Peroxisome deficiency but not the defect in ether lipid synthesis causes activation of the innate immune system and axonal loss in the central nervous system
© Bottelbergs et al; licensee BioMed Central Ltd. 2012
Received: 22 November 2011
Accepted: 29 March 2012
Published: 29 March 2012
Mice with peroxisome deficiency in neural cells (Nestin-Pex5 −/− ) develop a neurodegenerative phenotype leading to motor and cognitive disabilities and early death. Major pathologies at the end stage of disease include severe demyelination, axonal degeneration and neuroinflammation. We now investigated the onset and progression of these pathological processes, and their potential interrelationship. In addition, the putative role of oxidative stress, the impact of plasmalogen depletion on the neurodegenerative phenotype, and the consequences of peroxisome elimination in the postnatal period were studied.
Immunohistochemistry in association with gene expression analysis was performed on Nestin-Pex5 −/− mice to document demyelination, axonal damage and neuroinflammation. Also Gnpat −/− mice, with selective plasmalogen deficiency and CMV-Tx-Pex5 −/− mice, with tamoxifen induced generalized loss of peroxisomes were analysed.
Activation of the innate immune system is a very early event in the pathological process in Nestin-Pex5 −/− mice which evolves in chronic neuroinflammation. The complement factor C1q, one of the earliest up regulated transcripts, was expressed on neurons and oligodendrocytes but not on microglia. Transcripts of other pro- and anti-inflammatory genes and markers of phagocytotic activity were already significantly induced before detecting pathologies with immunofluorescent staining. Demyelination, macrophage activity and axonal loss co-occurred throughout the brain. As in patients with mild peroxisome biogenesis disorders who develop regressive changes, demyelination in cerebellum and brain stem preceded major myelin loss in corpus callosum of both Nestin-Pex5 −/− and CMV-Tx-Pex5 −/− mice. These lesions were not accompanied by generalized oxidative stress throughout the brain. Although Gnpat −/− mice displayed dysmyelination and Purkinje cell axon damage in cerebellum, confirming previous observations, no signs of inflammation or demyelination aggravating with age were observed.
Peroxisome inactivity triggers a fast neuroinflammatory reaction, which is not solely due to the depletion of plasmalogens. In association with myelin abnormalities this causes axon damage and loss.
KeywordsPeroxisomes Mouse models Plasmalogens Complement Demyelination Axonal degeneration Inflammation Macrophage
Patients with peroxisomal dysfunction present with severe and diverse neurological anomalies, including neuronal migration defects, dysmyelination and inflammatory demyelination and axon damage, proving that these organelles are indispensible for the normal development and maintenance of the central nervous system (CNS) [1–3]. According to the genetic causes these diseases can be categorized in peroxisome biogenesis defects (PBDs) and in single enzyme or transporter deficiencies. The PBDs are due to a mutation in a PEX gene, encoding a peroxin involved in the assembly of the organelles. The enzyme/transporter defects mostly involve 1) the peroxisomal α-oxidation pathway, necessary for the breakdown of phytanic acid and long chain 2-hydroxy fatty acids, 2) β-oxidation, which is required for degradation of very long chain fatty acids and pristanic acid, as well as the synthesis of polyunsaturated fatty acids and bile acids and 3) ether phospholipid synthesis which include plasmalogens.
To investigate the postnatal pathologies in the CNS, a mouse model with neural selective peroxisome dysfunction was generated by breeding Nestin-Cre mice with Pex5-loxP mice. In the latter mice the gene encoding the import receptor of peroxisomal matrix proteins is floxed . This model shows a mild and temporary delay in neurodevelopment  but from 3 weeks on Nestin-Pex5 knockout mice display motor and later on cognitive abnormalities, aggravating with increasing age and evolving in immobility and death before the age of 6 months. In brain, severe dys- and demyelination, astro- and microgliosis and axonal damage were observed . However, the relationship between these anomalies and the precise onset and progression of pathologies in different brain areas were not elucidated. It was further demonstrated that a similar but less aggressive phenotype develops in mice with oligodendrocyte selective inactivation of Pex5, whereas mice with neuron or astrocyte selective deletion of functional peroxisomes were spared from demyelination, axon damage and astro- and microgliosis .
Gnpat −/− mice , which lack a crucial enzyme of ether lipid synthesis, also exhibit a brain phenotype. The cerebellar fibers display hypomyelination at the age of 3 weeks which does not aggravate at 6 weeks, based on microscopical investigations and western blot analysis. Significant dysmyelination was also observed in the outer neocortical fibers of juvenile and adult (aged 8 months) Gnpat−/− mice. Furthermore, they display disturbed axoglial contacts resulting in abnormal paranodal organisation and axonal swellings . In lipid raft microdomains (LRMs) isolated from myelin a significant reduction of LRM proteins was found, which was ascribed to the severe lack of plasmalogens .
Also impaired peroxisomal β-oxidation causes a postnatal phenotype in the CNS. Abcd1 knockout mice, a model for the adrenomyeloneuropathy (AMN) form of X-linked adrenoleukodystrophy (X-ALD), cannot transport a subset of substrates over the peroxisomal membrane, presumably saturated and/or unsaturated very long chain fatty acid CoA esters. They develop a late onset axonopathy in the spinal cord, but no brain defects. Recently, oxidative stress was detected in spinal cord of X-ALD mice, long before these mice develop motor abnormalities . Treatment with anti-oxidants inhibited the development of oxidative stress and prevented the development of motor disability and axonal damage . Furthermore, in brain-selective Pex13 knockout mice  increased superoxides and MnSOD were detected in cerebellar cell cultures and up regulation of MnSOD in the Purkinje cell layer of the cerebellum in vivo.
Mice deficient in MFP-2 (also denoted as D-bifunctional protein), carry a broader defect in peroxisomal β-oxidation, as this enzyme is necessary for the degradation of both straight and branched chain substrates . They bear several similarities with Nestin-Pex5 knockout mice in view of their motor defects and early death. Marked astro- and microgliosis were described  but no thorough study of myelinated axons was performed.
Although we already reported on the pathology in the end phase of disease of mice with peroxisome deficiency in brain, the aim of the present investigation was to better define the onset and progression of pathological events in different brain areas of Nestin-Pex5 −/− mice. Therefore, immunofluorescent studies were performed to (co)-localize myelin, axonal damage and neuroinflammatory markers. Furthermore, to better characterize the inflammatory process and the status of microglial cells, the mRNA expression of pro- and anti-inflammatory markers was monitored. We also examined whether oxidative stress could be a causative factor in disease onset and progression. Finally, in order to define the role of plasmalogen deficiency in the pathological events, Gnpat−/− mice were directly compared with Nestin-Pex5 −/− mice.
Nestin-Pex5 knockout mice and Gnpat −/− mice were generated as previously described [4, 8] and bred into a Swiss Webster background. Tamoxifen inducible mice in which the Cre-ERTM fusion protein is under the control of the ubiquitously active CMV promoter  were obtained from The Jackson Laboratory. Tamoxifen was i.p. injected in CMV-Cre-ER TM -Pex5-loxP mice at the age of 4 weeks at a dose of 0.2 mg/g body weight. All mice received 5 injections, with one day intervals. Mice were bred in the animal housing facility of the KULeuven, had ad libitum access to water and standard rodent food, and were kept on a 12 h light and dark cycle. All animal experiments were performed in accordance with the “Guidelines for Care and Use of Experimental Animals” and fully approved by the Research Advisory Committee (Research Ethical committee) of the K.U.Leuven (#159/2008).
Treatment of the mice
Nestin-Pex5 mice were treated with anti-oxidants as described . N-acetylcysteine (1%) (Acros, Geel, Belgium) was administered via the drinking water (pH adjusted to 3.5) and α-lipoic acid (0.5% w/w) (Sigma-Aldrich, Bornem, Belgium) via the food chow. Treatment was started at the age of 3 weeks for a period of 9 weeks.
Paraffin sections were used for immunological detection of almost all antigens, except for C1q, CC-1 and CNP, for which cryosections were used. Stainings were done according to . The sources and concentrations of antibodies are listed in Additional file 1: Table S1. For each age, 3 to 5 individual knockout mice were analyzed and compared with control littermates.
After incubation with primary antibodies overnight, secondary HRP-labeled antibodies were applied during 1 h, followed by fluorescent labeling by the use of the cyanine 2 (FITC) TSA kit (Perkin Elmer Life sciences, Boston, USA). When double or triple immunolabeling was performed, sets of primary and secondary antibodies were sequentially applied. As a second and third fluorescent marker the cyanine 3 and cyanine 5 TSA kits (Perkin-Elmer) were used.
Images were analyzed with a Zeiss CLSM510 confocal laser scanning microscope equipped with a Zeiss axiocam camera.
After perfusion fixation of the corpus callosum with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer pH 7.2, samples were kept in fixative at 4 ° C overnight. After 1 h post-fixation in 2% osmium tetroxide/0.1 mol/L phosphate buffer pH 7.2 at 4°C, the samples were dehydrated in graded series of alcohol and embedded in epoxy resin. Ultra-thin sections (50 to 60 nm) were cut, stained with uranyl acetate and lead citrate and examined at 50 kV using a Zeiss EM 900 electron microscope (Oberkochen, Germany). Images were recorded digitally with a Jenoptik Progress C14 camera system (Jena, Germany) and operated using Image-Pro express software (Media Cybernetics, USA).
qRT-PCR on inflammatory markers was performed as previously described . The assay ID (Applied Biosystems, Halle, Belgium) or the sequence of primers and probes (when custom-made) are listed in Additional file 2: Table S2. FIZZ1 (Mm00445109_m1) and IL10 (Mm00439616_m1) were designed and synthesized by Applied Biosystems and purchased as a premix.
For microarray analysis, the transcriptional profiles of hypothalamus of 10-week-old wild type and Nestin-Pex5 −/− mice (n = 4 per genotype) were analyzed by using the whole genome Affymetrix GeneChip® Mouse Genome 430 2.0 Array as described previously . Labeling of the samples, hybridization, washing and scanning of the chips was carried out at the MicroArray Facility in Leuven (MAF, Leuven, Belgium). The bioinformatics analysis was performed as previously described . The complete dataset is available under GEO record GSE1938.
The TBARS assay to quantify the oxidative stress marker malondialdehyde was performed as decribed . Lipid hydroperoxides were measured with xylenol orange adapted from  Dried lipid extracts were redissolved in 0.2 ml chloroform, followed by addition of 0.8 ml reaction mixture (water/methanol 1/3, v/v), containing 32.25 mM H2SO4, 5 mM FeSO4, 250 μM xylenol orange and 31.25 mM butyl hydroxytoluene. After incubation for 30 min, absorbance was read at 560 nm and standardized against cumene hydroperoxide.
Demyelination and axon loss in cerebellum of young Nestin-Pex5−/− mice is accompanied with mild gliosis
It is well known that paranodes and juxtaparanodes get disorganized along demyelinated axons which according to recent reports might even be an early event in Multiple Sclerosis [19, 20]. For this reason, paranodes and juxtaparanodes were visualized by antibodies against caspr and potassium channels respectively. Already at the age of 3 weeks, potassium channels were more spread out over cerebellar fibers, in comparison to the characteristic juxtaparanodal distribution in control mice (Figure 1N–O).
Also in the cerebellar peduncles demyelination (Additional file 4: Figure S2 A–E) and axonal degeneration (Additional file 4: Figure S2C–D, arrows and F–H) takes place, respectively starting at 6 and 9 weeks and aggravating with age. In contrast to the cerebellar folia, marked microgliosis and macrophage activity was observed at 12 weeks (Additional file 4: Figure S2E–F).
Cortex: Loss of myelin in the absence of a strong microglial response
Brain stem and spinal cord: Strong correlation between microglia activation and demyelination
Inflammatory demyelination in corpus callosum
Activation of the immune system in nestin-Pex5 knockout mice
Microarray analysis on hypothalamus of 10-week-old Nestin-Pex5 −/− mice
Cytokine-cytokine receptor interaction
complement component 3a receptor 1
complement component 1, q subcomponent, beta polypeptide
complement component 4B
complement component 1, q subcomponent, alpha polypeptide
chemokine (C-X-C motif) ligand 13
chemokine (C-C motif) ligand 6
chemokine (C-C motif) ligand 3
Leukocyte transedothelial migration
Integrin beta 2
Integrin alpha M
Intercellular adhesion molecule 1
Vascular cell adhesion molecule 1
Antigen processing and presentation
Histocompatibility 2, K1, K region
Histocompatibility 2, D region locus 1
Fc receptor, IgG, low affinity IIb
Fc receptor, IgG, low affinity III
Pyrimidinergic receptor P2Y, G-protein coupled 6
Toll-like receptor signaling pathway
Toll-like receptor 2
Toll-like receptor 1
Toll-like receptor 7
Toll-like receptor 4
Transforming growth factor β
Manganese superoxide dismutase
Glutathione peroxidase 1
Fold change of mRNA levels of inflammatory mediators incorpus callosum and cerebellum of 6-week-old Nestin-Pex5 mice
Nestin-Pex5 6 weeks
28.96 ± 6.27***
17.15 ± 8.85
8.40 ± 1.43***
2.94 ± 1.45
11.91 ± 3.26***
12.42 ± 6.10
13.64 ± 6.38***
10.63 ± 0.34***
2.83 ± 0.18***
3.38 ± 0.5**
30.92 ± 10.65*
3.18 ± 3.10
1.78 ± 0.08**
2.78 ± 1.11
13.31 ± 8.59
1.62 ± 0.50
Importantly, the microarray data also pointed to phagocytotic activity of inflammatory cells in the brain of 10-week-old Nestin-Pex5−/− mice (Table 1), confirming histological results. Receptors involved in the binding and ingestion of pathogens, including Fc receptors, complement receptors and P2Y6 were highly up regulated (Table 1). The latter receptor recognizes the nucleotide UDP released from injured neurons and stimulates phagocytosis . Furthermore, expression of Mpeg1, a macrophage marker  and of lysosomal proteins, were significantly increased. Examples are cathepsin D (2.54 fold up regulated), Lamp2 (1.57 fold) and hexosaminidase B (3.17 fold). This was further confirmed by qRT-PCR on Mpeg1 in other brain regions and at earlier time points (Figure 7A and B; Table 2).
In a normal inflammatory process, a pro-inflammatory phase is followed by a resolution phase, during which microglial cells produce anti-inflammatory markers [23, 24]. According to microarray analysis only TGFβ and Heme oxygenase-1 were up regulated (2.54 and 2.37 fold respectively) (Table 2). However, additional anti-inflammatory markers  were analysed by qRT-PCR analysis in corpus callosum of 3- and 6-week-old mice. As Arginase 1, Fizz1, IL10 and TGFβ were all significantly up regulated in 6-week-old Nestin-Pex5 −/− mice, it appears that the pro-inflammatory response is accompanied by an anti-inflammatory reaction. In cerebellum of 6-week-old Nestin-Pex5 −/− mice, only TGFβ is significantly elevated, the other anti-inflammatory markers were not (Table 2).
The microarray results further revealed the up regulation of genes involved in transendothelial migration of blood-derived inflammatory cells into the brain. To verify increased infiltration, immunohistochemistry with an antibody recognizing T-cells (CD3) was performed. At the age of 12 weeks a limited number of T-cells were noticed in corpus callosum of Nestin-Pex5−/− mice (Figure 7C–D).
Together, these data indicate that the innate immune system is activated at a very early stage in peroxisome deficient brain, generating a strong and persistent pro-inflammatory response which could not be stopped by anti-inflammatory mechanisms. Secondarily, the adaptive immune system is activated.
As it was reported that immunohistochemistry is a more sensitive procedure to detect markers of oxidative stress in brain , stainings were performed with antibodies reacting with 4-hydroxynonenal (4-HNE). Slightly higher 4-HNE immunoreactivity was detected in Purkinje cells already at postnatal day 14 (Figure 8B–C) which increased with age (Figure 8D–E). In addition, 4-HNE staining was sporadically observed along cortical fibers and in the corpus callosum but not in all mice and not at all ages tested (not shown). In all the other brain regions and cell types, there was no difference in immunoreactivity between Nestin-Pex5 −/− and control mice. To confirm these findings, oxidative damage to proteins was examined by staining with antibodies to 3-nitrotyrosine . Immunoreactivity was also elevated in Purkinje cells of Nestin-Pex5 −/− mice, starting at 3 weeks (Figure 8F–G) and increasing with age (Figure 8H–I), but not in other brain regions. Interestingly, according to microarray data there is no up regulation of the cytokine iNOS in Nestin-Pex5 −/− mice, which was confirmed by qRT-PCR analysis in corpus callosum of 6-week-old mice (not shown). In agreement with iNOS, also mRNA levels of NADPH oxidase were not altered in the microarray. However, at the age of 5 months significantly higher levels of iNOS were detected in corpus callosum (2.35 fold, p < 0.05). At this age, also 4-HNE immunoreactivity was observed in corpus callosum of a subset of Nestin-Pex5 −/− mice.
Up regulation of anti-oxidative enzymes is another indicator of increased oxidative stress. According to microarray analysis the peroxiredoxins 1 and 6, catalase and glutathione peroxidase 1 were mildly but significantly up regulated, whereas several other ones such as MnSOD were not. MnSOD immunoreactivity was also not different between brains of Nestin-Pex5 −/− mice and control littermates (Table 1). Strongly increased catalase immunoreactivity in Nestin-Pex5 knockouts was already reported .
To rule out that a low level of oxidative stress, that was not detectable with the used methods, contributes to the pathology, Nestin-Pex5 −/− mice were treated from the age of 3 weeks for a period of 9 weeks with an anti-oxidant mixture of α-lipoic acid (0.5% w/w in food chow) and N-acetylcysteine (1% in drinking water). This treatment did not lead to improvement of the motor performance and health status of the mice. Microscopically, catalase levels were not reduced and 4-HNE was still detected in Purkinje cells of treated Nestin-Pex5 −/− mice. Thus, anti-oxidative treatment did not affect the phenotype of Nestin-Pex5 knockout mice.
No neuroinflammation in the gnpat knockout model
One of the major peroxisomal metabolic pathways is the synthesis of ether lipids, including plasmalogens which are very enriched in brain myelin. It was previously reported that cerebellar and neocortical dysmyelination occurs in juvenile Gnpat knockout mice, a model with selective ether lipid deficiency . In order to examine the contribution of deficient ether lipid synthesis to the pathology of Nestin-Pex5 knockout mice, the two models were directly compared. These two models show a similar premature death rate, with a critical period between P1 (postnatal day 1) and weaning when 30 – 50% of the knockouts die. However, Gnpat knockout mice can survive more than 20 months, whereas Nestin-Pex5−/− mice always die before the age of 6 months. First we analyzed the levels of plasmalogens in the brains of both models at 3 weeks. Whereas plasmalogen levels are 15-fold lower in the Nestin-Pex5 knockout brain in comparison to controls (0.6 nmol/mg tissue vs 9.8 nmol/mg) plasmalogen levels were even further reduced below the detection level (< 0.03 nmol/mg tissue) in Gnpat−/− mice.
In the cortex, some Gnpat knockout mice (aged 3 weeks – 5 months) also displayed reduced MBP immunoreactivity (Figure 9G–H), which however never reached the level of cortical demyelination of 12-week-old Nestin-Pex5 −/− mice. In general, the lack of myelin and axonal loss in the Gnpat−/− is clearly less drastic, compared to the Nestin-Pex5 −/− mice and restricted to the neocortex and cerebellum at all ages analyzed. Microgliosis was not observed in the knockout mice whereas a slightly increased number of astrocytes was noticed at 3 months in the white matter of the cerebellar folia and at 3 and 5 months in the brain stem (not shown). To investigate whether the innate immune system was activated, inflammatory markers were analyzed in the forebrain and brain stem of 4-month-old Gnpat−/− mice, but, remarkably, pro-inflammatory cytokines were not induced (Figure 9M).
Markers of oxidative stress were investigated by immunohistochemistry. Similar to the Nestin-Pex5 −/− mice, an increased immunoreactivity for 3-nitrotyrosine (Figure 9I–J) and a slightly higher reactivity for 4-HNE was detected in Purkinje cells of 3-week-old Gnpat −/− mice (Figure 10 K-L). At 12 weeks and 5 months, only a subset of knockout brains stained positive for 3-nitrotyrosine and 4-HNE in Purkinje cells. Higher protein levels of catalase were also detected in the Gnpat knockout, but this was only observed in the white matter and Bergmann glial cells of the cerebellum and not in the cerebrum. This is in contrast to Nestin-Pex5 −/− where higher catalase levels were seen throughout the brain . Summarizing, despite a more severe deficiency of plasmalogens as compared to Nestin-Pex5 −/− mice, Gnpat−/− mice do not develop an inflammatory phenotype, nor extensive demyelination and axonal loss.
Inactivation of peroxisomes after development causes similar pathology as the nestin-Pex5−/−
To exclude that the brain phenotype of Nestin-Pex5 −/− mice is somehow a consequence of mild developmental delays , a mouse model was investigated in which CNS peroxisomes were inactivated at a later time point. To this end, CMV-Cre-ERTM mice, in which the expression of Cre-recombinase fused to a mutated estrogen receptor ligand binding domain is driven by the ubiquitously active CMV promoter, were bred with Pex5-loxP mice. After administration of tamoxifen at the age of 4 weeks, the Cre recombinase fusion protein becomes activated causing Pex5 recombination. As previously shown (paper submitted, supplemental Figure 3), wild type PEX5p was not completely lost in all tissues, but inactivation was particularly effective in brain (less than 20% wild type Pex5 mRNA). Based on the cytosolic pattern of catalase, predominantly astrocytes and oligodendrocytes and some microglia were devoid of functional peroxisomes, whereas most neurons still contained a peroxisomal catalase staining (Additional file 5: Figure S3, arrows).
These mice developed significant motor problems from the age of 5 months which gradually aggravated with age, leading to death from the age of 8 months. This is similar to Nestin-Pex5 −/− mice although with a later onset and a slower progression (Additional file 6: Figure S4A). Immunohistochemistry at 3, 5 and 8 months of age revealed that brain pathologies developed in the same sequence and in the same brain regions as in Nestin-Pex5 −/− mice but with later onset. Microgliosis was mildly present in a subset of knockouts at 3 months, mainly in the cerebellum and the brain stem (not shown). However, it became more pronounced at 5 and 8 months in those brain regions (cerebellum, Additional file 4: Figure S4Ba-c). Microgliosis coincided with demyelination (Additional file 4: Figure S4Ba-c) and axonal damage (Additional file 4: Figure S4 Bb), axonal loss (Additional file 4: Figure S4 Bd–e) and swellings (Additional file 4: Figure S4Bf, arrow), which is the most pronounced at 8 months of age in these areas. Demyelination was confirmed by the presence of deMBP in the cerebellar folia (not shown). As in the Nestin-Pex5 −/− mice the corpus callosum was affected later than other brain regions, displaying microgliosis between 5 and 8 months, but no demyelination or axonal damage (Additional file 4: Figure S4Bg–i).
Thus, loss of functional peroxisomes from brain at any age induces a neurodegenerative program accompanied with a strong inflammatory response.
Absence of functional peroxisomes in the postnatal mouse CNS causes a neurodegenerative phenotype that ultimately leads to extensive axon loss and early death. This closely mimics pathologies in mild peroxisome biogenesis disorders and the cerebral form of X-ALD. We here investigated the involvement of micro- and astroglia activation, metabolic factors and oxidative stress in the onset and progression of myelin and axon loss triggered by peroxisome inactivity.
In all demyelinating areas, increased numbers of microglia were present. Remarkably, in cerebellum and cortex microglia were never very abundant, whereas they were strongly represented in brain stem starting in the juvenile period and they massively invaded the demyelinating corpus callosum of 12-week-old mice. Also of note is that astroglial activation lagged behind microglia proliferation, as described before in other neurodegenerative diseases . Many microglia displayed features of phagocytosing cells including a swollen appearance, sometimes containing MBP-positive myelin debris and surrounding a demyelinating fiber. This was also confirmed by increased transcripts for macrophage markers such as Mpeg1 and lysosomal enzymes.
According to recent insights, the number and shape of microglia does not predict the type of immunological reaction that is developing. Besides the classical pro-inflammatory microglia, macrophages occur that are involved in the resolution of inflammation which have been named “alternatively activated” or “deactivated” . In all brain regions, markers of a pro-inflammatory response, such as TNFα, IL6 and IL1β were highly up regulated . Although both markers of alternative microglial activation (Arginase 1, Fizz1) and of acquired deactivation (IL10, TGFβ) were up regulated in peroxisome deficient corpus callosum before the peak of microglial activation occurred, this could not halt the progression of inflammation.
It was remarkable that the induction of markers of the innate immune system occurred at a very early stage in the disease process, within 3 weeks after birth. Strikingly, it was recently reported that peroxisome deficiency in Pex1 mutant Drosophila larvae also caused up regulation of genes involved in innate immunity, further supporting that peroxisome deficiency modulates immunological responses . In addition, peroxisomes were shown to be involved in the generation of defense factors following a viral infection and thus participate in antiviral signaling . The precise role of peroxisomes in shaping the innate immune responses will need to be further elucidated.
With regard to neuronal morphology, we did not find much evidence for cell death  but there was progressive axonal loss throughout the CNS which was already very obvious at the age of 12 weeks based on neurofilament staining with antibody SMI31. At this and earlier ages, axonal swellings (detected by staining with anti-APP) and damage (detected by staining unphosphorylated neurofilaments with SMI32) could be visualized, but these stainings strongly underestimated ongoing axonal degeneration. By EM analysis, some axonal irregularities were already observed at the age of 3 weeks, illustrating the very early onset of the degenerative phenotype. The progressive axonal loss coincided with severe and progressive motor and cognitive decline, which we reported before . In this respect, it is interesting to mention that axonal injury is currently considered as the most important cause of clinical disabilities in MS [33, 34].
With the exception of a variable onset of lesions in the cortex, the development of brain pathology was consistent from mouse to mouse. It was also recapitulated, but delayed in time, in mice in which peroxisomes were deleted from the CNS in the juvenile period after completion of myelination. This ruled out the possibility that the observed anomalies were a late consequence of peroxisome ablation during development. Our present findings that demyelination in cerebellum precedes white matter abnormalities in cerebrum is in striking agreement with reports in mildly affected peroxisome biogenesis patients [35, 36] in which regressive changes predominate over developmental anomalies. By MRI analysis the first foci of white matter abnormalities were found in the central cerebellar area, additional lesions were often seen in the brain stem whereas the cerebral hemispheres were affected later. This distribution of white matter lesions is clearly different from the typical pattern in X-ALD in which the splenium of the corpus callosum is affected first. Remarkably, the leukoencephalopathy in PBD patients was not predictive for the clinical outcome  suggesting that additional pathology causes the psychomotor retardation. In addition, patients have been identified with milder PEX mutations, displaying normal mental capacities, but developing progressive ataxia, caused by cerebellar atrophy [36–38]. Why cerebellar neurons are selectively affected in these longer surviving patients is not clear.
A direct comparison of the lesions in Nestin-Pex5 −/− mice with those in Gnpat−/− mice allowed to determine the contribution of hampered ether phospholipid synthesis to the observed pathologies. Ether phospholipids are quintessential products of peroxisomal metabolism that are very enriched in the CNS and myelin under the form of plasmalogens. Gnpat−/− mice were previously shown to have myelin deficits in cerebellum and swellings on Purkinje cell axons . Although the pattern of hypomyelination, axonal swellings and axonal loss was very similar in cerebellum of juvenile Gnpat and Nestin-Pex5 knockout mice, with increasing age, Gnpat−/− mice did not further lose myelin in cerebellum, nor in other brain areas. Also in rhizomelic chondrodysplasia punctata RCDP patients, abnormalities in white matter were reported which were however rather located in supratentorial areas . In sharp contrast with Nestin-Pex5 knockout mice no phagocytotic microglia nor reactive astrocytes were detected at the age of 6 months throughout the Gnpat−/− brain which was further confirmed by the absence of pro-inflammatory markers. These findings are in accordance with the pathology observed in the Pex7 −/− mouse model for RCDP type 1. At the age of 9 – 11 months the latter mice do not display a reactive response of microglia  but they show mild astrocytosis. Likewise, in RCDP patients non-inflammatory dysmyelination and occasionally astrocytosis, but no inflammatory demyelination has been reported . A mild degree of gliosis is observed in a case of GNPAT deficiency . In view of the inflammatory demyelination in patients with peroxisomal β-oxidation deficiency , the latter metabolic defect is likely facilitating the inflammatory response in brain lacking peroxisomes. Extensive gliosis was indeed observed in Mfp2 knockout mice although the pro- versus anti-inflammatory character was not investigated yet. A role for plasmalogen shortage as a factor synergizing with peroxisomal β-oxidation defects to induce inflammation can however not be excluded. Indeed, microglia activation was observed in the Pex7:Abcd1 double knockout mouse model but not in the single knockouts .
An important question is whether the axon injury culminating in axonal loss is due to myelin abnormalities, to neuroinflammation, to other factors, or a combination of these. We can indeed exclude that this is the consequence of peroxisome deficiency within neurons, as we previously showed that neuron selective inactivation of Pex5 did not cause a neurodegenerative phenotype . Based on fluorescence microscopy, and particularly obvious in cerebellum, axonal swellings and degeneration mostly appear after demyelination, indicating that the lack of myelin evokes loss of the denuded axon. On the other hand, as previously reported and now confirmed at younger ages, ultrastructural analysis showed degenerating axons that were still surrounded by a full myelin sheath, indicating that alternative mechanisms may also be operating. The milder phenotype of Gnpat knockout mice, in which an inflammatory reaction is absent, strongly indicates that the early and severe activation of the innate immune system contributes to the neurodegenerative phenotype in Nestin-Pex5 −/− mice. The detrimental impact of neuroinflammation on axonal survival is well established. In MS patients, axonal loss is closely related to the degree of inflammation in the active lesions [34, 44]. In addition, it should be kept in mind that peroxisomes play an essential role in oligodendrocytes, because the selective loss of functional peroxisomes from these cells also results in inflammatory demyelination . In this respect the early expression of the complement component C1q on oligodendrocytes and neurons might be indicative of an endangered response which secondarily evokes inflammation. Indeed, although the complement system can be stimulated by both the innate and the adaptive immune system, it here seems to play a role in the early activation of innate immunity as the adaptive system is only involved much later with infiltrating T cells. Also in a mouse model for spinal cord injury, C1q is expressed on axons and oligodendrocytes . In Alzheimer disease, C1q causes activation of C3d, and via the complement pathway it can activate microglia with a damaging effect on myelin and axons . Although the relationship between peroxisomes and innate immunity needs further investigation, we speculate that metabolic abnormalities, likely related to peroxisomal β-oxidation defects, initiate an early activation of the innate immune system, which together with abnormalities in the formation and maintenance of myelin creates an environment which is detrimental for axons.
The absence of oxidative stress in young Nestin-Pex5 −/− mice is in contrast with recent findings in Abcd1 knockout mice, a model for the AMN form of X-ALD. In these mice oxidative damage to proteins was detected in spinal cord but not in brain, preceding by several months axonal damage in spinal cord. The crucial role of this oxidative stress for the pathogenesis was further proven as anti-oxidative therapy could prevent the degenerative phenotype. Increased concentrations of the very long chain fatty acid, C26:0 is thought to be the metabolic factor initiating oxidative stress . It remains unclear why this occurs in spinal cord and not in brain with Abcd1 deficiency and likewise, why C26:0 accumulation in Nestin-Pex5 knockout mice  does not trigger an oxidative stress response. In fact, in patients with ABCD1 deficiency, lipid peroxidation in plasma was higher in the AMN phenotype than in cerebral ALD and in asymptomatic patients . On the other hand, in PBD patients oxidative stress markers were not increased in plasma or urine . Taken together, the causal relationship between oxidative stress and inflammatory demyelination is currently unclear and we have no evidence that degeneration in the peroxisome deficient brain is triggered or boosted by the development of oxidative stress.
A lack of plasmalogens contributes to abnormalities in the formation and stability of myelin but not to the uncontrolled pro-inflammatory response in the brain of Nestin-Pex5 −/− mice. This neurotoxic environment leads to axonal damage and a chronic progression of neurodegeneration. It remains to be investigated whether the neurodegenerative phenotype can be halted by blocking the inflammatory response.
ATP-binding cassette, subfamily D, member 1
amyloid precursor protein
cyclic nucleotide phosphodiesterase
central nervous system
chemokine (C-C motif) ligand
chemokine (C-X-C motif) ligand
Found in inflammatory zone
glial fibrillary acidic protein
inducible nitric oxide synthase
Kyoto Encyclopedia of Genes and Genomes
lysosome associated membrane protein
lipid raft microdomain
myelin basic protein
multifunctional protein 2
manganese superoxide dismutase
macrophage expressed gene
nicotinamide adenine dinucleotide phosphate
peroxisome biogenesis disorder
rhizomelic chondrodysplasia punctata
standard error of the mean
Sternberger monoclonals inc
thiobarbituric acid reactive substances
transforming growth factor
Toll like receptor
tumor necrosis factor
tyramide signal amplification
The authors wish to thank Benno Das, Lies Pauwels and Els Meyhi for excellent technical assistance. This work was funded by grants from Fonds Wetenschappelijk Onderzoek Vlaanderen (G.0760.09), OT Leuven (08/40), the European Union (LSHG-CT-2004-512018, FP6) and ELA2007-0004I4. Astrid Bottelbergs is a predoctoral fellow of the IWT Vlaanderen.
- Steinberg S, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW: Peroxisome biogenesis disorders. Biochim Biophys Acta. 2006, 1763: 1733-1748. 10.1016/j.bbamcr.2006.09.010.View ArticlePubMed
- Wanders RJ, Waterham HR: Peroxisomal disorders: the single peroxisomal enzyme deficiencies. Biochim Biophys Acta. 2006, 1763: 1707-1720. 10.1016/j.bbamcr.2006.08.010.View ArticlePubMed
- Baes M, Aubourg P: Peroxisomes, myelination, and axonal integrity in the CNS. Neuroscientist. 2009, 15: 367-379. 10.1177/1073858409336297.View ArticlePubMed
- Hulshagen L, Krysko O, Huyghe S, Klein R, Van Veldhoven PP, De Deyn PP, D’hooge R, Hartmann D, Baes M: Absence of functional peroxisomes from mouse central nervous system causes dysmyelination and axon degeneration. J Neurosci. 2008, 28: 4015-4027. 10.1523/JNEUROSCI.4968-07.2008.View ArticlePubMed
- Krysko O, Hulshagen L, Janssen A, Schutz G, Klein R, De Bruycker M, Espeel M, Gressens P, Baes M: Neocortical and cerebellar developmental abnormalities in conditions of selective elimination of peroxisomes from brain or from liver. J Neurosci Res. 2007, 85: 58-72. 10.1002/jnr.21097.View ArticlePubMed
- Kassmann CM, Lappe-Siefke C, Baes M, Brügger B, Mildner A, Werner HB, Natt O, Michaelis T, Prinz M, Frahm J, Nave K-A: Axonal loss and neuroinflammation caused by peroxisome-deficient oligodendrocytes. Nat Genet. 2007, 39: 969-976. 10.1038/ng2070.View ArticlePubMed
- Bottelbergs A, Verheijden S, Hulshagen L, Gutmann DH, Goebbels S, Nave KA, Kassmann C, Baes M: Axonal integrity in the absence of functional peroxisomes from projection neurons and astrocytes. GLIA. 2010, 58 (13): 1532-1543.PubMed
- Rodemer C, Thai TP, Brugger B, Kaercher T, Werner H, Nave KA, Wieland F, Gorgas K, Just WW: Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum Mol Genet. 2003, 12: 1881-1895. 10.1093/hmg/ddg191.View ArticlePubMed
- Teigler A, Komijenovic D, Draguhn A, Gorgas K, Just WW: Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse cerebrum. Hum Mol Genet. 2009, 18: 1897-1908. 10.1093/hmg/ddp110.PubMed CentralView ArticlePubMed
- Fourcade S, Lopez-Erauskin J, Galino J, Duval C, Naudi A, Jove M, Kemp S, Villarroya F, Ferrer I, Pamplona R, Portero-Otin M, Pujol A: Early oxidative damage underlying neurodegeneration in X-adrenoleukodystrophy. Hum Mol Genet. 2008, 17: 1762-1773. 10.1093/hmg/ddn085.View ArticlePubMed
- Galino J, Ruiz M, Fourcade S, Schluter A, Lopez-Erauskin J, Guilera C, Jove M, Naudi A, Garcia-Arumi E, Andreu AL, Starkov AA, Pamplona R, Ferrer I, Portero-Otin M, Pujol A: Oxidative damage compromises energy metabolism in the axonal degeneration mouse model of X-adrenoleukodystrophy. Antioxid Redox Signal. 2011, 15 (8): 2095-2107. 10.1089/ars.2010.3877.PubMed CentralView ArticlePubMed
- Muller CC, Nguyen TH, Ahlemeyer B, Meshram M, Santrampurwala N, Cao S, Sharp P, Fietz PB, Baumgart-Vogt E, Crane DI: PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress. Dis Model Mech. 2011, 4 (1): 104-119. 10.1242/dmm.004622.PubMed CentralView ArticlePubMed
- Huyghe S, Mannaerts GP, Baes M, Van Veldhoven PP: Peroxisomal multifunctional protein-2: the enzyme, the patients and the knockout mouse model. Biochim Biophys Acta. 2006, 1761: 973-994. 10.1016/j.bbalip.2006.04.006.View ArticlePubMed
- Huyghe S, Schmalbruch H, Hulshagen L, Van Veldhoven PP, Baes M, Hartmann D: Peroxisomal multifunctional protein-2 deficiency causes motor deficits and glial lesions in the adult CNS. Am J Pathol. 2006, 168: 1321-1334. 10.2353/ajpath.2006.041220.PubMed CentralView ArticlePubMed
- Hayashi S, McMahon AP: Efficient recombination in diverse tissues by a tamoxifen-iniducible form of Cre: a tool for temporally regulated Cre activation/inactivation in the mouse. Dev Biol. 2002, 244: 305-318. 10.1006/dbio.2002.0597.View ArticlePubMed
- Martens K, Ver Loren Van Themaat E, van Batenburg MF, Heinaniemi M, Huyghe S, Van Hummelen P, Carlberg C, van Veldhoven PP, Van Kampen A, Baes M: Coordinate induction of PPAR alpha and SREBP2 in multifunctional protein 2 deficient mice. Biochim Biophys Acta. 2008, 1781 (11–12): 694-702.View ArticlePubMed
- Fernandes CG, Leipnitz G, Seminotti B, Amaral AU, Zanatta A, Vargas CR, Dutra Filho CS, Wajner M: Experimental evidence that phenylalanine provokes oxidative stress in hippocampus and cerebral cortex of developing rats. Cell Mol Neurobiol. 2010, 30 (2): 317-326. 10.1007/s10571-009-9455-6.View ArticlePubMed
- Gay C, Collins J, Gebicki JM: Hydroperoxide assay with the ferric-xylenol orange complex. Anal Biochem. 1999, 273 (2): 149-155. 10.1006/abio.1999.4208.View ArticlePubMed
- Howell OW, Palser A, Polito A, Melrose S, Zonta B, Scheiermann C, Vora AJ, Brophy PJ, Reynolds R: Disruption of neurofascin localization reveals early changes preceding demyelination and remyelination in multiple sclerosis. Brain. 2006, 129 (Pt 12): 3173-3185.View ArticlePubMed
- Coman I, Aigrot MS, Seilhean D, Reynolds R, Girault JA, Zalc B, Lubetzki C: Nodal, paranodal and juxtaparanodal axonal proteins during demyelination and remyelination in multiple sclerosis. Brain. 2006, 129: 3186-3195. 10.1093/brain/awl144.View ArticlePubMed
- Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K, Shinozaki Y, Ohsawa K, Tsuda M, Joshi BV, Jacobson KA, Kohsaka S, Inoue K: UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature. 2007, 446: 1091-1095. 10.1038/nature05704.PubMed CentralView ArticlePubMed
- Ellett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ: mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood. 2011, 117 (4): e49-e56. 10.1182/blood-2010-10-314120.PubMed CentralView ArticlePubMed
- Min KJ, Yang MS, Kim SU, Jou I, Joe EH: Astrocytes induce hemeoxygenase-1 expression in microglia: a feasible mechanism for preventing excessive brain inflammation. J Neurosci. 2006, 26 (6): 1880-1887. 10.1523/JNEUROSCI.3696-05.2006.View ArticlePubMed
- Innamorato NG, Lastres-Becker I, Cuadrado A: Role of microglial redox balance in modulation of neuroinflammation. Curr Opin Neurol. 2009, 22 (3): 308-314. 10.1097/WCO.0b013e32832a3225.View ArticlePubMed
- Colton CA, Wilcock DM: Assessing activation states in microglia. CNS Neurol Disord Drug Targets. 2010, 9 (2): 174-191.View ArticlePubMed
- Moreira PI, Sayre LM, Zhu X, Nunomura A, Smith MA, Perry G: Detection and localization of markers of oxidative stress by in situ methods: application in the study of Alzheimer disease. Methods Mol Biol. 2010, 610: 419-434. 10.1007/978-1-60327-029-8_25.View ArticlePubMed
- Rubbo H, Radi R: Protein and lipid nitration: role in redox signaling and injury. Biochim Biophys Acta. 2008, 1780 (11): 1318-1324. 10.1016/j.bbagen.2008.03.007.View ArticlePubMed
- Liu W, Tang Y, Feng J: Cross talk between activation of microglia and astrocytes in pathological conditions in the central nervous system. Life Sci. 2011, 89 (5–6): 141-146.View ArticlePubMed
- Schwartz M, Butovsky O, Bruck W, Hanisch UK: Microglial phenotype: is the commitment reversible?. Trends Neurosci. 2006, 29 (2): 68-74. 10.1016/j.tins.2005.12.005.View ArticlePubMed
- Rojo AI, Innamorato NG, Martin-Moreno AM, De Ceballos ML, Yamamoto M, Cuadrado A: Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson’s disease. Glia. 2010, 58 (5): 588-598.PubMed
- Mast FD, Li J, Virk MK, Hughes SC, Simmonds AJ, Rachubinski RA: A Drosophila model for the Zellweger spectrum of peroxisome biogenesis disorders. Dis Model Mech. 2011, 4 (5): 659-672. 10.1242/dmm.007419.PubMed CentralView ArticlePubMed
- Dixit E, Boulant S, Zhang Y, Lee AS, Odendall C, Shum B, Hacohen N, Chen ZJ, Whelan SP, Fransen M, Nibert ML, Superti-Furga G, Kagan JC: Peroxisomes are signaling platforms for antiviral innate immunity. Cell. 2010, 141 (4): 668-681. 10.1016/j.cell.2010.04.018.PubMed CentralView ArticlePubMed
- Bjartmar C, Kidd G, Mork S, Rudick R, Trapp BD: Neurological disability correlates with spinal cord axonal loss and reduced N-acetyl aspartate in chronic multiple sclerosis patients. Ann Neurol. 2000, 48 (6): 893-901. 10.1002/1531-8249(200012)48:6<893::AID-ANA10>3.0.CO;2-B.View ArticlePubMed
- Frischer JM, Bramow S, Dal-Bianco A, Lucchinetti CF, Rauschka H, Schmidbauer M, Laursen H, Sorensen PS, Lassmann H: The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain. 2009, 132 (Pt 5): 1175-1189.PubMed CentralView ArticlePubMed
- Barth PG, Majoie CB, Gootjes J, Wanders RJ, Waterham HR, van der Knaap MS, de Klerk JB, Smeitink J, Poll-The BT: Neuroimaging of peroxisome biogenesis disorders (Zellweger spectrum) with prolonged survival. Neurology. 2004, 62: 439-444. 10.1212/01.WNL.0000106943.40848.03.View ArticlePubMed
- Ebberink MS, Csanyi B, Chong WK, Denis S, Sharp P, Mooijer PA, Dekker CJ, Spooner C, Ngu LH, De Sousa C, Wanders RJ, Fietz MJ, Clayton PT, Waterham HR, Ferdinandusse S: Identification of an unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene. J Med Genet. 2010, 47 (9): 608-615. 10.1136/jmg.2009.074302.View ArticlePubMed
- Sevin C, Ferdinandusse S, Waterham HR, Wanders RJ, Aubourg P: Autosomal recessive cerebellar ataxia caused by mutations in the PEX2 gene. Orphanet J Rare Dis. 2011, 6: 8-10.1186/1750-1172-6-8.PubMed CentralView ArticlePubMed
- Regal L, Ebberink MS, Goemans N, Wanders RJ, De ML, Jaeken J, Schrooten M, Van Coster R, Waterham HR: Mutations in PEX10 are a cause of autosomal recessive ataxia. Ann Neurol. 2010, 68 (2): 259-263.PubMed
- Bams-Mengerink AM, Majoie CB, Duran M, Wanders RJ, Van Hove J, Scheurer CD, Barth PG, Poll-The BT: MRI of the brain and cervical spinal cord in rhizomelic chondrodysplasia punctata. Neurology. 2006, 66: 798-803. 10.1212/01.wnl.0000205594.34647.d0.View ArticlePubMed
- Brites P, Mooyer PAW, el Mrabet L, Duran M, Waterham HR, Wanders RJA: Plasmalogens participate in very-long-chain fatty acid-induced pathology. Brain. 2009, 132: 482-492.View ArticlePubMed
- Powers JM, Moser HW: Peroxisomal disorders : genotype, phenotype, major neuropathologic lesions, and pathogenesis. Brain Pathol. 1998, 8: 101-120.View ArticlePubMed
- Viola A, Confort-Gouny S, Ranjeva JP, Chabrol B, Raybaud C, Vintila F, Cozzone PJ: MR imaging and MR spectroscopy in rhizomelic chondrodysplasia punctata. AJNR Am J Neuroradiol. 2002, 23: 480-483.PubMed
- Powers JM: Demyelination in peroxisomal diseases. J Neurol Sci. 2005, 228: 206-207. 10.1016/j.jns.2004.10.012.View ArticlePubMed
- Kuhlmann T, Lingfeld G, Bitsch A, Schuchardt J, Bruck W: Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time. Brain. 2002, 125 (Pt 10): 2202-2212.View ArticlePubMed
- Anderson AJ, Robert S, Huang W, Young W, Cotman CW: Activation of complement pathways after contusion-induced spinal cord injury. J Neurotrauma. 2004, 21 (12): 1831-1846. 10.1089/neu.2004.21.1831.View ArticlePubMed
- Yasojima K, Schwab C, McGeer EG, McGeer PL: Up-regulated production and activation of the complement system in Alzheimer’s disease brain. Am J Pathol. 1999, 154 (3): 927-936. 10.1016/S0002-9440(10)65340-0.PubMed CentralView ArticlePubMed
- Lopez-Erauskin J, Fourcade S, Galino J, Ruiz M, Schluter A, Naudi A, Jove M, Portero-Otin M, Pamplona R, Ferrer I, Pujol A: Antioxidants halt axonal degeneration in a mouse model of X-adrenoleukodystrophy. Ann Neurol. 2011, 70 (1): 84-92. 10.1002/ana.22363.PubMed CentralView ArticlePubMed
- Deon M, Sitta A, Barschak AG, Coelho DM, Pigatto M, Schmitt GO, Jardim LB, Giugliani R, Wajner M, Vargas CR: Induction of lipid peroxidation and decrease of antioxidant defenses in symptomatic and asymptomatic patients with X-linked adrenoleukodystrophy. Int J Dev Neurosci. 2007, 25 (7): 441-444. 10.1016/j.ijdevneu.2007.08.008.View ArticlePubMed
- Ferdinandusse S, Finckh B, de Hingh YC, Stroomer LE, Denis S, Kohlschutter A, Wanders RJ: Evidence for increased oxidative stress in peroxisomal D-bifunctional protein deficiency. Mol Genet Metab. 2003, 79: 281-287. 10.1016/S1096-7192(03)00108-2.View ArticlePubMed
- Pujol A, Hindelang C, Callizot N, Bartsch U, Schachner M, Mandel JL: Late onset neurological phenotype of the X-ALD gene inactivation in mice : a mouse model for adrenomyeloneuropathy. Hum Mol Genet. 2002, 11: 499-505. 10.1093/hmg/11.5.499.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the gveative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.