In Situ Analysis of N-Linked Glycans as Potential Biomarkers of Clinical Course in Human Prostate Cancer.

Prostate cancer is the most common cancer in men worldwide. Despite its prevalence, there is a critical knowledge gap in understanding factors driving disparities in survival among different cohorts of patients with prostate cancer. Identifying molecular features separating disparate populations is an important first step in prostate cancer research that could lead to fundamental hypotheses in prostate biology, predictive biomarker discovery, and personalized therapy. N-linked glycosylation is a cotranslational event during protein folding that modulates a myriad of cellular processes. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Herein, we performed direct on-tissue analysis of N-linked glycans using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) from tissue microarrays of over 100 patient tumors with over 10 years of follow-up metadata. We successfully identified a panel of N-glycans that are unique between benign and prostate tumor tissue. Specifically, high-mannose as well as tri-and tetra-antennary N-glycans were more abundant in tumor tissue and increase proportionally with tumor grade. Further, we expanded our analyses to examine the N-glycan profiles of Black and Appalachian patients and have identified unique glycan signatures that correlate with recurrence in each population. Our study highlights the potential applications of MALDI-MSI for digital pathology and biomarker discovery for prostate cancer. IMPLICATIONS: MALDI-MSI identifies N-glycan perturbations in prostate tumors compared with benign tissue. This method can be utilized to predict prostate cancer recurrence and study prostate cancer disparities.

Antibody-Mediated Enzyme Therapeutics and Applications in Glycogen Storage Diseases.

The use of antibodies as targeting molecules or cell-penetrating tools has emerged at the forefront of pharmaceutical research. Antibody-directed therapies in the form of antibody-drug conjugates, immune modulators, and antibody-directed enzyme prodrugs have been most extensively utilized as hematological, rheumatological, and oncological therapies, but recent developments are identifying additional applications of antibody-mediated delivery systems. A novel application of this technology is for the treatment of glycogen storage disorders (GSDs) via an antibody-enzyme fusion (AEF) platform to penetrate cells and deliver an enzyme to the cytoplasm, nucleus, and/or other organelles. Exciting developments are currently underway for AEFs in the treatment of the GSDs Pompe disease and Lafora disease (LD). Antibody-based therapies are quickly becoming an integral part of modern disease therapeutics.

Central Nervous System Delivery and Biodistribution Analysis of an Antibody-Enzyme Fusion for the Treatment of Lafora Disease.

Lafora disease (LD) is a fatal juvenile epilepsy characterized by the accumulation of aberrant glucan aggregates called Lafora bodies (LBs). Delivery of protein-based therapeutics to the central nervous system (CNS) for the clearance of LBs remains a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on the efficacy and CNS delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase, in a mouse model of LD. An enzyme-linked immunosorbent assay has been developed to detect VAL-0417 post-treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using this method, we measured biodistribution in different methods of delivery. We found that intracerebroventricular administration provided robust CNS delivery when compared to intrathecal administration. These data define critical steps in the translational pipeline of VAL-0417 for the treatment of LD.

Targeting Pathogenic Lafora Bodies in Lafora Disease Using an Antibody-Enzyme Fusion.

Lafora disease (LD) is a fatal childhood epilepsy caused by recessive mutations in either the EPM2A or EPM2B gene. A hallmark of LD is the intracellular accumulation of insoluble polysaccharide deposits known as Lafora bodies (LBs) in the brain and other tissues. In LD mouse models, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Therefore, LBs have become a therapeutic target for ameliorating LD. Herein, we demonstrate that human pancreatic α-amylase degrades LBs. We fused this amylase to a cell-penetrating antibody fragment, and this antibody-enzyme fusion (VAL-0417) degrades LBs in vitro and dramatically reduces LB loads in vivo in Epm2a-/- mice. Using metabolomics and multivariate analysis, we demonstrate that VAL-0417 treatment of Epm2a-/- mice reverses the metabolic phenotype to a wild-type profile. VAL-0417 is a promising drug for the treatment of LD and a putative precision therapy platform for intractable epilepsy.

Mitochondrial Metabolism in Major Neurological Diseases.

Mitochondria are bilayer sub-cellular organelles that are an integral part of normal cellular physiology. They are responsible for producing the majority of a cell's ATP, thus supplying energy for a variety of key cellular processes, especially in the brain. Although energy production is a key aspect of mitochondrial metabolism, its role extends far beyond energy production to cell signaling and epigenetic regulation⁻functions that contribute to cellular proliferation, differentiation, apoptosis, migration, and autophagy. Recent research on neurological disorders suggest a major metabolic component in disease pathophysiology, and mitochondria have been shown to be in the center of metabolic dysregulation and possibly disease manifestation. This review will discuss the basic functions of mitochondria and how alterations in mitochondrial activity lead to neurological disease progression.

Microdose Lithium NP03 Diminishes Pre-Plaque Oxidative Damage and Neuroinflammation in a Rat Model of Alzheimer's-like Amyloidosis.

BACKGROUND: Microdose lithium is protective against Alzheimer's disease (AD), although the precise mechanisms through which its protective effects are conferred remain unclear. OBJECTIVE: To further examine the effects during the earliest stages of Aß pathology, we evaluated whether NP03, a microdose lithium formulation, modulates Aß-mediated oxidative damage and neuroinflammation when applied to a rat transgenic model of AD-like amyloidosis overexpressing amyloid precursor protein (APP). METHOD: McGill-R-Thy1-APP transgenic rats and wild-type littermates were treated with NP03 or vehicle formulation for 8 weeks beginning at 3 months of age - a phase preceding Aß plaque deposition in the transgenic rats. RESULTS: Oxidative and nitrosative stress markers, protein-bound 4-hydroxynonenal (HNE) and proteinresident 3-nitrotyrosine (3-NT), inflammatory cytokines production, as well as microglial recruitment towards Aß-burdened neurons were assayed. NP03 significantly decreased cerebral HNE and 3-NT, and reduced production of pro-inflammatory cytokines in McGill-R-Thy1-APP transgenic rats. NP03 further reduced expression of microglia surface receptor Trem2 and led to a corresponding reduction in microglia recruitment towards Aß-burdened neurons in the CA1 region of the hippocampus. CONCLUSION: These results suggest that NP03 may function to slow the AD-like pathology in part by modifying oxidative/nitrosative damage and neuroinflammation, raising the possibility that low doses of microencapsulated lithium might be of therapeutic-preventive value during very early or preclinical AD.