Hepatocyte-specific deletion of peroxisomal protein PEX13 results in disrupted iron homeostasis.

Peroxisomes are organelles, abundant in the liver, involved in a variety of cellular functions, including fatty acid metabolism, plasmalogen synthesis and metabolism of reactive oxygen species. Several inherited disorders are associated with peroxisomal dysfunction; increasingly many are associated with hepatic pathologies. The liver plays a principal role in regulation of iron metabolism. In this study we examined the possibility of a relationship between iron homeostasis and peroxisomal integrity. We examined the effect of deleting Pex13 in mouse liver on systemic iron homeostasis. We also used siRNA-mediated knock-down of PEX13 in a human hepatoma cell line (HepG2/C3A) to elucidate the mechanisms of PEX13-mediated regulation of hepcidin. We demonstrate that transgenic mice lacking hepatocyte Pex13 have defects in systemic iron homeostasis. The ablation of Pex13 expression in hepatocytes leads to a significant reduction in hepatic hepcidin levels. Our results also demonstrate that a deficiency of PEX13 gene expression in HepG2/C3A cells leads to decreased hepcidin expression, which is mediated through an increase in the signalling protein SMAD7, and endoplasmic reticulum (ER) stress. This study identifies a novel role for a protein involved in maintaining peroxisomal integrity and function in iron homeostasis. Loss of Pex13, a protein important for peroxisomal function, in hepatocytes leads to a significant increase in ER stress, which if unresolved, can affect liver function. The results from this study have implications for the management of patients with peroxisomal disorders and the liver-related complications they may develop.

Impaired neurogenesis and associated gliosis in mouse brain with PEX13 deficiency.

Zellweger syndrome (ZS), a neonatal lethal disorder arising from defective peroxisome biogenesis, features profound neuroanatomical abnormalities and brain dysfunction. Here we used mice with brain-restricted inactivation of the peroxisome biogenesis gene PEX13 to model the pathophysiological features of ZS, and determine the impact of peroxisome dysfunction on neurogenesis and cell maturation in ZS. In the embryonic and postnatal PEX13 mutant brain, we demonstrate key regions with altered brain anatomy, including enlarged lateral ventricles and aberrant cortical, hippocampal and hypothalamic organization. To characterize the underlying mechanisms, we show a significant reduction in proliferation, migration, differentiation, and maturation of neural progenitors in embryonic E12.5 through to P3 animals. An increasing reactive gliosis in the PEX13 mutant brain started at E14.5 in association with the pathology. Together with impaired neurogenesis and associated gliosis, our data demonstrate increased cell death contributing to the hallmark brain anatomy of ZS. We provide unique data where impaired neurogenesis and migration are shown as critical events underlying the neuropathology and altered brain function of mice with peroxisome deficiency.

Peroxisomal protein PEX13 functions in selective autophagy.

PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with Zellweger syndrome spectrum (ZSS) disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurological, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy) and that disease-associated PEX13 mutants I326T and W313G are defective in mitophagy. The mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13-mediated mitophagy may contribute to ZSS pathogenesis.

Mitochondrial changes and oxidative stress in a mouse model of Zellweger syndrome neuropathogenesis.

Zellweger syndrome (ZS) is a peroxisome biogenesis disorder that involves significant neuropathology, the molecular basis of which is still poorly understood. Using a mouse model of ZS with brain-restricted deficiency of the peroxisome biogenesis protein PEX13, we demonstrated an expanded and morphologically modified brain mitochondrial population. Cultured fibroblasts from PEX13-deficient mouse embryo displayed similar changes, as well as increased levels of mitochondrial superoxide and membrane depolarization; this phenotype was rescued by antioxidant treatment. Significant oxidative damage to neurons in brain was indicated by products of lipid and DNA oxidation. Similar overall changes were observed for glial cells. In toto, these findings suggest that mitochondrial oxidative stress and aberrant mitochondrial dynamics are associated with the neuropathology arising from PEX13 deficiency.

Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress.

A new disaster victim identification management strategy targeting "near identification-threshold" cases: Experiences from the Boxing Day tsunami.

The international disaster victim identification (DVI) response to the Boxing Day tsunami, led by the Royal Thai Police in Phuket, Thailand, was one of the largest and most complex in DVI history. Referred to as the Thai Tsunami Victim Identification operation, the group comprised a multi-national, multi-agency, and multi-disciplinary team. The traditional DVI approach proved successful in identifying a large number of victims quickly. However, the team struggled to identify certain victims due to incomplete or poor quality ante-mortem and post-mortem data. In response to these challenges, a new 'near-threshold' DVI management strategy was implemented to target presumptive identifications and improve operational efficiency. The strategy was implemented by the DNA Team, therefore DNA kinship matches that just failed to reach the reporting threshold of 99.9% were prioritized, however the same approach could be taken by targeting, for example, cases with partial fingerprint matches. The presumptive DNA identifications were progressively filtered through the Investigation, Dental and Fingerprint Teams to add additional information necessary to either strengthen or conclusively exclude the identification. Over a five-month period 111 victims from ten countries were identified using this targeted approach. The new identifications comprised 87 adults, 24 children and included 97 Thai locals. New data from the Fingerprint Team established nearly 60% of the total near-threshold identifications and the combined DNA/Physical method was responsible for over 30%. Implementing the new strategy, targeting near-threshold cases, had positive management implications. The process initiated additional ante-mortem information collections, and established a much-needed, distinct "end-point" for unresolved cases.

Low dose tubulin-binding drugs rescue peroxisome trafficking deficit in patient-derived stem cells in Hereditary Spastic Paraplegia.

Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS) cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine) that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials.

Revisiting the neuropathogenesis of Zellweger syndrome.

Zellweger syndrome (ZS) is a neonatal-lethal genetic disease that affects all tissues, and features neuropathology that involves primary developmental defects as well as neurodegeneration. Neuropathological changes include abnormal neuronal migration affecting the cerebral hemispheres, cerebellum and inferior olivary complex, abnormal Purkinje cell arborisation, demyelination and post-developmental neuronal degeneration. ZS is caused by mutations in peroxisome biogenesis, or PEX, genes which lead to defective peroxisome biogenesis and the resultant loss of peroxisomal metabolic function. The molecular and cellular bases of ZS neuropathology are still not completely understood. Attempts to explain the neuropathogenesis have implicated peroxisomal metabolic dysfunction, and more specifically the loss of peroxisomal products, such as plasmalogens and docosahexaenoic, and the accumulation of peroxisomal substrates, such as very-long-chain-fatty acids. In this review, consideration is also given to recent findings that implicate other candidate pathogenetic factors, such as mitochondrial dysfunction, oxidative stress, protein misfolding, aberrant cell signalling, and inflammation - factors that have also been identified as important in the pathogenesis of other neurological diseases.

Excess iron modulates endoplasmic reticulum stress-associated pathways in a mouse model of alcohol and high-fat diet-induced liver injury.

Endoplasmic reticulum (ER) stress is an important pathogenic mechanism for alcoholic (ALD) and nonalcoholic fatty liver disease (NAFLD). Iron overload is an important cofactor for liver injury in ALD and NAFLD, but its role in ER stress and associated stress signaling pathways is unclear. To investigate this, we developed a murine model of combined liver injury by co-feeding the mildly iron overloaded, the hemochromatosis gene-null (Hfe(-/)) mouse ad libitum with ethanol and a high-fat diet (HFD) for 8 weeks. This co-feeding led to profound steatohepatitis, significant fibrosis, and increased apoptosis in the Hfe(-/-) mice as compared with wild-type (WT) controls. Iron overload also led to induction of unfolded protein response (XBP1 splicing, activation of IRE-1α and PERK, as well as sequestration of GRP78) and ER stress (increased CHOP protein expression) following HFD and ethanol. This is associated with a muted autophagic response including reduced LC3-I expression and impaired conjugation to LC3-II, reduced beclin-1 protein, and failure of induction of autophagy-related proteins (Atg) 3, 5, 7, and 12. As a result of the impaired autophagy, levels of the sequestosome protein p62 were most elevated in the Hfe(-/-) group co-fed ethanol and HFD. Iron overload reduces the activation of adenosine monophosphate protein kinase associated with ethanol and HFD feeding. We conclude that iron toxicity may modulate hepatic stress signaling pathways by impairing adaptive cellular compensatory mechanisms in alcohol- and obesity-induced liver injury.

The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies.

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.

RhoA regulation of cardiomyocyte differentiation.

Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac α -actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes.

Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins.

Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca(2+) or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX's suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER.

PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress.

Delayed cerebellar development is a hallmark of Zellweger syndrome (ZS), a severe neonatal neurodegenerative disorder. ZS is caused by mutations in PEX genes, such as PEX13, which encodes a protein required for import of proteins into the peroxisome. The molecular basis of ZS pathogenesis is not known. We have created a conditional mouse mutant with brain-restricted deficiency of PEX13 that exhibits cerebellar morphological defects. PEX13 brain mutants survive into the postnatal period, with the majority dying by 35 days, and with survival inversely related to litter size and weaning body weight. The impact on peroxisomal metabolism in the mutant brain is mixed: plasmalogen content is reduced, but very-long-chain fatty acids are normal. PEX13 brain mutants exhibit defects in reflex and motor development that correlate with impaired cerebellar fissure and cortical layer formation, granule cell migration and Purkinje cell layer development. Astrogliosis and microgliosis are prominent features of the mutant cerebellum. At the molecular level, cultured cerebellar neurons from E19 PEX13-null mice exhibit elevated levels of reactive oxygen species and mitochondrial superoxide dismutase-2 (MnSOD), and show enhanced apoptosis together with mitochondrial dysfunction. PEX13 brain mutants show increased levels of MnSOD in cerebellum. Our findings suggest that PEX13 deficiency leads to mitochondria-mediated oxidative stress, neuronal cell death and impairment of cerebellar development. Thus, PEX13-deficient mice provide a valuable animal model for investigating the molecular basis and treatment of ZS cerebellar pathology.

alpha-Synuclein abnormalities in mouse models of peroxisome biogenesis disorders.

alpha-Synuclein (alphaS) is a presynaptic protein implicated in Parkinson's disease (PD). Growing evidence implicates mitochondrial dysfunction, oxidative stress, and alphaS-lipid interactions in the gradual accumulation of alphaS in pathogenic forms and its deposition in Lewy bodies, the pathological hallmark of PD and related synucleinopathies. The peroxisomal biogenesis disorders (PBD), with Zellweger syndrome serving as the prototype of this group, are characterized by malformed and functionally impaired peroxisomes. Here we utilized the PBD mouse models Pex2-/-, Pex5-/-, and Pex13-/- to study the potential effects of peroxisomal dysfunction on alphaS-related pathogenesis. We found increased alphaS oligomerization and phosphorylation and its increased deposition in cytoplasmic inclusions in these PBD mouse models. Furthermore, we show that alphaS abnormalities correlate with the altered lipid metabolism and, specifically, with accumulation of long chain, n-6 polyunsaturated fatty acids that occurs in the PBD models.

Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR.

The Cre/loxP-system has become an invaluable tool for the generation of tissue-specific gene disruption in mice. However, because Cre recombinase excision of individual genes can be variable, an accurate and sensitive method is necessary to determine the ultimate level of gene disruption. The analysis of gene disruption is particularly difficult for tissue that has been fixed for (immuno)histochemical analysis with paraformaldehyde. Here, we describe a simple, rapid and cost effective method for measurement of gene disruption using quantitative real-time PCR, through application to the analysis of PEX13 gene disruption in a brain-specific PEX13 mouse mutant. We show that this general protocol is suitable for both normal and paraformaldehyde-fixed tissue.

Fuzzy scaling analysis of a mouse mutant with brain morphological changes.

Scaling behavior inherently exists in fundamental biological structures, and the measure of such an attribute can only be known at a given scale of observation. Thus, the properties of fractals and power-law scaling have become attractive for research in biology and medicine because of their potential for discovering patterns and characteristics of complex biological morphologies. Despite the successful applications of fractals for the life sciences, the quantitative measure of the scale invariance expressed by fractal dimensions is limited in more complex situations, such as for histopathological analysis of tissue changes in disease. In this paper, we introduce the concept of fuzzy scaling and its analysis of a mouse mutant with postnatal brain morphological changes.

A region analysis approach for segmenting neural-cell images.

In this paper we present new algorithms based on region analysis of grey and distance differences of images that successfully circumvent these problems. Two key parameters of this analysis, window width and logical threshold, are automatically extracted for use in logical thresholding, and spurious regions are detected and removed through use of a hierarchical window filter. The efficacy of the developed algorithms is demonstrated here through an analysis of cultured brain neurons from newborn mice.

Segmentation of cultured neurons using logical analysis of grey and distance difference.

The molecular and cellular bases of neuronal cell death that underpin a wide range of neurodegenerative disorders are still not well understood. One approach to investigating neuronal death is through systematic studies of the changing morphology of cultured brain neurons in response to cellular challenges. Image segmentation methods developed to date to analyze such changes have been limited by the low contrast of cells in unstained neuronal cultures and the unimodal histograms generated by these analyses. In this paper we present new algorithms based on logical analysis of grey and distance difference of images that successfully circumvent these problems. Two key parameters of this analysis, window width and logical threshold, are automatically extracted for use in logical level technique, and spurious regions are detected and removed through use of a hierarchical window filter. The efficacy of the developed algorithms is demonstrated here through an analysis of cultured brain neurons from newborn mice.

Failure of microtubule-mediated peroxisome division and trafficking in disorders with reduced peroxisome abundance.

In contrast to peroxisomes in normal cells, remnant peroxisomes in cultured skin fibroblasts from a subset of the clinically severe peroxisomal disorders that includes the biogenesis disorder Zellweger syndrome and the single-enzyme defect D-bifunctional protein (D-BP) deficiency, are enlarged and significantly less abundant. We tested whether these features could be related to the known role of microtubules in peroxisome trafficking in mammalian cells. We found that remnant peroxisomes in fibroblasts from patients with PEX1-null Zellweger syndrome or D-BP deficiency exhibited clustering and loss of alignment along peripheral microtubules. Similar effects were observed for both cultured embryonic fibroblasts and brain neurons from a PEX13-null mouse with a Zellweger-syndrome-like phenotype, and a less-pronounced effect was observed for fibroblasts from an infantile Refsum patient who was homozygous for a milder PEX1 mutation. By contrast, such changes were not seen for patients with peroxisomal disorders characterized by normal peroxisome abundance and size. Stable overexpression of PEX11beta to induce peroxisome proliferation largely re-established the alignment of peroxisomal structures along peripheral microtubules in both PEX1-null and D-BP-deficient cells. In D-BP-deficient cells, peroxisome division was apparently driven to completion, as induced peroxisomal structures were similar to the spherical parental structures. By contrast, in PEX1-null cells the majority of induced peroxisomal structures were elongated and tubular. These structures were apparently blocked at the division step, despite having recruited DLP1, a protein necessary for peroxisome fission. These findings indicate that the increased size, reduced abundance, and disturbed cytoplasmic distribution of peroxisomal structures in PEX1-null and D-BP-deficient cells reflect defects at different stages in peroxisome proliferation and division, processes that require association of these structures with, and dispersal along, microtubules.

PEX1 mutations in the Zellweger spectrum of the peroxisome biogenesis disorders.

Diseases of the Zellweger spectrum represent a major subgroup of the peroxisome biogenesis disorders, a group of autosomal-recessive diseases that are characterized by widespread tissue pathology, including neurodegeneration. The Zellweger spectrum represents a clinical continuum, with Zellweger syndrome (ZS) having the most severe phenotype, and neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) having progressively milder phenotypes. Mutations in the PEX1 gene, which encodes a 143-kDa AAA ATPase protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The PEX1 mutations identified to date comprise insertions, deletions, nonsense, missense, and splice site mutations. Mutations that produce premature truncation codons (PTCs) are distributed throughout the PEX1 gene, whereas the majority of missense mutations segregate with the two essential AAA domains of the PEX1 protein. Severity at the two ends of the Zellweger spectrum correlates broadly with mutation type and impact (i.e., the severe ZS correlates with PTCs on both alleles, and the milder phenotypes correlate with missense mutations), but exceptions to these general correlations exist. This article provides an overview of the currently known PEX1 mutations, and includes, when necessary, revised mutation nomenclature and genotype-phenotype correlations that may be useful for clinical diagnosis.