Developmental and Degenerative Cerebellar Pathologies in Peroxisomal ß-Oxidation Deficiency.

The integrity of the cerebellum is exquisitely dependent on peroxisomal ß-oxidation metabolism. Patients with peroxisomal ß-oxidation defects commonly develop malformation, leukodystrophy, and/or atrophy of the cerebellum depending on the gene defect and on the severity of the mutation. By analyzing mouse models lacking the central peroxisomal ß-oxidation enzyme, multifunctional protein-2 (MFP2), either globally or in selected cell types, insights into the pathomechanisms could be obtained. All mouse models developed ataxia, but the onset was earlier in global and neural-selective (Nestin) Mfp2-/- knockout mice as compared to Purkinje cell (PC)-selective Mfp2 knockouts.At the histological level, this was associated with developmental anomalies in global and Nestin-Mfp2-/- mice, including aberrant wiring of PCs by parallel and climbing fibers and altered electrical properties of PCs. In all mouse models, dystrophy of PC axons with swellings initiating in the deep cerebellar nuclei and evolving to the proximal axon, preceded death of PCs. These degenerative features are in part mediated by deficient peroxisomal ß-oxidation within PCs but are accelerated when MFP2 is also absent from other neural cell types. The metabolic causes of the diverse cerebellar pathologies remain unknown.In conclusion, peroxisomal ß-oxidation is required both for the development and for the maintenance of the cerebellum. This is mediated by PC autonomous and nonautonomous mechanisms.

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 ß-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 ß-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 ß-oxidation pathway is required within Purkinje neurons to maintain their axonal integrity, independent of glial dysfunction.

Early-onset Purkinje cell dysfunction underlies cerebellar ataxia in peroxisomal multifunctional protein-2 deficiency.

The cerebellar pathologies in peroxisomal diseases underscore that these organelles are required for the normal development and maintenance of the cerebellum, but the mechanisms have not been resolved. Here we investigated the origins of the early-onset coordination impairment in a mouse model with neural selective deficiency of multifunctional protein-2, the central enzyme of peroxisomal ß-oxidation. At the age of 4weeks, Nestin-Mfp2(-/-) mice showed impaired motor learning on the accelerating rotarod and underperformed on the balance beam test. The gross morphology of the cerebellum and Purkinje cell arborization were normal. However, electrophysiology revealed a reduced Purkinje cell firing rate, a decreased excitability and an increased membrane capacitance. The distribution of climbing and parallel fiber synapses on Purkinje cells was immature and was accompanied by an increased spine length. Despite normal myelination, Purkinje cell axon degeneration was evident from the occurrence of axonal swellings containing accumulated organelles. In conclusion, the electrical activity, axonal integrity and wiring of Purkinje cells are exquisitely dependent on intact peroxisomal ß-oxidation in neural cells.

Peroxisomal Disorders: A Review on Cerebellar Pathologies.

Peroxisomes are organelles with diverse metabolic tasks including essential roles in lipid metabolism. They are of utmost importance for the normal functioning of the nervous system as most peroxisomal disorders are accompanied with neurological symptoms. Remarkably, the cerebellum exquisitely depends on intact peroxisomal function both during development and adulthood. In this review, we cover all aspects of cerebellar pathology that were reported in peroxisome biogenesis disorders and in diseases caused by dysfunction of the peroxisomal α-oxidation, ß-oxidation or ether lipid synthesis pathways. We also discuss the phenotypes of mouse models in which cerebellar pathologies were recapitulated and search for connections with the metabolic abnormalities. It becomes increasingly clear that besides the most severe forms of peroxisome dysfunction that are associated with developmental cerebellar defects, milder impairments can give rise to ataxia later in life.

Precise Anatomic Localization of Accumulated Lipids in Mfp2 Deficient Murine Brains Through Automated Registration of SIMS Images to the Allen Brain Atlas.

Mass spectrometry imaging (MSI) is a powerful tool for the molecular characterization of specific tissue regions. Histochemical staining provides anatomic information complementary to MSI data. The combination of both modalities has been proven to be beneficial. However, direct comparison of histology based and mass spectrometry-based molecular images can become problematic because of potential tissue damages or changes caused by different sample preparation. Curated atlases such as the Allen Brain Atlas (ABA) offer a collection of highly detailed and standardized anatomic information. Direct comparison of MSI brain data to the ABA allows for conclusions to be drawn on precise anatomic localization of the molecular signal. Here we applied secondary ion mass spectrometry imaging at high spatial resolution to study brains of knock-out mouse models with impaired peroxisomal ß-oxidation. Murine models were lacking D-multifunctional protein (MFP2), which is involved in degradation of very long chain fatty acids. SIMS imaging revealed deposits of fatty acids within distinct brain regions. Manual comparison of the MSI data with the histologic stains did not allow for an unequivocal anatomic identification of the fatty acids rich regions. We further employed an automated pipeline for co-registration of the SIMS data to the ABA. The registration enabled precise anatomic annotation of the brain structures with the revealed lipid deposits. The precise anatomic localization allowed for a deeper insight into the pathology of Mfp2 deficient mouse models.

Central nervous system pathology in MFP2 deficiency: insights from general and conditional knockout mouse models.

Multifunctional protein-2 (MFP2), also known as D-bifunctional protein, is a central enzyme of the peroxisomal ß-oxidation pathway. Defects in this enzyme are associated with a spectrum of neurological disorders encompassing developmental and degenerative pathologies. In order to investigate the cellular and molecular mechanisms of these neuropathologies, mouse models with general and cell type selective loss of MFP2 were generated. In this review the distinct anomalies in the CNS of adult Mfp2 knockout mice are discussed, in particular the cerebellar degeneration and neuroinflammation. The potential underlying mechanisms are considered with regard to the cellular origin and biochemical causes. Finally, the similarities and differences between the CNS phenotypes of mice lacking MFP2 and mice with peroxisome biogenesis disorders are assessed.

Systemic G-CSF attenuates cerebral inflammation and hypomyelination but does not reduce seizure burden in preterm sheep exposed to global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) is common in preterm infants, but currently no curative therapy is available. Cell-based therapy has a great potential in the treatment of hypoxic-ischemic preterm brain injury. Granulocyte-colony stimulating factor (G-CSF) is known to mobilize endogenous hematopoietic stem cells (HSC) and promotes proliferation of endogenous neural stem cells. On these grounds, we hypothesized that systemic G-CSF would be neuroprotective in a large translational animal model of hypoxic-ischemic injury in the preterm brain. Global hypoxia-ischemia (HI) was induced by transient umbilical cord occlusion in instrumented preterm sheep. G-CSF treatment (100µg/kg intravenously, during five consecutive days) was started one day before the global HI insult to ascertain mobilization of endogenous stem cells within the acute phase after global HI. Mobilization of HSC and neutrophils was studied by flow cytometry. Brain sections were stained for microglia (IBA-1), myelin basic protein (MBP) and myeloperoxidase (MPO) to study microglial proliferation, white matter injury and neutrophil invasion respectively. Electrographic seizure activity was analyzed using amplitude-integrated electroencephalogram (aEEG). G-CSF effectively mobilized CD34-positive HSC in the preterm sheep. In addition, G-CSF caused marked mobilization of neutrophils, but did not influence enhanced invasion of neutrophils into the preterm brain after global HI. Microglial proliferation and hypomyelination following global HI were reduced as a result of G-CSF treatment. G-CSF did not cause a reduction of the electrographic seizure activity after global HI. In conclusion, G-CSF induced mobilization of endogenous stem cells which was associated with modulation of the cerebral inflammatory response and reduced white matter injury in an ovine model of preterm brain injury after global HI. G-CSF treatment did not improve neuronal function as shown by seizure analysis. Our study shows that G-CSF treatment has neuroprotective potential following hypoxic-ischemic injury in the preterm brain.

Alveolar nerve unfolding technique for synoptic analysis: visualization and quantification of inferior alveolar nerve tracings in three-dimensional cone-beam computed tomography.

The aim of the technique presented here is to visualize the anatomical context of the inferior alveolar nerve (IAN) canal. For 2 cases, cone-beam computed tomography images of the mandible were obtained from patient files together with the manual preoperative IAN canal tracings. For both cases, similar to simulated panoramic images, a two-dimensional image is extracted from a three-dimensional cone-beam computed tomography image. Unlike panoramic images, the unfolding does not follow the general curvature of the mandible but follows the nerve tracing closely and places the traced nerve track on a horizontal central line. Because of the centering of the nerve tracing together with the nerve canal and its surroundings in a two-dimensional representation, the technique (ANUTSA [Alveolar Nerve Unfolding Technique for Synoptic Analysis]) allowed the first case to evidence the adjacency of root tips along the IAN, whereas in the second case the degree of penetration of the IAN by an implant is revealed. The global aspect of the representation through unfolding allowed for the detection of the anomalies and the IAN-penetrating lesion along the IAN canal at a glance.

Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation.

Although peroxisome biogenesis and ß-oxidation disorders are well known for their neurodevelopmental defects, patients with these disorders are increasingly diagnosed with neurodegenerative pathologies. In order to investigate the cellular mechanisms of neurodegeneration in these patients, we developed a mouse model lacking multifunctional protein 2 (MFP2, also called D-bifunctional protein), a central enzyme of peroxisomal ß-oxidation, in all neural cells (Nestin-Mfp2(-/-)) or in oligodendrocytes (Cnp-Mfp2(-/-)) and compared these models with an already established general Mfp2 knockout. Nestin-Mfp2 but not Cnp-Mfp2 knockout mice develop motor disabilities and ataxia, similar to the general mutant. Deterioration of motor performance correlates with the demise of Purkinje cell axons in the cerebellum, which precedes loss of Purkinje cells and cerebellar atrophy. This closely mimics spinocerebellar ataxias of patients affected with mild peroxisome ß-oxidation disorders. However, general knockouts have a much shorter life span than Nestin-Mfp2 knockouts which is paralleled by a disparity in activation of the innate immune system. Whereas in general mutants a strong and chronic proinflammatory reaction proceeds throughout the brain, elimination of MFP2 from neural cells results in minor neuroinflammation. Neither the extent of the inflammatory reaction nor the cerebellar degeneration could be correlated with levels of very long chain fatty acids, substrates of peroxisomal ß-oxidation. In conclusion, MFP2 has multiple tasks in the adult brain, including the maintenance of Purkinje cells and the prevention of neuroinflammation but this is not mediated by its activity in oligodendrocytes nor by its role in very long chain fatty acid degradation.

Cerebral inflammation and mobilization of the peripheral immune system following global hypoxia-ischemia in preterm sheep.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI. METHODS: Preterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis. RESULTS: Global HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function. CONCLUSIONS: Our data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.