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BRAIN UPTAKE AND UTILIZATION OF FATTY ACIDS: 

Applications to Peroxisomal Biogenesis Diseases

(An International Workshop)

March 2-4, 2000, Holiday Inn Bethesda, Bethesda, Maryland

Session 4 (Afternoon, March 4, 2000) 

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The Role of DHA in Zellweger Syndrome, a Representative Peroxisomal Biogenesis Disorder

(Saturday, March 4, 2000 Afternoon Session Summary)

Co-Chairs: Hugo W. Moser, M.D., Manuella Martinez, M.D.

Discussant: Michael Noetzel, M.D.

Prepared by: Hugo W. Moser and Michael Noetzel

Neuropathology of Peroxisomal Biogenesis Disorders.

Disturbances of fatty acid metabolism play a prominent role in the Disorders of Peroxisome Biogenesis (PBD). Very long chain fatty acids (VLCFA) and branched fatty acids, such as phytanic and pristanic acid accumulate, due to impaired capacity to degrade these substances. PBD patients also have an impaired capacity to synthesize plasmalogens. Most recently it was shown that the last step of the synthesis of docosahexaneoic acid (DHA) is deficient in PBD patients. Fatty acids and the PBD disorders are intertwined for several reasons. Historically, recognition of the key role of peroxisomes in VLCFA, branched chain fatty acids and plasmalogen was a direct consequence of the study of PBD patients, and might not have occurred otherwise. Measurements of the levels of these lipids form the basis of the most commonly used diagnostic assays. Finally, studies of fatty acids in PBD may lead to better understanding of pathogenesis and of normal cellular function, and provide leads for therapy. These latter aspects formed the topic of this session. The interest of the topic and the potential to learn more about these disease states as well as normal cell function is heightened by the fact that animal models of PBD are now available and that one of the biochemical abnormalities, namely the deficiency of DHA, can be corrected easily by oral administration of this substance.

Dr. James M Powers presented an overview of neuropathological changes in the peroxisomal disorders. Two types of pathology dominate: developmental and post-developmental or degenerative. The developmental lesions are most severe in the Zellweger syndrome and result in a characteristic parasylvian or centrosylvian pachygyria-polymicrogyria. Large subcortical heterotopias are characteristic of this unique cerebral malformation. Powers considers the elevated levels of VLCFA to be the most likely cause of the neuronal migration defect, based upon the identification of lamellar-lipid profiles (considered to be indicative of VLCFA excess) in neurites and the experimental evidence that VLCFA excess is toxic and alters membrane structure and function. Post-developmental degenerative changes in the peroxisomal disorders are most conspicuous in adrenomyeloneuropathy (AMN), the adult variant of X-linked adrenoleukodystrophy. The major neuropathological lesion is a dying-back axonopathy that affects mainly the gracile fibers in the upper cervical cord and the corticospinal tract in the lumbar region. The parent neurons of the gracile tracts are located in the lumbar dorsal root ganglia were atrophic, but there was only minimal neuronal loss and no evidence of apoptosis, consistent with the hypothesis that axonal damage is the primary pathology. Powers speculates that incorporation of VLCFA into axonal membranes, particularly into gangliosides, results in the inability of the target neurons and their axons to respond to neurotrophic factors it is likely that the other biochemical abnormalities observed in the peroxisomal disorders also contribute to the pathogenesis of the neurologic deficits. Cerebellar atrophy associated with severe loss of Purkinje cells and granule neurons occurs in patients with rhizomelic chondrodysplasia punctata (RCDP). It is of interest that this feature is present only in those RCDP patients with relatively long survival. This suggests that this pathology represents a post-developmental degenerative change. The demonstration of apoptosis in the Purkinje cells, and particularly in the granule cells is consistent with this interpretation. The main biochemical abnormalities in RCDP are elevated levels of phytanic acid and a deficiency of plasmalogens.  VLCFA levels are not increased in RCDP patients. Phytanic acid is of dietary origin exclusively, and elevated levels of this substance do not occur until the postnatal period. Powers proposes that phytanic acid excess may lead to distortion of calcium channels within Purkinje cell membranes. The plasmalogen deficit may render cells more susceptible to the damaging effects of oxygen radicals.

Animal models of DHA deficiency for the study of neuronal function in health and disease: the Zellweger mouse and DHA depleted rat models

-Dr. Phyllis L. Faust and associates reported on the neuropathological changes in a mouse model of Zellweger syndrome, created by the targeted deletion of the PEX2 peroxisome assembly gene. The utility of the model was increased greatly when the PEX2 null mutation was placed on a Swiss Webster x 129 Svev murine background. Mice with this background survive to 7-10 postnatal day, and occasionally to the 15^th or 18^th postnatal day. The affected mice show significant growth retardation, hypotonia, spasticity and microcephaly. They have a significant delay in the arrival of neurons in the cerebral cortical plate, but they do not display the numerous white matter heterotopias that are characteristic of human Zellweger syndrome. Thus, the neuronal migration defect is present in the PEX2 -/- mouse but it is not as severe as in the human disease. The affected mice also show significant foliation defects in the vermis and the posterior folia of the cerebellum, which probably are the cause of the ataxic gait displayed by these animals. The Purkinje cells showed severe deficits of dendritic arborization. As in the human disease the neurons also showed lamellar cytoplasmic inclusions, probably related to VLCFA excess as well as another type of lipid that has not yet been identified. Biochemical studies revealed a severe deficit of plasmalogens as in the human disease. VLCFA levels were also increased, but not as much as in the human disease. DHA levels in the brain were reduced in comparison to littermates, consistent with findings in the human disease, but unlike the human disease, they were normal in liver, red blood cells, and higher than normal in plasma. These findings suggest that in the mice transport of DHA from the placenta is sufficient to maintain normal levels in fetal visceral organs, it fails to maintain normal levels of this substance in brain. The reduced DHA level in brain may be due to a defect in DHA transport or synthesis or both. The anatomical and biochemical studies of the PEX2 -/- mouse model thus show many points of resemblance to human Zellweger syndrome, but on careful analysis also show differences which are still unexplained.

-Dr. Norman Salem, Jr. and colleagues developed a rat model of brain DHA deficiency. The   model was obtained through careful control of the diet so that n-3 fatty acids were deprived through two or three generations. The demonstrated that DHA deficiency achieved in this way results in a loss of learning/memory related performance in a spatial task, the Morris water maze. Both the escape latency and memory retention performance was significantly worse in the n-3 deficient group in comparison to that in the n-3 adequate group. They also presented preliminary data that the n-3 deficient animals showed defects in an olfactory-based reversal learning task. The n-3 deficient animals had significantly more errors to criterion than the n-3 adequate animals over five reversals. These data, when taken together with those in the literature, indicate that loss of brain DHA leads to a mild cognitive deficit.

DHA-based therapy of Zellweger syndrome

Dr. Manuella Martinez reported results of DHA therapy in 20 patients with PBD disorders. The rationale for this approach was based upon her previous original observations that DHA levels are reduced in the brain, retina and other tissues of patients with PBD. The therapeutic program includes the administration of DHA ethyl ester, at daily doses of 100-500 mg. accompanied by a nutritious diet, normal for age, in order to provide all necessary nutrients and avoid a polyunsaturated fatty acid (PUFA) deficiency. The diet was begun as soon as diagnosis was established. Special care was taken with nutrition in small infants. Here, a whole milk formula enriched in DHA and arachidonic acid in proportion similar to that in mother's milk was used. Results were monitored biochemically and clinically. DHA normalization in plasma and erythrocytes was obtained in all patients in only a few weeks. In addition, plasma plasmalogen levels increased in the erythrocytes of most patients, the levels of VLCFA levels in the plasma of most patients diminished. Plasma phytanic acid levels increased initially when the complete diet was introduced, but then decreased again in few months, and never reached levels that are considered harmful.

Nutritional status improved in most patients, particularly the small children. In three infants who began therapy at 2, 4 and 5 months, marked liver dysfunction and failure to thrive improved dramatically in a few days. In one patient liver function and myelination normalized rapidly and the patient is now capable of walking, climbing steps and even riding a bicycle, and is communicating with elemental language, and the other children whose therapy started early made parallel gains. In the patients who started treatment after one year, improvement was less constant, and some stabilized after an initial period of amelioration, however, neurologic deterioration was observed in only one patient who initiated therapy at 15 months and had a severe from of neonatal adrenoleukodystrophy. Patients who are less affected made some progress even starting treatment as late as at 5 years of age. In this patient vision improved, demyelination halted, she became more social and active, and muscle tone improved quite constantly. The changes in MRI are considered to be particularly important. In the 12 patients in whom MRI follow-up could be achieved, progress in myelination could be detected in nine, and in four the images are now normal for age.

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The Role of DHA in Zellweger Syndrome, a Representative Peroxisomal Biogenesis Disorder.

(Saturday, March 4, 2000 Afternoon Session Recommendations for Future Research)

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?  

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