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Brain Uptake and Utilization of Fatty Acids: Application to Peroxisomal Biogenesis Diseases


International Workshop on
Brain Uptake and Utilization of Fatty Acids: Application to Peroxisomal Biogenesis Diseases

Recommendations for Future Research

Table of Content:

I. Agenda

Thursday, March 2, 2000

6 p.m.Registration - Pick up badges and programs - Versailles IV
Opening Reception and Poster Session - Versailles IV
Co-Chairs: Paul A. Watkins and Robert Katz
7:00-7:30 p.m.Reception
7:30-8:30 p.m.Viewing of posters
8:30-9:30 p.m.Viewing with authors by odd-numbered posters
9:30-10:30 p.m.Viewing with authors by even-numbered posters.
(Posters will remain displayed through 10:00 p.m. on March 3.)

Friday, March 3, 2000

7:30 a.m.Continental Breakfast - Versailles IV
7:55 a.m.Oral presentations - Versailles III
Morning Session :Fatty acid uptake by and transport in the brain.
Co-Chairs: Jacques H. Veerkamp and James A. Hamilton
7:55 a.m.Welcome. Paul A. Watkins
(All presentations are 20 minutes long and are followed by a 5 minute Q & A session directly relevant to the presentation)
8:00-8:25 a.m.Structure of the microvascular endothelium. Lester R. Drewes
8:25-8:50 a.m.The diffusion mechanism in model and biological membranes. James A. Hamilton
8:50-9:15 a.m.Molecular barriers to lipid transport. Henry J. Pownall
9:15-9:40 a.m.A model of coupled fatty acid transport and metabolism. Jean A. Schaffer
9:40-10:05 a.m.The role of CD36 in fatty acid transport by various tissues. Nada Abumrad
10:05-10:30 a.m.Coffee break - Versailles IV
10:30-10:55 a.m.The role of membrane-associated proteins in cellular fatty acid uptake. Jan F. Glatz
10:55-11:20 a.m.Structural and functional properties of eight FABP types. Jacques H.Veerkamp
11:20-11:45 a.m.Mechanisms of fatty acid transport and targeting. Judith Storch
11:45 a.m.-12:30 p.m.Round table discussion and recommendations.
Discussant: Alexander Leaf
12:30-1:30 p.m.Lunch - on your own
Afternoon Session :Brain uptake, transport and metabolism of PUFA: In vivo and in vitro studies.
Co-chairs: Arthur A. Spector and Steven A. Moore
1:30-1:55 p.m.Sources of PUFA in the plasma. Arthur A. Spector
1:55-2:20 p.m.Measurement of brain PUFA uptake by perfusion techniques. Quentin R. Smith
2:20-2:45 p.m.NMR and isotope ratio MS studies of in vivo uptake and metabolism of PUFA by the rodent brain. Stephen C. Cunnane
2:45-3:10 p.m.PUFA transport and utilization in the developing brain. John Edmond
3:10-3:40 p.m.Coffee break - Versailles IV
3:40-4:05 p.m.PUFA synthesis, transfer and metabolism by brain-derived cells in vitro. Steven A. Moore
4:05-4:30 p.m.Lysophosphatidylcholine as a DHA carrier to the brain. Michel Lagarde
4:30-4:55 p.m.Uptake, synthesis and metabolism of PUFA by the retina and retina-derived cells in vitro. Robert E. Anderson
5:00-5:45 p.m.Round table discussion and recommendations.
Discussant: Howard Sprecher
Evening Session :Workshop Dinner and Dinner Lecture - Washington Room
Hosts: Paul A. Watkins and Robert Katz
7:00-7:30 p.m.Reception and cash bar
7:30-9:30 p.m.Dinner
Dinner Speaker: Alexander Leaf "Reflections on the effects of polyunsaturated fatty acids on brain, heart and coronary heart disease"

Saturday: March 4, 2000

7:30 a.m.Continental Breakfast - Versailles IV
8:00 a.m.Oral presentations - Versailles III
Morning Session :The regulation and functions of DHA in neurons and neuronal membranes.
Co-Chairs: Stanley I. Rapoport and Norman Salem, Jr.
8:00-8:25 a.m.The protective effect of DHA in neuronal apoptosis. Hee-Yong Kim
8:25-8:50 a.m.DHA accumulation and overaccumulation before birth. Impact on oxidative stress. Ephraim Yavin
8:50-9:15 a.m.The role of DHA-containing phospholipids in modulating G-protein-coupled signaling pathways: The visual transduction pathway. Burton J. Litman
9:15-9:40 a.m.Quantifying in vivo fatty acid signaling and turnover in the central nervous system. Stanley I. Rapoport
9.40-10:10 a.m.Coffee break - Versailles IV
10:10-10:35 a.m.Plasmalogens, phospholipases and DHA turnover. Lloyd A. Horrocks
10:35-11:00 a.m.Do DHA or other long-chain PUFA have gene regulatory roles? James M. Ntambi
11:00-11:45 a.m.Round table discussions and recommendations.
Discussant: Robert E. Anderson
11:45 a.m.-12:45 p.m.Lunch - on your own
Afternoon Session:The roles of DHA in Zellweger syndrome, a representative peroxisomal biogenesis disorder.
Co-chairs: Hugo W. Moser and Manuella Martinez
12:45-1:10 p.m.Normal and defective neuronal membranes: Structure and function. James M. Powers
1:10-1:35 p.m.The Zellweger syndrome animal model. Phillis L. Faust
1:35-2:00 p.m.The DHA deficiency animal model. Norman Salem, Jr.
2:00-2:25 p.m.Restoring DHA levels in the brains of Zellweger patients. Manuela Martinez
2:25-2:45 p.m.Coffee break - Versailles IV
2:45-3:10 p.m.DHA in other peroxisomal deficiency disorders. Hugo W. Moser
3:10-3:55 p.m.Round table discussions and recommendations.
Discussant: Michael J. Noetzel
4:00 p.m.Adjournment

II. Recommendations from March 3, 2000 Afternoon Session

Plasma sources of PUFA

Although data from serum lipid composition suggest that a potential major source of brain PUFA is lipoprotein-associated complex lipids, few studies have as yet addressed specific details of PUFA transport and uptake by this mechanism. Numerous lipoprotein classes and glycerolipid pools may be involved. Thus, investigations directed at the identification and functional characterization of PUFA-selective lipoprotein classes and PUFA-enriched glycerolipid pools would be enlightening. It may be possible to utilize stable PUFA isotopes (13C or perdeuterated) to identify and follow these lipoproteins/glycerolipids in feeding studies.

Role of barrier cells and astrocytes in the uptake and processing of PUFA or lipoprotein-associated PUFA

Studies that parallel the focus on serum lipid composition (particularly lipoproteins) should also be directed at cells carrying out barrier functions between blood and neurons. Foremost among these barrier cells are cerebral endothelium of the blood-brain barrier, choroid plexus epithelium that produce the bulk of cerebrospinal fluid, and astrocytes that closely associate with both cerebral blood vessels and neurons. The distribution, molecular nature, and function of lipoprotein lipase, lipoprotein receptors, and intracellular mechanisms for lipoprotein processing or trafficking in these cells are some of the major areas that require attention.

Once PUFAs are free of their carrier proteins and/or glycerolipids, a different group of binding and transport proteins are likely to be important in directing the flow of PUFA across barrier cells or within the brain. In this regard molecular and cell biological approaches should be aimed at understanding how fatty acid binding proteins (FABP) and fatty acid transport proteins (FATP) are distributed and how they function. Attention should also be directed at the specificity of enzymes that esterify PUFA, since the selectivity observed in membrane lipids may be regulated significantly at the esterification level.

Finally, additional carrier proteins or glycerolipids are likely to be involved in the intercellular distribution of PUFA within the brain parenchyma. For example, astrocytes are known to be a source of apolipoprotein synthesis in the brain, but little is known about the function of brain apolipoproteins in the intercellular exchange of PUFA. The fact that the E4 allele of apolipoprotein E is a major risk factor for Alzheimer's disease provides a significant allure to this area of study.

Local synthesis of PUFA

Although there is strong evidence for local synthesis of long-chain PUFA by brain-derived cells, recent advances in the molecular and cell biology of desaturase enzymes and peroxisomal assembly have yet to be applied systematically to the brain. The application of this new knowledge, particularly using genetic engineering approaches, should provide the clearest

evidence yet on the role of local synthesis in the accretion of PUFA by brain. It might also provide a basis for developing molecular approaches to therapy in neurological disorders where long-chain PUFAs are deficient.


III. Recommendations from March 4, 2000 Morning Session

Uptake rates of nutritionally supplied fatty acids by the brain

The method, model and "operational equations" presented at the workshop, for quantifying in vivo brain turnover rates and half-lives of fatty acids during uptake from plasma to the brain, can be applied to the elucidation of uptake rates of nutritionally -provided omega-6 PUFA (e.g. linoleic acid, LA; gamma-linolenic acid, GLA, arachidonic acid, AA), omega-3 PUFA (e.g. alpha-linolenic acid, ALA; eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) and of dietary saturated and monounsaturated fatty acids. In the case of saturated and monounsaturated fatty acids, studies could help clarify the possible differences between their uptake versus de novo synthesis in the developing and in the adult brains.

Effects of centrally acting drugs on the steady state of fatty acids

The above approach can also be applied to the assessment of changes in steady states in response to centrally acting drugs and pathological changes in bipolar and other disorders. When combined with neuroimaging intravenously injected radiolabeled PUFA can also be utilized to examine neuroplastic remodeling of brain fatty acids and lipid membranes.

Role of plasmalogens in glial and neuronal tissue

  • It was noted that AA- and DHA-containing plasmalogens (the glycerophospholipids containing an enol-ether group at the sn-1 position) play important roles as potential protectors of neurons from oxidative stress and suppliers of free AA and DHA to the cell. They can function also as modulators of neuronal membrane physical state and permeability both in the developing and in the mature brain. Plasmalogens are formed in peroxisomes, therefore they could be involved in a wide range of pathological states such as peroxisomal biogenesis disorders (Zellweger's syndrome and neonatal leukodystrophy) as well as ischemia or neurodegerative diseases, such as Alzheimer's disease, and even spinal cord trauma. All of these issues warrant rapid further elucidation.
  • It would also be important to understand the contribution of plasmalogens to the free fatty acid pool and therefore their contributions to prostaglandin-mediated processes in the developing and mature neuron in health and disease.

The role of DHA in the developing and adult brain

  • Results reported in Session 2 and 3 settle neither the question whether saturated and monounsaturated fatty acids are taken up by the brain or whether they are synthesized de novo by the brain, nor the question whether developing brain is similar or identical with adult brain in this regard. Studies on the gene regulatory effects of EPA and DHA in the liver and other organs on lipogenic enzymes do not extend to the brain in a satisfactory fashion. A strong need exists in elucidating the molecular biology and genetics of brain lipogenesis and its regulation by long-chain, omega-3 fatty acids (EPA and DHA) in both the developing and the adult brain.
  • The accelerated in utero uptake of DHA (by the rat embryo) appears characteristic both to animals and humans. Since the mother is the major supplier of DHA to the developing rat brain, a low circulating level of plasma DHA in the mother could lower the embryo's DHA brain levels. Direct intra-amniotic injection of DHA ethyl ester restores embryonic brain uptake of DHA to normal levels. Additional research into the mechanisms of DHA accretion and depletion in embryonic and developing rat brain and into the potential behavioral or cognitive consequences of below normal DHA levels in the embryonic and postnatal brain should shed light on why DHA and possibly AA supplementation of infant formulas would be important for normal brain development. Elucidating the consequences of DHA deficiency during the developmental period is especially important due to the reported potential role of DHA to protect the developing brain from oxidative stress.
  • The extension of these in vivo studies to humans would render the development of appropriate non-invasive techniques for quantitation of neuronal changes due to DHA presence or absence including, but not limited to the development of new imaging and tracer approaches highly desirable.
  • The question of the role of DHA in the management of environmental stress and oxidative stress in the adult brain is also worthy of exploration.
  • A major breakthrough in understanding the role of DHA in signal transduction was recently reported in the outer rod segments (ROS) of the retina. It appears that the role of DHA in ROS membranes is attributable to its six double bonds that provide and ensure a "most favored" microenvironment in which rhodopsin can absorb a photon of light and undergo the rapid conformational changes that result in the activation of a G-protein. This activation initiates the chain of biochemical events in the visual transduction pathway that ultimately leads to hyperpolarization of the plasma membrane. This observation, based on solid methodological determinations indicates that the high concentrations of DHA in the rhodopsin the outer rod segments might be of paramount importance to efficient signal transduction. DHA might have similar functions in the transduction of the neuronal signals through the membrane and synapses. Validating this potential role of DHA in the different neurons of different areas of the brain and should contribute significantly to our understanding of the functioning of the central nervous system.
  • It is also important to understand what implications does DHA deficiency, during brain development, have on neuronal signal transduction and transmission.

Intermediary metabolism of PUFA and brain disorders

Answers should be sought to questions involving post-uptake shuttling and metabolism of AA, EPA and DHA. Such questions are exemplified in the following: How does DHA, taken up by the brain through the BBB or synthesized from its precursors by astrocytic peroxisomes, reach its neuronal locations? Are there special DHA transporters in the brain? Are astrocytes mostly devoid of DHA and function primarily as elongation/ desaturation tools of EPA and its higher homolog docosapentaenoic acid (DPA)? What are the functions of EPA and DPA in astrocytes? Are EPA and DHA naturally segregated between astrocytes (EPA) and neurons (DHA)? What are the eicosanoids produced from EPA and what are their functions in the brain? What are docosanoids and what are their functions in the brain? Are these functions related to the pathophysiology of neuropsychobehavioral disorders such as: bipolar disorder, unipolar depression and schizophrenia? Do EPA, DPA and DHA exert the same therapeutic effect in these disorders? Etc.

DHA, EPA and apoptosis of glial and neuronal cells

-The finding that DHA can potentially inhibit neuronal apoptosis in two cell lines and that this inhibitory effect appears related to an increase in PS concentration raises several questions on yet another potential physiological role of DHA namely that of long-term survival of the neuronal cell. Does depletion of DHA result in neuronal death? If that effect of DHA is proven, is this effect reversible? Is there a connection between DHA depletion and the pathophysiology of inherited neurodegenerative diseases or Alzheimer's disease? Is the anti-apoptotic effect of DHA responsible for its apparent segregation to the neuron? What would be the consequences of long-term DHA storage in astrocytes, which appear to eliminate free DHA as it is formed from its precursors. How would the anti-apoptotic effect persist in animal models? All of these questions deserve to be answered.


IV. Recommendations from March 4, 2000 Afternoon Session

Therapeutic interventions in peroxisomal biogenesis disorders

  • Therapeutic trials in peroxisomal disorders will form an increasingly important future priority. This represents an obvious priority, since these disorders cause such severe disability. In addition, the tools to carry out and evaluate therapies are becoming available to an increasing extent. The existence of animal models is a key feature, which opens also the possibility of evaluating in utero therapy. Another key tool is the increasing availability of neuroimaging studies, such as MRI, magnetic resonance spectroscopy and diffusion fiber tracking techniques. Improved understanding of the molecular and enzymatic defects has led to new therapeutic approaches, of which DHA therapy of peroxisomal biogenesis disorders (PBD) is a prime example. Evaluation of therapeutic interventions in peroxisomal disorders is complicated by the marked and only partially predictable variability in natural history, and also the desirability for early initiation of therapy at time before irreversible damage has occurred. Discussion took place about the use of randomized placebo-controlled studies. Some participants took the position that such trials were not ethically justifiable in circumstances when the consequences of the disease are severe and the therapy is essentially free of side effects, as is the case with DHA. Others took the position that carefully designed placebo controlled trials supervised by an independent treatment effects monitoring committee represented the most rapid method of determining effectiveness and safety of therapy and could be conducted in a way that safeguarded the best interests of the patients and their families.
  • The application and evaluation of DHA therapy in PBD disorders exemplifies another important principle and opportunity. PBD patients (and animal models of these disorders) have profound disturbances in DHA metabolism and they show profound neurologic deficits. This combination provides the opportunity to determine relatively quickly the extent to which DHA therapy can be of benefit and when and how it should be administered. However, there is increasing evidence that more subtle DHA deficits, traceable in most instances to environmental circumstances that could be altered, are of clinical significance in much larger groups of individuals.

Understanding the functions of fatty acids in peroxisomal biogenesis disorders

The study of fatty acids in PBD disorders represents an important and promising field of investigation. The PBD disorders are associated with characteristic and severe handicaps and can be diagnosed early, including prenatally, by non-invasive and reliable diagnostic assays. Characteristic and striking abnormalities in fatty acid profiles and metabolism are present in all of these disorders. Some of these abnormalities can be normalized completely or in part, by dietary manipulations or by the administration of non-toxic natural compounds. Results on improvement in myelination of Zellweger patients following DHA supplementation therapy, over time, raises a slew of new questions. What is the mechanism by which DHA supplementation improves myelination? Since DHA is not abundant in white matter, is DHA present in oligodendrocytes where it exerts its effect indirectly through correction of reduced levels? Or rather does DHA lower VLCFA levels (which have been implicated in demyelination)? Does DHA raise plasmalogen levels known to be low in Zellweger syndrome thus protecting membranes from oxidative stress (see also recommendations in the March 4, 2000 Morning Session)? Animal models for most of these disorders are now available and new ones can be developed as needed.

Animal models for study of fatty acid function in glia and neurons

  • A variety of future research programs flow naturally from these observations, and several are already in progress. Many of the PBD patients, as well as the animal models, show characteristic defects of neuronal migration which take place during fetal life. Gaining an understanding of the mechanisms that lead to disturbances in this fundamental biological process may lead to therapies that can be applied during early phases of development, possibly even during fetal life, and will also contribute to an understanding of normal brain development. The availability of a mouse model that displays similar neuronal migration defects is of immense value here. It is likely that a variety of models can be developed in which genetic defects are more restricted and targeted and that these will permit delineation of the comparative roles of specific genetic and biochemical defects. Other neuropathological changes occur post-developmentally. Examples of these are changes secondary to the accumulation of branched chain fatty acids such as phytanic acid, since the abnormal accumulation of this substance does not commence until after birth. The most striking example of post-developmental pathology is the distal axonopathy that occurs in adults with AMN, and appears attributable in some way to the accumulation of very long chain fatty acids.
  • The studies in rodents who have been deprived of DHA for three generations exemplify the broad implications of this research approach and provide recommendations for future research. These studies have shown that DHA deprivation in rodents impairs spatial tasks and olfactory discrimination. Studies of behavioral tasks that are cued to olfactory modality are an ideal manner to examine higher levels of learning/memory related performance in rodents because they are macrosomatic. The reversal learning task may prove a useful approach for the study of DHA adequacy, and may prove of great value for the definition of the role of DHA in normal human brain development.

Omega-3 PUFA and the hepatic side of Zellweger syndrome

The peroxisomal defects of the Zellweger brain extend to the liver and kidney. The liver is known to be an important location for elongation and desaturation of alpha-linolenic acid (ALA) and other intermediates in the pathway to EPA and DHA. Several issues deserve further studies. For example: Is ALA elongated and desaturated in the livers of Zellweger patients? Is there partial production of DHA in the liver but not sufficient for satisfying needs? Can the existing animal models of Zellweger syndrome be used to develop methods that will allow a comparative assessment of plasma, erythrocyte and brain levels of EPA and DHA of healthy individuals and of Zellweger animal models? Could such methods be extended to other diseases of the central nervous system?


 V. Participants

Nada A. Abumrad, PhD
Dept. of Physiology and Biophysics
SUNY at Stony Brook School of Medicine

Robert E. Anderson, MD, PhD
Oklahoma Center for Neuroscience
Univ. of Oklahoma Health Science Center

Stephen C. Cunnane, PhD
Dept. of Nutrition Science
University of Toronto

Lester R. Drewes, PhD
Dept. of Biochemistry and Molecular Biology
University of Minnesota School of Medicine

John Edmond PhD
Dept. of Biological Chemistry
UCLA School of Medicine

Phyllis L. Faust, MD, PhD
Columbia University
Department of Pathology

Jan F.C. Glatz, PhD
Dept. of Physiology
Maastricht University

James A. Hamilton, PhD
Dept. of Biophysics
Boston Univ. School of Medicine

Lloyd A. Horrocks, PhD
The Ohio State University

Robert Katz, PhD
Omega-3 Research Institute, Inc.

Hee-Yong Kim, PhD

Michel Lagarde, PhD, DSc

Alexander Leaf, MD
Harvard Medical School
Massachusetts General Hospital, East

Burton J. Litman, PhD
Lab. of Membrane Biochem. & Biophys.

Manuela Martinez, MD
Hospital Materno-Infantil Vall D'Hebron

Steven A. Moore, MD, PhD
Dept. of Pathology
University of Iowa

Hugo W. Moser, MD
Kennedy Krieger Institute

Michael J. Noetzel, MD
Dept. of Pediatric Neurology
Washington Univ. School of Medicine

James M. Ntambi, PhD
Dept. of Biochemistry
University of Wisconsin, Madison

James M. Powers, MD
Dept. of Pathology
University of Rochester Medical Center

Henry J. Pownall, PhD
Methodist Hospital
Baylor College of Medicine

Stanley I. Rapoport MD
Chief, Laboratory of Neuroscience

Norman Salem, Jr. PhD
Lab. of Membrane Biochem. & Biophysics

Jean E. Schaffer, MD
Center for Cardiovascular Research
Washington University School of Medicine

Quentin R. Smith, PhD
Dept. of Pharmaceutical Science
Texas Tech University HSC
School of Pharmacy

Arthur A. Spector, MD
Dept. of Biochemistry
University of Iowa

Howard Sprecher, PhD
Dept. of Medical Biochemistry
The Ohio State University

Judith Storch, PhD
Dept. of Nutrition Science
Rutgers University

Jacques H. Veerkamp, PhD
Dept. of Biochemistry
University of Nijmegen

Paul A. Watkins MD, PhD
Kennedy Krieger Institute

Efraim Yavin, PhD
Weizmann Institute

 VI. Additional Link

Omega-3 Research Institute Workshop Summary


Last Modified April 15, 2011