The Role of Exosomes in Lysosomal Storage Disorders.

Exosomes, small membrane-bound organelles formed from endosomal membranes, represent a heterogenous source of biological and pathological biomarkers capturing the metabolic status of a cell. Exosomal cargo, including lipids, proteins, mRNAs, and miRNAs, can either act as inter-cellular messengers or are shuttled for autophagic/lysosomal degradation. Most cell types in the central nervous system (CNS) release exosomes, which serve as long and short distance communicators between neurons, astrocytes, oligodendrocytes, and microglia. Lysosomal storage disorders are diseases characterized by the accumulation of partially or undigested cellular waste. The exosomal content in these diseases is intrinsic to each individual disorder. Emerging research indicates that lysosomal dysfunction enhances exocytosis, and hence, in lysosomal disorders, exosomal secretion may play a role in disease pathogenesis. Furthermore, the unique properties of exosomes and their ability to carry cargo between adjacent cells and organs, and across the blood-brain barrier, make them attractive candidates for use as therapeutic delivery vehicles. Thus, understanding exosomal content and function may have utility in the treatment of specific lysosomal storage disorders. Since lysosomal dysfunction and the deficiency of at least one lysosomal enzyme, glucocerebrosidase, is associated with the development of parkinsonism, the study and use of exosomes may contribute to an improved understanding of Parkinson disease, potentially leading to new therapeutics.

Glucocerebrosidase and its relevance to Parkinson disease.

Mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, are among the most common known genetic risk factors for the development of Parkinson disease and related synucleinopathies. A great deal is known about GBA1, as mutations in GBA1 are causal for the rare autosomal storage disorder Gaucher disease. Over the past decades, significant progress has been made in understanding the genetics and cell biology of glucocerebrosidase. A least 495 different mutations, found throughout the 11 exons of the gene are reported, including both common and rare variants. Mutations in GBA1 may lead to degradation of the protein, disruptions in lysosomal targeting and diminished performance of the enzyme in the lysosome.Gaucher disease is phenotypically diverse and has both neuronopathic and non-neuronopathic forms. Both patients with Gaucher disease and heterozygous carriers are at increased risk of developing Parkinson disease and Dementia with Lewy Bodies, although our understanding of the mechanism for this association remains incomplete. There appears to be an inverse relationship between glucocerebrosidase and α-synuclein levels, and even patients with sporadic Parkinson disease have decreased glucocerebrosidase. Glucocerebrosidase may interact with α-synuclein to maintain basic cellular functions, or impaired glucocerebrosidase could contribute to Parkinson pathogenesis by disrupting lysosomal homeostasis, enhancing endoplasmic reticulum stress or contributing to mitochondrial impairment. However, the majority of patients with GBA1 mutations never develop parkinsonism, so clearly other risk factors play a role. Treatments for Gaucher disease have been developed that increase visceral glucocerebrosidase levels and decrease lipid storage, although they have yet to properly address the neurological defects associated with impaired glucocerebrosidase. Mouse and induced pluripotent stem cell derived models have improved our understanding of glucocerebrosidase function and the consequences of its deficiency. These models have been used to test novel therapies including chaperone proteins, histone deacetylase inhibitors, and gene therapy approaches that enhance glucocerebrosidase levels and could prove efficacious in the treatment of forms of parkinsonism. Consequently, this rare monogenic disorder, Gaucher disease, provides unique insights directly applicable to our understanding and treatment of Parkinson disease, a common and complex neurodegenerative disorder.

Using induced pluripotent stem cells derived neurons to model brain diseases.

The ability to use induced pluripotent stem cells (iPSC) to model brain diseases is a powerful tool for unraveling mechanistic alterations in these disorders. Rodent models of brain diseases have spurred understanding of pathology but the concern arises that they may not recapitulate the full spectrum of neuron disruptions associated with human neuropathology. iPSC derived neurons, or other neural cell types, provide the ability to access pathology in cells derived directly from a patient's blood sample or skin biopsy where availability of brain tissue is limiting. Thus, utilization of iPSC to study brain diseases provides an unlimited resource for disease modelling but may also be used for drug screening for effective therapies and may potentially be used to regenerate aged or damaged cells in the future. Many brain diseases across the spectrum of neurodevelopment, neurodegenerative and neuropsychiatric are being approached by iPSC models. The goal of an iPSC based disease model is to identify a cellular phenotype that discriminates the disease-bearing cells from the control cells. In this mini-review, the importance of iPSC cell models validated for pluripotency, germline competency and function assessments is discussed. Selected examples for the variety of brain diseases that are being approached by iPSC technology to discover or establish the molecular basis of the neuropathology are discussed.

Exosomes: Origins and Therapeutic Potential for Neurodegenerative Disease.

Exosomes, small lipid bilayer vesicles, are part of the transportable cell secretome that can be taken up by nearby recipient cells or can travel through the bloodstream to cells in distant organs. Selected cellular cytoplasm containing proteins, RNAs, and other macromolecules is packaged into secreted exosomes. This cargo has the potential to affect cellular function in either healthy or pathological ways. Exosomal content has been increasingly shown to assist in promoting pathways of neurodegeneration such as ß-amyloid peptide (Aß) accumulation forming amyloid plaques in the brains of patients with Alzheimer's disease, and pathological aggregates of proteins containing α-synuclein in Parkinson's disease transferred to the central nervous system via exosomes. In attempting to address such debilitating neuropathologies, one promising utility of exosomes lies in the development of methodology to use exosomes as natural delivery vehicles for therapeutics. Because exosomes are capable of penetrating the blood-brain barrier, they can be strategically engineered to carry drugs or other treatments, and possess a suitable half-life and stability for this purpose. Overall, analyses of the roles that exosomes play between diverse cellular sites will refine our understanding of how cells communicate. This mini-review introduces the origin and biogenesis of exosomes, their roles in neurodegenerative processes in the central nervous system, and their potential utility to deliver therapeutic drugs to cellular sites.

A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis.

Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and is largely responsible for evoking the inflammatory response. Antibiotic and anti-inflammatory therapy for treating Gram-negative infections remains suboptimal. Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived protein with antimicrobial and LPS-neutralizing properties. Select peptide derivatives of BPI are reported to retain these properties. The objective of this study was to evaluate the antimicrobial activity of a human BPI-derived synthetic peptide against clinical bovine mastitis isolates of Gram-negative bacteria. A hybrid peptide was synthesized corresponding to two regions of human BPI (amino acids 90-99 and 148-161), the former of which has bactericidal activity and the latter of which has LPS-neutralizing activity. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of this peptide against various genera of bacteria were determined using a broth microdilution assay. The MIC's were determined to be: 16-64 microg/ml against Escherichia coli; 32-128 microg/ml against Klebsiella pneumoniae and Enterobacter spp.; and 64-256 microg/ml against Pseudomonas aeruginosa. The MBC's were equivalent to or 1-fold greater than corresponding MIC's. The peptide had no growth inhibitory effect on Serratia marcescens. The antimicrobial activity of the peptide was retained in the presence of serum, but severely impaired in milk. Further functional evaluation of the peptide demonstrated its ability to completely neutralize LPS. Together, these data support additional investigations into the therapeutic application of BPI to the treatment of Gram-negative infections in cattle.

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice.

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.