Zellweger Syndrome: A Histochemical Diagnosis of Two Cases

Until 17 years ago the diagnosis of the cerebrohepatorenal Zellweger syndrome (ZS) rested largely on clinical grounds confirmed by pathologic findings at autopsy. The observation that peroxisomes are not detectable morphologically or histochemically in liver or kidney of patients with this syndrome gave histopathologists the opportunity to make the diagnosis of this complex syndrome at biopsy. Catalase reaction of the peroxisomes can be used as a rapid and accurate method to differentiate between ZS and other clinical conditions in which the peroxisomes are present in normal or reduced number. We describe two patients in whom the diagnosis of ZS was made by the absence of histochemical staining for catalase in a liver biopsy. The findings were subsequently confirmed using standard biochemical tests.     

Autonomous Purkinje cell axonal dystrophy causes ataxia in peroxisomal multifunctional protein‐2 deficiency

Peroxisomes play a crucial role in normal neurodevelopment and in the maintenance of the adult brain. This depends largely on intact peroxisomal β‐oxidation given the similarities in pathologies between peroxisome biogenesis disorders and deficiency of multifunctional protein‐2 (MFP2), the central enzyme of this pathway. Recently, adult patients diagnosed with cerebellar ataxia were shown to have mild mutations in the MFP2 gene, hydroxy‐steroid dehydrogenase (17 beta) type 4 (HSD17B4). Cerebellar atrophy also develops in MFP2 deficient mice but the cellular origin of the degeneration is unexplored. In order to investigate whether peroxisomal β‐oxidation is essential within Purkinje cells, the sole output neurons of the cerebellum, we generated and characterized a mouse model with Purkinje cell selective deletion of the MFP2 gene. We show that selective loss of MFP2 from mature cerebellar Purkinje neurons causes a late‐onset motor phenotype and progressive Purkinje cell degeneration, thereby mimicking ataxia and cerebellar deterioration in patients with mild HSD17B4 mutations. We demonstrate that swellings on Purkinje cell axons coincide with ataxic behavior and precede neurodegeneration. Loss of Purkinje cells occurs in a characteristic banded pattern, proceeds in an anterior to posterior fashion and is accompanied by progressive astro‐ and microgliosis. These data prove that the peroxisomal β‐oxidation pathway is required within Purkinje neurons to maintain their axonal integrity, independent of glial dysfunction.     

Ataxia with loss of Purkinje cells in a mouse model for Refsum disease

Refsum disease is caused by a deficiency of phytanoyl-CoA hydroxylase (PHYH), the first enzyme of the peroxisomal α-oxidation system, resulting in the accumulation of the branched-chain fatty acid phytanic acid. The main clinical symptoms are polyneuropathy, cerebellar ataxia, and retinitis pigmentosa. To study the pathogenesis of Refsum disease, we generated and characterized a Phyh knockout mouse. We studied the pathological effects of phytanic acid accumulation in Phyh^−/− mice fed a diet supplemented with phytol, the precursor of phytanic acid. Phytanic acid accumulation caused a reduction in body weight, hepatic steatosis, and testicular atrophy with loss of spermatogonia. Phenotype assessment using the SHIRPA protocol and subsequent automated gait analysis using the CatWalk system revealed unsteady gait with strongly reduced paw print area for both fore- and hindpaws and reduced base of support for the hindpaws. Histochemical analyses in the CNS showed astrocytosis and up-regulation of calcium-binding proteins. In addition, a loss of Purkinje cells in the cerebellum was observed. No demyelination was present in the CNS. Motor nerve conduction velocity measurements revealed a peripheral neuropathy. Our results show that, in the mouse, high phytanic acid levels cause a peripheral neuropathy and ataxia with loss of Purkinje cells. These findings provide important insights in the pathophysiology of Refsum disease.     

Lipoperoxidase activity of Pityrosporum: characterisation of by-products and possible rôle in pityriasis versicolor

Abstract Modification of pigmentation and damage of melanocytes are characteristic features of skin colonisation by Pityrosporum orbiculare hyphae in pityriasis versicolor (PV). The yeast is lipophylic and lipid-dependent, capable of oxidising unsaturated lipid components of skin surface, i.e. unsaturated fatty acids, cholesterol and squalene (SQ). The oxidation of unsaturated fatty acids gives rise to dicarboxylic acids (DA) which behave, in vitro, as competitive inhibitors of tyrosinase. In this work, we further investigate the oxidase activity of Pityrosporum in vitro, by evaluating (a) the generation of lipoperoxides in cultures supplemented with fatty acids at various degrees of unsaturation; (b) the mechanism of SQ oxidation; (c) the chemical characteristics of some by-products of lipoperoxidation; (d) the formation of peroxisomes in fungal cells. In cultures supplemented with the saturated palmitic acid (C16:0) and monounsaturated oleic acid (C18:1 n-9), low amounts of lipoperoxides were detected by a spectrophotometric test, whereas in cultures supplemented with di-unsaturated linoleic acid (C 18:2 n-6), significant concentrations were found. Gas chromatographymass spectrometry analyses showed the generation of linoleic acid hydroperoxides both in Pityrosporum cultures and following incubation of acetone powder of the fungus with the unsaturated fatty acid, indicating the presence of a lipoxygenase activity in the fungus. In cultures supplemented with linoleic acid plus SQ, and increase of lipoperoxide generation was observed and trans-trans farnesal and squalene epoxides have been identified. Electron microscopic examinations have evidenced peroxisomes in cells grown in the presence of linoleic acid, whereas they were not delected in cultures supplemented with oleic acid and palmitic acid. The metabolic activities of peroxisomes, through the formation of hydrogen peroxide and the subsequent generation of hydroxyl radicals, may account for the peroxidation of SQ, which is not a substrate of lipoxygenase. Following these results, we propose a mechanism for DA generation by Pityrosporum metabolism and hypothesize that the lipoperoxidation process induced by lipoxygenase activity of the fungus may be the key to understanding the clinical appearance of skin manifestation of PV.     

HYPOLIPIDEMIC EFFECT OF CLOFIBRATE AND FATTY ACID OXIDATION IN PEROXISOMES

Rat liver peroxisomes contain a fatty acid oxidizing system similar to that present in plant glyoxysomes. Treatment with clofibrate, a hypolipidemic drug, leads to marked stimulation of this system.     

Ultrastructure of the Liver in the Cerebrohepatorenal Syndrome of Zellweger

Three infants with clinical and biochemical features of Zellweger's cerebrohepatorenal syndrome are presented, and the ultrastructural features of successive biopsy and autopsy liver specimens are described. No hepatocellular peroxisomes were found in these patients on routine electron microscopy or electron microscopic histochemistry. In a control group of liver biopsies from 9 patients with other pediatric liver diseases, peroxisomes were readily identifiable in each hepatocyte. Apart from the absence of peroxisomes, the hepatocytes had a remarkably “normal” aspect, even in the final stages of the disease. Mitochondrial abnormalities, which have been the subject of some controversy in this syndrome, were a highly variable and inconstant finding in our cases.

We draw attention to another ultrastructural feature of the syndrome, namely the occurrence of large angulate lysosomes, containing conspicuous double lamellae, inside macrophages, which were especially abundant in later stages of the disease. These angulate lysosomes may be of additional value in the ultrastructural diagnosis of Zellweger's syndrome, especially when only poorly preserved liver tissue (e.g., paraffin-embedded or postmortem material) is available, and the absence of peroxisomes is difficult to assess. In these instances, the angulate lysosomes can still be identified with ease.     

Identification of a chronic non‐neurodegenerative microglia activation state in a mouse model of peroxisomal β‐oxidation deficiency

The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β‐oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2^?/? microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2^?/? microglia were compared with those from SOD1^G93A mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1^G93A spinal cord, neurons were intact in Mfp2^?/? brain and Mfp2^?/? microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2^?/? microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2^?/? microglia were immunologically activated but not polarized to a pro‐ or anti‐inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2^?/? brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity. GLIA 2015;63:1606–1620