Oscar A. Bizzozero - US grants

Our long term goal is to understand the mechanisms that control the formation and maintenance of the CNS and PNS myelin, PO, the major protein of PNS myelin, participates in the formation and compaction of the myelin lamellae. PO is esterified with long chain fatty acids, as is PLP, the major protein of CNS myelin. We hypothesize that acylation plays an important role in the process of myelin synthesis and maintenance. The present studies are aimed at determining fundamental aspects of the biology of PO acylation. Our specific aims are: 1. To study the metabolism of the fatty acid bound to PO using a nerve slice system. Double-label pulse and pulse-chase experiments, combined with subcellular fractionation, will be used to identify the membranes in which acylation of PO takes place. We will establish the relationship between acylation and the other PO posttranslational modifications by using inhibitors of protein N-glycosylation and phosphorylation. We will also correlate the extent of PO synthesis and acylation during nerve development to gain insights on the role of this modification. Our preliminary results indicate that acylation occurs only on newly-synthesized but also on a pre- existing PO, suggesting that acylation could also relate to myelin maintenance. 2. To determine the acylation site on the PO molecule. PO will be labeled with [3H] palmitic acid, digested with several proteases and the acyl- peptides isolated and sequenced. 3. To develop a cell-free system using deacylated PO and labeled acyl-CoA as substrates. We will carry out a detailed characterization of the acylating enzyme, localize the subcellular site of cylation unequivocally, isolate the acyltransferase and determine its protein substrate specificity, and determine the developmental and evolutionary changes of this enzyme. The studies proposed in this application will provide basic knowledge on the biology of PO acylation. Moreover, they will contribute to gain insights into normal myelination processes and ultimately to understand the pathophysiology of human peripheral nerve disorders.

Our long term goal is to understand the mechanisms that control the formation and maintenance of the CNS and PNS myelin, PO, the major protein of PNS myelin, participates in the formation and compaction of the myelin lamellae. PO is esterified with long chain fatty acids, as is PLP, the major protein of CNS myelin. We hypothesize that acylation plays an important role in the process of myelin synthesis and maintenance. The present studies are aimed at determining fundamental aspects of the biology of PO acylation. Our specific aims are: 1. To study the metabolism of the fatty acid bound to PO using a nerve slice system. Double-label pulse and pulse-chase experiments, combined with subcellular fractionation, will be used to identify the membranes in which acylation of PO takes place. We will establish the relationship between acylation and the other PO posttranslational modifications by using inhibitors of protein N-glycosylation and phosphorylation. We will also correlate the extent of PO synthesis and acylation during nerve development to gain insights on the role of this modification. Our preliminary results indicate that acylation occurs only on newly-synthesized but also on a pre- existing PO, suggesting that acylation could also relate to myelin maintenance. 2. To determine the acylation site on the PO molecule. PO will be labeled with [3H] palmitic acid, digested with several proteases and the acyl- peptides isolated and sequenced. 3. To develop a cell-free system using deacylated PO and labeled acyl-CoA as substrates. We will carry out a detailed characterization of the acylating enzyme, localize the subcellular site of cylation unequivocally, isolate the acyltransferase and determine its protein substrate specificity, and determine the developmental and evolutionary changes of this enzyme. The studies proposed in this application will provide basic knowledge on the biology of PO acylation. Moreover, they will contribute to gain insights into normal myelination processes and ultimately to understand the pathophysiology of human peripheral nerve disorders.

Our long-term goal is to understand the molecular mechanisms that control the formation and maintenance of CNS myelin and the factors that lead to its breakdown in multiple sclerosis (MS). Although the production of nitric oxide (NO) and peroxynitrite by activated macrophages and microglial cells is considered responsible for the destruction of myelin and oligodendrocytes in MS, the molecular targets of these agents in myelin have not yet been identified. We hypothesize that both the structure and fatty acid acylation of the abundant myelin proteolipid protein (PLP) are affected by NO and peroxynitrites, and that this may lead to myelin instability. Our specific aims are: 1- To characterize the mechanism of fatty acylation of PLP using endogenously generated 18/O-labeled fatty acids. This recently developed isotopomeric technique measures not only the acylation rate but also the minimal amount of proteins that are modified. Brain white matter slices from rapidly myelinating rats will be incubated with [3H]palmitate and H2/18/O, in the presence of a variety of metabolic poisons and enzyme inhibitors to ascertain whether PLP acylation (a) needs ATP, (b) requires the formation acyl-CoA, (c) using primarily fatty acids synthesized de novo, and (d) is catalyzed by a separate protein fatty acyltransferase. 2- To assess the effects of nitric oxide and peroxynitrite on the fatty acylation of PLP. We will determine the effects of pathological concentrations of endogenously-generated NO, exogenously-produced NO and peroxynitrite on acylation on PLP and lipids using tissue slices and the double-label technique described above. In addition, a variety of metabolic and structural studies will be carried out to identify the mechanism(s) by which nitrogen and oxygen free radicals could alter protein acylation. 3- To determine whether or not the structure and fatty acylation of the various PLP species isolated from MS brains are normal. The major PLP, DM-20 and 16 KdA proteolipid present in myelin and non-myelin membranes prepared from control and MS brains will be isolated and subjected to a comprehensive chemical and mass-spectrometric analysis. The studies proposed in this application will provide direct information into the mechanisms of myelin destruction that takes place in MS, and at the same time, they will aid our understanding of the biology of PLP and its only post-translational modification.

DESCRIPTION (provided by applicant): Our long-term goal is to identify and characterize the mechanisms that lead to the destruction of myelin in inflammatory demyelinating disorders. Nitric oxide (NO) has been implicated in the pathophysiology of both multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), as high levels of this gas are released by macrophages/microglia following induction of nitric oxide synthetase by various proinflammatory cytokines. However, the mechanism by which NO leads to myelin breakdown is far from clear. Based on recent investigations regarding the molecular and cellular consequences of nitrosative damage in other systems, and a number of important findings from our laboratory, we hypothesize that NO can spread at considerable distances from inflammatory lesions to cause extensive myelin decompaction. We propose that underlying this process is the S-nitrosylation of important myelin proteins by N203, a strong nitrosylating agent that can be readily formed by NO autooxidation in a lipid-rich environment like the myelin sheath. The experiments described in this proposal will characterize in detail the mechanism of S-nitrosylation of myelin proteins in rat spinal cord slices incubated with a non-permeable NO-donor, which generates levels of NO comparable to those found in EAE and MS. These experiments will also (1) examine the existence of other NO-induced thiol-related modifications (S-thiolation and formation of protein disulfides), (2) identify the major S-nitrosylated myelin proteins by mass-spectrometry, and (3) test several compounds for their ability to prevent/revert these deleterious protein modifications. In addition, the occurrence of S-nitrosylated proteins in spinal cord during the course of EAE will be investigated and the identity of these species determined using a proteomic approach. Finally, the distribution of S-nitrosylated proteins in the affected tissue will be assessed by immunocytochemistry to directly test the hypothesis that nitrosative protein damage induced by free NO can take place at some distance from the inflammatory lesions and independently of peroxynitrite generation. The proposed studies will generate crucial information that could support a new pathway for the NO-mediated destruction of myelin during inflammatory demyelination. The elucidation of the mechanism of protein modification, the identification of the molecules in myelin that are affected by NO, and the occurrence of these modifications in the CNS of a widely-used animal model of MS are essential for understanding the pathophysiology of this devastating disorder.

[unreadable] DESCRIPTION (provided by applicant): Our long-term goal is to define and characterize the mechanisms underlying tissue injury in multiple sclerosis, the most common demyelinating disease of the central nervous system in humans. In recent years, oxidative stress has been implicated in the pathophysiology of both multiple sclerosis and its animal model, experimental allergic encephalomyelitis (EAE). Reactive oxygen species (ROS) are released by activated macrophages/microglia or are endogenously generated by dysfunctional mitochondria in the nerve cells. Although there is unquestionable experimental evidence demonstrating that oxidative stress plays a causal role in these disorders, the precise mechanism(s) by which ROS produces tissue damage is far from clear. A major consequence of ROS accumulation is the non-enzymatic introduction of aldehydes or ketones into specific amino acid residues of proteins (i.e. carbonylation). Based on recent reports regarding the molecular and cellular consequences of protein carbonylation in other systems and a number of important findings from our laboratory, we hypothesize that a major outcome of oxidative stress in EAE is the carbonylation of neuronal proteins, which contributes to tissue damage and axonal injury. We also put forth the idea that inhibition of protein carbonylation will be therapeutic in this disease. To test our hypothesis, we will measure the levels of protein carbonyls in the spinal cord and brain of remitting/relapsing EAE mice in relationship to well-established pathological hallmarks of the disease (inflammation, neuronal death, axonal damage and demyelination). These studies will employ state-of the-art biochemical and immunocytochemical techniques. We will then identify the oxidized proteins in the inflammatory and degenerative stages of the disease by redox proteomics, and will ascertain both the chemical nature and origin of the oxidizing species from the type of carbonylated amino acid residues produced. Finally, we will examine the ability of various carbonyl scavengers and metabolic inhibitors to prevent tissue injury and axonal damage in EAE animals. If successful, these studies will uncover a novel molecular mechanism by which oxidative stress causes chronic disability in demyelinating disorders. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a neurological disorder that affects approximately 1 in 700 young adults in the US. We have recently observed that a special type of oxidative process called carbonylation modifies several brain proteins from MS patients. These modifications affect protein function and likely contribute to tissue injury in this devastating disease. Using a mouse model of MS, we will determine whether drugs that reduce protein carbonylation can effectively prevent tissue damage and neurological deficits. We envision that in the future these agents could be administered in combination with antioxidants, anti-inflammatory or neuroactive substances for an improved clinical management of chronic MS. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our laboratory has previously shown that multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are characterized by severe oxidative stress and the accumulation of oxidized proteins in the CNS, which in turn reduces cell viability and leads to tissue damage. Build-up of oxidized/misfolded proteins often occurs as a consequence of deficiencies in the proteasome, the enzymatic machinery responsible for their proteolytic removal. We have also discovered that in both MS and chronic EAE there is a significant reduction in the peptidolytic activities of the proteasome, which may explain the accumulation of oxidized proteins observed in the neurodegenerative stages of these diseases. While very important, all of these studies were carried out in tissue homogenates using fluorescent peptide substrates, and a critical issue that remains to be established is whether these deficits are localized to specific cell types or affect all CNS cells o the same extent. Furthermore, the proteolytic activity of the standard (s) and immuno (i) proteasome core particles (i.e. s-20S and i-20S) are greatly affected by the binding of regulatory caps (19S and 11S) and inhibitors, which may be expressed differentially in the various CNS cells during the course of the disease. In this proposal, we hypothesize that as a result of disease activity neurons, oligodendrocytes, astrocytes and microglia express unique patterns of proteasome complexes, making some CNS cells less equipped than others to remove abnormal proteins and thereby more susceptible to stress. To test this idea, we will first determine by double-immunohistochemistry the temporal/spatial pattern of proteasome core (s-20S and i-20S) and proteasome cap (19S and 11S) expression and their relationship to the extent of inflammation and oxidative stress during the course of EAE. We will then isolate neurons, oligodendrocytes, astrocytes and microglial cells from the CNS of control and EAE mice and measure the levels of each complex (20S, 11S- 20S-11S, 19S-20S-19S and 19S-20S-11S) by blue native-polyacrylamide gel electrophoresis and quantitative western blot analysis. Finally, we will compare the ability of the various proteasome complexes isolated from control and EAE tissues to digest oxidized and ubiquitinated proteins. These exploratory studies are essential for understanding how the various CNS cells regulate the expression and assembly of functional proteasome complexes as EAE progresses from the inflammatory to the neurodegenerative stages, and for determining the molecular basis for proteasome impairment in chronic EAE. Identification of the cell(s) with altered proteasome function will ultimately serve to design a therapeutic approach geared to decrease the toxic consequence of oxidative burden in EAE and MS.

DESCRIPTION (provided by applicant): Our long-term goal is to define and characterize the mechanisms underlying tissue injury in multiple sclerosis (MS), the most common demyelinating disease of the central nervous system in humans. In recent years, oxidative stress has been implicated in the pathophysiology of both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Previous studies from our laboratory have shown that MS and EAE, like several other neurological disorders, are characterized by the accumulation of carbonylated (oxidized) proteins within CNS cells. We have also demonstrated that the build-up of oxidized and other misfolded proteins in these disorders is a consequence of decreased activity of the proteasome, the proteolytic machinery responsible for their removal. Carbonylation and other oxidative protein modifications are known to cause inappropriate inter- and intra-protein cross-links as well as protein misfolding. This, in turn, results in the formation of protein aggregates that we believe contribute to the demise of neural cells and ultimately tissue damage. Indeed, we have obtained preliminary evidence that aggregates containing oxidized proteins accumulate in the CNS of MS patients and EAE mice and that inhibition of protein aggregation prevents neuronal cell death in culture. Based on these novel findings, we hypothesize that protein aggregation plays a pathogenic role in EAE. To test this idea, we will (1) quantify the temporal/spatial pattern of protein aggregation and its relationship to the extent of neuronal and oligodendroglial cell death in the spinal cord during the course of MOG peptide-induced EAE, and (2) examine the prophylactic and therapeutic efficacy of two chemically- distinct protein aggregation inhibitors at preventing cell death and tissue injury and at reducing neurological symptoms of EAE. If successful, these studies will not only demonstrate that protein aggregation is pathogenic in inflammatory demyelinating disease but also will provide the basis for developing new or testing previously identified non-toxic aggregation inhibitors for an improved clinical management of MS.