Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin from peripheral blood and using them as a biomarker discovery platform for neurological disorders. This methodology has demonstrated excellent diagnostic and predictive performance for Alzheimer’s and other neurodegenerative diseases in multiple studies, providing a strong proof of concept for this approach. Here, we describe our methodology in detail and offer further evidence that isolated EVs are enriched for neuronal origin. In addition, we present evidence that EVs enriched for neuronal origin represent a more sensitive and accurate base for biomarkers than plasma, serum or non-enriched total plasma EVs. Finally, we proceed to investigate the protein content of EV enriched for neuronal origin and compare it with other relevant enriched and non-enriched populations of plasma EVs. Neuronal-origin enriched plasma EVs contain higher levels of signaling molecules of great interest for cellular metabolism, survival, and repair, which may be useful as biomarkers and to follow response to therapeutic interventions in a mechanism-specific manner.

In a previous study, we showed that the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP ₇ ) physiologically phosphorylates mammalian and yeast proteins. We now report that this phosphate transfer reflects pyrophosphorylation. Thus, proteins must be prephosphorylated by ATP to prime them for IP ₇ phosphorylation. IP ₇ phosphorylates synthetic phosphopeptides but not if their phosphates have been masked by methylation or pyrophosphorylation. Moreover, IP ₇ phosphorylated peptides are more acid-labile and more resistant to phosphatases than ATP phosphorylated peptides, indicating a different type of phosphate bond. Pyrophosphorylation may represent a novel mode of signaling to proteins.

To characterize associations between antiepileptic drugs with sedating or anesthetic effects (third-line antiepileptic drugs) vs. other antiepileptic agents, and short-term outcomes, in status epilepticus. Furthermore, to evaluate the role of adverse hemodynamic and respiratory effects of these agents in status epilepticus treatment. Retrospective comparative analysis. Tertiary academic medical center with two emergency departments and two neurologic intensive care units. Adults admitted with a diagnosis of status epilepticus defined as seizures lasting continuously >5 mins, or for discrete periods in succession. None. Of 126 patients with 144 separate status epilepticus admissions, 57 were female (45%) with mean age 54.7 ± 15.7 yrs. Status epilepticus was convulsive in 132 cases (92%). Status epilepticus etiologies included subtherapeutic antiepileptic drugs (43%), alcohol or other nonantiepileptic drug (13%), and acute central nervous system disease (12%). Third-line antiepileptic drugs were administered in 47 cases (33%). Seventy-eight status epilepticus episodes (54%) had good outcomes (Glasgow Outcome Score = 1, 2) at the time of hospital discharge. On univariate analysis, poor outcome (Glasgow Outcome Score > 2) was associated with older age (mean 59.8 ± 15.5 vs. 50.5 ± 13.8 yrs, p < .001), acute central nervous system disease (21% vs. 4%, p = .001), mechanical ventilation (76% vs. 53%, p = .004), longer duration of ventilation (median 10 days [range 1-56] vs. 2 days [range 1-10], p < .001), treatment with vasopressors (35% vs. 5%, p < .001), and treatment with third-line antiepileptic drugs (51% vs. 17%, p < .001). Death was associated with acute central nervous system disease, prolonged ventilation, treatment with vasopressors, and treatment with third-line antiepileptic drugs. Predictors of poor outcome among all status epilepticus episodes were older age (odds ratio 1.06; 95% confidence interval 1.03-1.09; p < .001), treatment with third-line antiepileptic therapy (odds ratio 5.64; 95% confidence interval 2.31-13.75; p < .001), and first episode of status epilepticus (odds ratio 3.73; 95% confidence interval 1.38-10.10; p = .010). Among status epilepticus episodes treated by third-line antiepileptic drugs, predictors of poor outcome were older age (odds ratio, 1.09; 95% confidence interval 1.01-1.18; p = .038) and longer ventilation (odds ratio, 1.47; 95% confidence interval 1.08-2.00; p = .015). Predictors of mortality among all status epilepticus episodes were treatment with third-line antiepileptic drugs (odds ratio, 12.08; 95% confidence interval 2.30-63.39; p = .003) and older age (odds ratio, 1.06; 95% confidence interval 1.00-1.12; p = .045). Third-line antiepileptic drug therapies with sedating or anesthetic effects predicted poor outcome and death in status epilepticus. Hypotension requiring vasopressor therapy and duration of mechanical ventilation induced by these agents may be contributing factors, especially when pentobarbital is used. These findings may inform decision making on drug therapy in status epilepticus and help develop safer and more effective treatment strategies to improve outcome.

Inositol pyrophosphates have been implicated in numerous biological processes. Inositol hexakisphosphate kinase-2 (IP6K2), which generates the inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), influences apoptotic cell death. The tumor suppressor p53 responds to genotoxic stress by engaging a transcriptional program leading to cell-cycle arrest or apoptosis. We demonstrate that IP6K2 is required for p53-mediated apoptosis and modulates the outcome of the p53 response. Gene disruption of IP6K2 in colorectal cancer cells selectively impairs p53-mediated apoptosis, instead favoring cell-cycle arrest. IP6K2 acts by binding directly to p53 and decreasing expression of proarrest gene targets such as the cyclin-dependent kinase inhibitor p21.

There is an urgent need for effective therapies to restore neurologic function and decrease disability following traumatic brain injury (TBI). Here, emerging findings on the mechanisms of post-TBI neural repair and regeneration, as well as therapeutic implications, are selectively reviewed. Recent discoveries include the characterization of the inhibitory signaling systems within the injury site, postinjury stem cell niche activation, the role of serotonin signaling in repair, and environment enrichment. A potentially transformative finding has been the identification of exosomes, nano-sized extracellular vesicles which have key roles in cell signaling, and might serve as novel biomarkers and as vehicles for targeted delivery of repair-inducing molecules. In the experimental setting, post-TBI repair can be promoted by modulation of inhibitory signaling, neurotrophic factor administration, and amplified serotonin signaling; additional strategies include mobilization of endogenous stem cell populations, exogenous cell-based therapies, and environmental enhancement. Feasibility, safety, and efficacy of these approaches need further investigation in humans. Studies are also needed to evaluate biomarkers based on molecular traces of neural repair and regeneration, which could transform prognostic and predictive modeling of post-TBI recovery trajectories.

The inositol pyrophosphate, diphosphoinositol pentakisphosphate, regulates p53 and protein kinase Akt signaling, and its aberrant increase in cells has been implicated in apoptosis and insulin resistance. Inositol hexakisphosphate kinase-2 (IP6K2), one of the major inositol pyrophosphate synthesizing enzymes, mediates p53-linked apoptotic cell death. Casein kinase-2 (CK2) promotes cell survival and is upregulated in tumors. We show that CK2 mediated cell survival involves IP6K2 destabilization. CK2 physiologically phosphorylates IP6K2 at amino acid residues S347 and S356 contained within a PEST sequence, a consensus site for ubiquitination. HCT116 cells depleted of IP6K2 are resistant to cell death elicited by CK2 inhibitors. CK2 phosphorylation at the degradation motif of IP6K2 enhances its ubiquitination and subsequent degradation. IP6K2 mutants at the CK2 sites that are resistant to CK2 phosphorylation are metabolically stable.