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Traumatic brain injury (TBI) is often complicated by long-lasting disabilities, including headache, fatigue, insomnia, hyperactivity, and cognitive deficits. In a previous study in mice, we showed that persistent non-goal-directed hyperactivity is a characteristic post-TBI behavior that was associated with low levels of endocannabinoids in the perilesional cortex. We now analyzed lipidome patterns in the brain and plasma in TBI versus sham mice in association with key behavioral parameters and endocannabinoids. Lipidome profiles in the plasma and subcortical ipsilateral and contralateral brain were astonishingly equal in sham and TBI mice, but the ipsilateral perilesional cortex revealed a strong increase in neutral lipids represented by 30 species of triacylglycerols (TGs) of different chain lengths and saturation. The accumulation of TG was localized predominantly to perilesional border cells as revealed by Oil Red O staining. In addition, hexosylceramides (HexCer) and phosphatidylethanolamines (PE and ether-linked PE-O) were reduced. They are precursors of gangliosides and endocannabinoids, respectively. High TG, low HexCer, and low PE/PE-O showed a linear association with non-goal-directed nighttime hyperactivity but not with the loss of avoidance memory. The analyses suggest that TG overload and HexCer and PE deficiencies contributed to behavioral dimensions of post-TBI psychopathology.
Background: There is a need for early therapeutic interventions after traumatic brain injury (TBI) to prevent neurodegeneration. Microglia/macrophage (M/M) depletion and repopulation after treatment with colony stimulating factor 1 receptor (CSF1R) inhibitors reduces neurodegeneration. The present study investigates short- and long-term consequences after CSF1R inhibition during the early phase after TBI.
Methods: Sex-matched mice were subjected to TBI and CSF1R inhibition by PLX3397 for 5 days and sacrificed at 5 or 30 days post injury (dpi). Neurological deficits were monitored and brain tissues were examined for histo- and molecular pathological markers. RNAseq was performed with 30 dpi TBI samples.
Results: At 5 dpi, CSF1R inhibition attenuated the TBI-induced perilesional M/M increase and associated gene expressions by up to 50%. M/M attenuation did not affect structural brain damage at this time-point, impaired hematoma clearance, and had no effect on IL-1β expression. At 30 dpi, following drug discontinuation at 5 dpi and M/M repopulation, CSF1R inhibition attenuated brain tissue loss regardless of sex, as well as hippocampal atrophy and thalamic neuronal loss in male mice. Selected gene markers of brain inflammation and apoptosis were reduced in males but increased in females after early CSF1R inhibition as compared to corresponding TBI vehicle groups. Neurological outcome in behaving mice was almost not affected. RNAseq and gene set enrichment analysis (GSEA) of injured brains at 30 dpi revealed more genes associated with dendritic spines and synapse function after early CSF1R inhibition as compared to vehicle, suggesting improved neuronal maintenance and recovery. In TBI vehicle mice, GSEA showed high oxidative phosphorylation, oxidoreductase activity and ribosomal biogenesis suggesting oxidative stress and increased abundance of metabolically highly active cells. More genes associated with immune processes and phagocytosis in PLX3397 treated females vs males, suggesting sex-specific differences in response to early CSF1R inhibition after TBI.
Conclusions: M/M attenuation after CSF1R inhibition via PLX3397 during the early phase of TBI reduces long-term brain tissue loss, improves neuronal maintenance and fosters synapse recovery. Overall effects were not sex-specific but there is evidence that male mice benefit more than female mice.
Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN-/- deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN-/- deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN-/- mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.
Genes encoding endocannabinoid and sphingolipid metabolism pathways were suggested to contribute to the genetic risk towards attention deficit hyperactivity disorder (ADHD). The present pilot study assessed plasma concentrations of candidate endocannabinoids, sphingolipids and ceramides in individuals with adult ADHD in comparison with healthy controls and patients with affective disorders. Targeted lipid analyses of 23 different lipid species were performed in 71 mental disorder patients and 98 healthy controls (HC). The patients were diagnosed with adult ADHD (n = 12), affective disorder (major depression, MD n = 16 or bipolar disorder, BD n = 6) or adult ADHD with comorbid affective disorders (n = 37). Canonical discriminant analysis and CHAID analyses were used to identify major components that predicted the diagnostic group. ADHD patients had increased plasma concentrations of sphingosine-1-phosphate (S1P d18:1) and sphinganine-1-phosphate (S1P d18:0). In addition, the endocannabinoids, anandamide (AEA) and arachidonoylglycerol were increased. MD/BD patients had increased long chain ceramides, most prominently Cer22:0, but low endocannabinoids in contrast to ADHD patients. Patients with ADHD and comorbid affective disorders displayed increased S1P d18:1 and increased Cer22:0, but the individual lipid levels were lower than in the non-comorbid disorders. Sphingolipid profiles differ between patients suffering from ADHD and affective disorders, with overlapping patterns in comorbid patients. The S1P d18:1 to Cer22:0 ratio may constitute a diagnostic or prognostic tool.
Rodent models of Parkinson’s disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1−/−SNCAA53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.
Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood–brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.
Nucleoredoxin (NXN) is a redox regulator of Disheveled and thereby of WNT signaling. Deficiency in mice leads to cranial dysmorphisms and defects of heart, brain, and bone, suggesting defects of cell fate determination. We used shRNA-mediated knockdown of NXN in SH-SY5Y neuroblastoma cells to study its impact on neuronal cells. We expected that shNXN cells would easily succumb to redox stress, but there were no differences in viability on stimulation with hydrogen peroxide. Instead, the proliferation of naïve shNXN cells was increased with a higher rate of mitotic cells in cell cycle analyses. In addition, basal respiratory rates were higher, whereas the relative change in oxygen consumption upon mitochondrial stressors was similar to control cells. shNXN cells had an increased expression of redox-sensitive heat shock proteins, Hsc70/HSPA8 and HSP90, and autophagy markers suggested an increase in autophagosome formation upon stimulation with bafilomycin and higher flux under low dose rapamycin. A high rate of self-renewal, autophagy, and upregulation of redox-sensitive chaperones appears to be an attractive anti-aging combination if it were to occur in neurons in vivo for which SH-SY5Y cells are a model.
Aims: Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD-associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1−/−SNCAA53T double mutant mice. Methods: We studied nociceptive and olfactory behaviour and the neuropathology of dorsal root ganglia (DRGs), including ultrastructure, mitochondrial respiration, transcriptomes, outgrowth and calcium currents of primary neurons, and tissue ceramides and sphingolipids before the onset of a PD-like disease that spontaneously develops in Pink1−/−SNCAA53T double mutant mice beyond 15 months of age. Results: Similar to PD patients, Pink1−/−SNCAA53T mice developed a progressive prodromal sensory neuropathy with a loss of thermal sensitivity starting as early as 4 months of age. In analogy to human plasma, lipid analyses revealed an accumulation of glucosylceramides (GlcCer) in the DRGs and sciatic nerves, which was associated with pathological mitochondria, impairment of mitochondrial respiration, and deregulation of transient receptor potential channels (TRPV and TRPA) at mRNA, protein and functional levels in DRGs. Direct exposure of DRG neurons to GlcCer caused transient hyperexcitability, followed by a premature decline of the viability of sensory neurons cultures upon repeated GlcCer application. Conclusions: The results suggest that pathological GlcCer contribute to prodromal sensory disease in PD mice via mitochondrial damage and calcium channel hyperexcitability. GlcCer-associated sensory neuron pathology might be amenable to GlcCer lowering therapeutic strategies.
Trehalose, a sugar from fungi, mimics starvation due to a block of glucose transport and induces Transcription Factor EB- mediated autophagy, likely supported by the upregulation of progranulin. The pro-autophagy effects help to remove pathological proteins and thereby prevent neurodegenerative diseases such as Alzheimer’s disease. Enhancing autophagy also contributes to the resolution of neuropathic pain in mice. Therefore, we here assessed the effects of continuous trehalose administration via drinking water using the mouse Spared Nerve Injury model of neuropathic pain. Trehalose had no effect on drinking, feeding, voluntary wheel running, motor coordination, locomotion, and open field, elevated plus maze, and Barnes Maze behavior, showing that it was well tolerated. However, trehalose reduced nerve injury-evoked nociceptive mechanical and thermal hypersensitivity as compared to vehicle. Trehalose had no effect on calcium currents in primary somatosensory neurons, pointing to central mechanisms of the antinociceptive effects. In IntelliCages, trehalose-treated mice showed reduced activity, in particular, a low frequency of nosepokes, which was associated with a reduced proportion of correct trials and flat learning curves in place preference learning tasks. Mice failed to switch corner preferences and stuck to spontaneously preferred corners. The behavior in IntelliCages is suggestive of sedative effects as a 'side effect' of a continuous protracted trehalose treatment, leading to impairment of learning flexibility. Hence, trehalose diet supplements might reduce chronic pain but likely at the expense of alertness.
Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30–100 µM and 5–20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.