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

Background: 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. Methods: 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. Results: 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. Conclusions: 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

Neural oscillations during cognitive processes in an <i>App</i> knock-in mouse model of Alzheimer's disease pathology

Multiple animal models have been created to gain insight into Alzheimer's disease (AD) pathology. Among the most commonly used models are transgenic mice overexpressing human amyloid precursor protein (APP) with mutations linked to familial AD, resulting in the formation of amyloid beta plaques, one of the pathological hallmarks observed in AD patients. However, recent evidence suggests that the overexpression of APP by itself can confound some of the reported observations. Therefore, we investigated in the present study the App(NL-G-F)model, an App knock-in (App-KI) mouse model that develops amyloidosis in the absence of APP-overexpression. Our findings at the behavioral, electrophysiological, and histopathological level confirmed an age-dependent increase in A beta 1-42 levels and plaque deposition in these mice in accordance with previous reports. This had apparently no consequences on cognitive performance in a visual discrimination (VD) task, which was largely unaffected in App(NL-G-F) mice at the ages tested. Additionally, we investigated neurophysiological functioning of several brain areas by phase-amplitude coupling (PAC) analysis, a measure associated with adequate cognitive functioning, during the VD task (starting at 4.5 months) and the exploration of home environment (at 5 and 8 months of age). While we did not detect age-dependent changes in PAC during home environment exploration for both the wild-type and the App(NL-G-F) mice, we did observe subtle changes in PAC in the wild-type mice that were not present in the App(NL-G-F) mice

Time Trends of Cerebrospinal Fluid Biomarkers of Neurodegeneration in Idiopathic Normal Pressure Hydrocephalus

Background: Longitudinal changes in cerebrospinal fluid (CSF) biomarkers are seldom studied. Furthermore, data on biomarker gradient between lumbar (L-) and ventricular (V-) compartments seems to be discordant. Objective: To examine alteration of CSF biomarkers reflecting Alzheimer's disease (AD)-related amyloid-beta (A beta) aggregation, tau pathology, neurodegeneration, and early synaptic degeneration by CSF shunt surgery in idiopathic normal pressure hydrocephalus (iNPH) in relation to AD-related changes in brain biopsy. In addition, biomarker levels in L- and V-CSF were compared. Methods: L-CSF was collected prior to shunt placement and, together with V-CSF, 3-73 months after surgery. Thereafter, additional CSF sampling took place at 3, 6, and 18 months after the baseline sample from 26 iNPH patients with confirmed A beta plaques in frontal cortical brain biopsy and 13 iNPH patients without A beta pathology. CSF Amyloid-beta(42) (A beta(42)), total tau (T-tau), phosphorylated tau (P-tau(181)), neurofilament light (NFL), and neurogranin (NRGN) were analyzed with customized ELISAs. Results: All biomarkers but A beta(42) increased notably by 140-810% in L-CSF after CSF diversion and then stabilized. A beta(42) instead showed divergent longitudinal decrease between A beta-positive and -negative patients in L-CSF, and thereafter increase in A beta-negative iNPH patients in both L- and V-CSF. All five biomarkers correlated highly between V-CSF and L-CSF (A beta(42) R = 0.87, T-tau R = 0.83, P-tau R = 0.92, NFL R = 0.94, NRGN R = 0.9; all p < 0.0001) but were systematically lower in V-CSF (A beta(42) 14 %, T-tau 22%, P-tau 20%, NFL 32%, NRGN 19%). With APOE genotype-grouping, only A beta(42) showed higher concentration in non-carriers of allele epsilon 4. Conclusion: Longitudinal follow up shows that after an initial post-surgery increase, T-tau, P-tau, and NRGN are stable in iNPH patients regardless of brain biopsy A beta pathology, while NFL normalized toward its pre-shunt levels. A beta(42) as biomarker seems to be the least affected by the surgical procedure or shunt and may be the best predictor of AD risk in iNPH patients. All biomarker concentrations were lower in V-than L-CSF yet showing strong correlations.Peer reviewe

The role of peroxisomes in the formation and maintenance of myelinated axons.

Patients with Zellweger syndrome or other peroxisomal disorders underscore that peroxisomes are indispensible organelles for the development of the CNS. However, milder affected patients do not only display developmental problems, like a neuronal migration defect, hypotonia and seizures, but also degenerative changes, including inflammatory demyelination. Mouse models, mimicking human pathology, are valuable tools to investigate the pathomechanisms triggered by peroxisomal dysfunction. The Nestin-Pex5 mouse model, lacking functional peroxisomes in neural cells, was developed to circumvent the very early death of the general Pex5 knockout model, which hampered postnatal research. As described earlier, Nestin-Pex5 knockout mice do not display pronounced developmental problems, but show remarkable regressive pathology after birth. Three major findings have been described at the microscopic level, including demyelination, axonal degeneration and neuroinflammation, comprising astro- and microgliosis. Microscopic brain pathology is associated with an abnormal phenotype characterized by disturbed motor performance, progressively worsening with increasing age and evolving in lethargy and early death. As expected, metabolic alterations were also observed in these mice, including an accumulation of VLCFAs and a depletion of plasmalogens. Until now, the link between all these changes is still not elucidated. The Cnp-Pex5 model, with dysfunctional peroxisomes specifically in oligodendrocytes, proved that functional peroxisomes in these myelinating cells are of major importance for the preservation of normal myelinated axons. This mouse model developed similar pathology as the Nestin-Pex5 knockout, although with a later onset and a slower progression. The main goal of this thesis was to clarify the role of peroxisomes in the maintenance of healthy myelinated nerve fibers, which are crucial for the normal functioning of the brain. In view of the less severe phenotype of the oligodendrocyte specific Cnp-Pex5-/- compared with the neural Nestin-Pex5-/- mice, the role of peroxisomes in neurons and astrocytes was investigated, by the use of the NEX- and GFAP-Pex5 mouse models. Based on the normal phenotype of the NEX-Pex5-/-, peroxisomes are not important in neurons, provided that there are still peroxisomes in other brain cell types. Several abnormalities were observed in the astrocyte selective GFAP-Pex5 knockout, including an accumulation of VLCFAs, a small reduction of plasmalogens, an accumulation of lipid droplets and elevated levels of catalase. However, this had no major repercussion on the health and survival of these mice. Taking into account the phenotype of the three cell type selective Pex5 knockouts, we can conclude that peroxisomes in oligodendrocytes are the most important for normal brain functioning and that peroxisomes in astrocytes may have a supportive function. The second aim of this manuscript was to decipher the onset and relationship between neuroinflammation, demyelination and axonal degeneration. Immunohistochemical investigations revealed that demyelination and microgliosis are observed in young Nestin-Pex5 knockout mice and in close association with each other. Axonal degeneration was mostly observed after demyelination, indicating that this is presumably a consequence of demyelination. In order to rule out that these defects are a consequence of developmental abnormalities, a mouse model was generated and analyzed with deletion of functional peroxisomes after the myelination period. This CMV-Tx-Pex5 knockout model developed similar pathologies of inflammation, demyelination and axon degeneration. The analysis of mRNA levels of inflammatory markers like TNF&#945;, interleukins and chemokines showed that pro-inflammatory markers were highly up regulated and clearly before demyelination was observed by fluorescence microscopy. Interestingly, also anti-inflammatory markers were up regulated in Nestin-Pex5 knockout but this could not prevent the evolvement in a chronic inflammatory reaction. As activated microglial cells may have different shapes and functions, we demonstrated their phagocytotic activity by their swollen morphology, the presence of myelin debris within several microglial cells, and the up regulation of mRNA levels of macrophage markers. This proves that inflammatory demyelination is ongoing in the Nestin-Pex5 knockout. C1q, the first component of the complement system, was detected on neurons and oligodendrocytes before significant microgliosis was observed. This component is considered to be present on damaged cells as an alert signal towards inflammatory cells. We hypothesize that dysfunction of peroxisomes in oligodendrocytes triggers the activation of the innate immune system. If peroxisomes are also absent from other neural cell types, this reaction occurs much earlier and to a larger extent. Direct comparison of the Nestin-Pex5 model with the Gnpat knockout model, characterized by plasmalogen depletion, demonstrated that the absence of plasmalogens per se does not trigger an inflammatory reaction. Markers of oxidative stress were detected in both models in Purkinje cells and only in a late phase in the corpus callosum of Nestin-Pex5-/- mice. Thus, oxidative stress is not considered to be a trigger for neuroinflammation. The latter is confirmed by the observation that treatment of Nestin-Pex5 knockout mice with anti-oxidant drugs did not improve pathology.status: publishe

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

Abstract Background 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. Methods 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. Results 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. Conclusions 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.</p

Direct nose to brain delivery of small molecules: critical analysis of data from a standardized in vivo screening model in rats

The blood–brain barrier (BBB) is often a limiting factor for getting drugs in the brain. Bypassing the BBB by intranasal (IN), or also called nose to brain (NTB), route is an interesting and frequently investigated concept for brain drug delivery. However, despite the body of evidence for IN drug delivery in literature over the last decades, reproducibility and interpretation of animal data remain challenging. The objective of this project was to assess the feasibility and value of a standardized IN screening model in rats for the evaluation of direct brain delivery. A chemically diverse set of commercial and internal small molecules were tested in the in vivo model with different doses and/or formulations. Data were analyzed using different ways of ratio calculations: blood concentration at time of sacrifice, total exposure in blood (area under the curve, AUC) and the brain or olfactory bulb concentrations. The IN route was compared to another parenteral route to decide if there is potential direct brain transport. The results show that blood and tissue concentrations and ratios are highly variable and not always reproducible. Potential direct brain delivery was concluded for some compounds, however, sometimes depending on the analysis: using blood levels at sacrifice or AUC could lead to different conclusions. We conclude that a screening model for the evaluation of direct brain transport of small molecules is very difficult to achieve and a conclusion based on a limited number of animals with this variability is questionable