries, all of which involve rotational acceleration/deceleration of the brain. During these injuries, the brain endures a multitude of primary insults including compression of brain tissue, damaged vasculature, and diffuse axonal injury. All of these deleterious effects can contribute to secondary brain ischemia, cellular death, and neuroinflammation that progress for weeks to months after injury and impede neurological recovery. While the linear effects of head trauma have been extensively modeled, less is known about how rotational injuries mediate neuronal damage following injury. Here, we developed a new model of rotational head trauma in rodents and extensively characterized the pathological, behavioral, and electrophysiological effects of rotational TBI (rTBI). We identify aberrant cyclin dependent kinase 5 (Cdk5) activity as a principal mediator of rTBI and show pharmacological inhibition of Cdk5 reduces the cognitive and pathological consequences of injury. Finally, we utilize Cdk5-enriched phosphoproteomics to uncover potential downstream mediators of rTBI. These studies contribute meaningfully to our understanding of the mechanisms of rTBI and how they may be effectively treated.

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.

Proteomic studies often employ multi-dimensional analytical methods such as nano-LC-ESI-MS/MS to simplify the sample complexity. The time and expertise required to implement LCMS methods can be a barrier to integrating targeted proteomics within translational research programs. We present a robust method that combines multiplexed immuno-affinity capture with MALDI MS, called Affi-BAMS. This platform enables development of highly specific assays for simultaneous profiling of multiple analytes. The workflow utilizes enrichment on single beads that contain one antibody, having enough binding capacity to quantify within 3 orders of magnitude. Multiplexing is achieved by combining assay beads with different specificities. Assay beads are spatially arrayed and captured peptides are eluted into individual micro-wells. The resulting array of micro-spots contain concentrated analytes for direct measurement by MALDI MS. While both intact proteins and protein fragments can be monitored by Affi-BAMS, we focused this work for bottom-up and middle-down proteomics. We illustrate Affi-BAMS assays to several protein targets associated with progression of Alzheimer's disease. Examples include a multiplexed assay for beta-amyloid to monitor fifteen C-terminal fragments, localized within the region of aa672 - aa711 (including AB1-38, AB1-40, AB1-42 & APP669-711). We have also configured assays to monitor twelve different regions of tau (MAPT), spanning from the N-terminus to the C-terminus, including assays for both total and known phosphorylation sites. Lastly, we demonstrate how Affi-BAMS can be used to monitor epigenetic marks on Histone H3 in normal and disease brain. The unique features of this technology include multiplexing capacity that exceeds the current limit of bead-based sandwich immunoassay platforms and the ability to independently screen multiple sites within a single protein. This platform should aid protein panel profiling across a wide range of research applications.

Injury to the CNS induces astrogliosis, an astrocyte-mediated response that has both beneficial and detrimental impacts on surrounding neural and non-neural cells. The precise signaling events underlying astrogliosis are not fully characterized. Here, we show that astrocyte activation was altered and proliferation was reduced in Semaphorin 4B (Sema4B)-deficient mice following injury. Proliferation of cultured Sema4B(-/-) astrocytes was also significantly reduced. In contrast to its expected role as a ligand, the Sema4B ectodomain was not able to rescue Sema4B(-/-) astrocyte proliferation but instead acted as an antagonist against Sema4B(+/-) astrocytes. Furthermore, the effects of Sema4B on astrocyte proliferation were dependent on phosphorylation of the intracellular domain at Ser825. Our results suggest that Sema4B functions as an astrocyte receptor, defining a novel signaling pathway that regulates astrogliosis after CNS injury.

Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers because of its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when overexpressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor's cell surface expression. Disrupting NR2B-Cdk5 interaction via a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers.