RESUMEN
The neurometabolic disorder succinic semialdehyde dehydrogenase (SSADH) deficiency leads to great neurochemical imbalances and severe neurological manifestations. The cause of the disease is loss of function of the enzyme SSADH, leading to impaired metabolism of the principal inhibitory neurotransmitter GABA. Despite the known identity of the enzymatic deficit, the underlying pathology of SSADH deficiency remains unclear. To uncover new mechanisms of the disease, we performed an untargeted integrative analysis of cerebral protein expression, functional metabolism, and lipid composition in a genetic mouse model of SSADH deficiency (ALDH5A1 knockout mice). Our proteomic analysis revealed a clear regional vulnerability, as protein alterations primarily manifested in the hippocampus and cerebral cortex of the ALDH5A1 knockout mice. These regions displayed aberrant expression of proteins linked to amino acid homeostasis, mitochondria, glial function, and myelination. Stable isotope tracing in acutely isolated brain slices demonstrated an overall maintained oxidative metabolism of glucose, but a selective decrease in astrocyte metabolic activity in the cerebral cortex of ALDH5A1 knockout mice. In contrast, an elevated capacity of oxidative glutamine metabolism was observed in the ALDH5A1 knockout brain, which may serve as a neuronal compensation of impaired astrocyte glutamine provision. In addition to reduced expression of critical oligodendrocyte proteins, a severe depletion of myelin-enriched sphingolipids was found in the brains of ALDH5A1 knockout mice, suggesting degeneration of myelin. Altogether, our study highlights that impaired astrocyte and oligodendrocyte function is intimately linked to SSADH deficiency pathology, suggesting that selective targeting of glial cells may hold therapeutic potential in this disease.
Asunto(s)
Astrocitos , Encéfalo , Ratones Noqueados , Oligodendroglía , Succionato-Semialdehído Deshidrogenasa , Ácido gamma-Aminobutírico , Animales , Oligodendroglía/metabolismo , Oligodendroglía/patología , Astrocitos/metabolismo , Astrocitos/patología , Succionato-Semialdehído Deshidrogenasa/deficiencia , Succionato-Semialdehído Deshidrogenasa/metabolismo , Succionato-Semialdehído Deshidrogenasa/genética , Ratones , Ácido gamma-Aminobutírico/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Ratones Endogámicos C57BL , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/patología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Discapacidades del DesarrolloRESUMEN
The sphingolipids galactosylceramide (GalCer), sulfatide (ST) and sphingomyelin (SM) are essential for myelin stability and function. GalCer and ST are synthesized mostly from C22-C24 ceramides, generated by Ceramide Synthase 2 (CerS2). To clarify the requirement for C22-C24 sphingolipid synthesis in myelin biosynthesis and stability, we generated mice lacking CerS2 specifically in myelinating cells (CerS2ΔO/ΔO ). At 6 weeks of age, normal-appearing myelin had formed in CerS2ΔO/ΔO mice, however there was a reduction in myelin thickness and the percentage of myelinated axons. Pronounced loss of C22-C24 sphingolipids in myelin of CerS2ΔO/ΔO mice was compensated by greatly increased levels of C18 sphingolipids. A distinct microglial population expressing high levels of activation and phagocytic markers such as CD64, CD11c, MHC class II, and CD68 was apparent at 6 weeks of age in CerS2ΔO/ΔO mice, and had increased by 10 weeks. Increased staining for denatured myelin basic protein was also apparent in 6-week-old CerS2ΔO/ΔO mice. By 16 weeks, CerS2ΔO/ΔO mice showed pronounced myelin atrophy, motor deficits, and axon beading, a hallmark of axon stress. 90% of CerS2ΔO/ΔO mice died between 16 and 26 weeks of age. This study highlights the importance of sphingolipid acyl chain length for the structural integrity of myelin, demonstrating how a modest reduction in lipid chain length causes exposure of a denatured myelin protein epitope and expansion of phagocytic microglia, followed by axon pathology, myelin degeneration, and motor deficits. Understanding the molecular trigger for microglial activation should aid the development of therapeutics for demyelinating and neurodegenerative diseases.
Asunto(s)
Microglía , Vaina de Mielina , Ratones , Animales , Microglía/metabolismo , Vaina de Mielina/metabolismo , Ceramidas/metabolismo , Esfingolípidos/metabolismoRESUMEN
Following ischemic stroke, substance P (SP)-mediated neurogenic inflammation is associated with profound blood-brain barrier (BBB) dysfunction, cerebral edema, and elevated intracranial pressure (ICP). SP elicits its effects by binding the neurokinin 1 tachykinin receptor (NK1-R), with administration of an NK1-R antagonist shown to ameliorate BBB dysfunction and cerebral edema in rodent and permanent ovine stroke models. Given the importance of reperfusion in clinical stroke, this study examined the efficacy of NK1-R antagonist treatment in reducing cerebral edema and ICP in an ovine model of transient middle cerebral artery occlusion (tMCAo). Anesthetized sheep (n = 24) were subject to 2-hours tMCAo and randomized (n = 6/group) to receive early NK1-R treatment (days 1-3 post-stroke), delayed NK1-R treatment (day 5 post-stroke), or saline vehicle. At 6-days post-stroke animals were re-anaesthetized and ICP measured, followed by MRI to evaluate infarction, edema and BBB dysfunction. Following both early and delayed NK1-R antagonist administration, ICP was significantly reduced on day 6 compared to vehicle animals (p < 0.05), accompanied by a reduction in cerebral edema, midline shift and BBB dysfunction (p < 0.05). This study demonstrates that NK1-R antagonist treatment is an effective novel therapy for cerebral edema and elevated ICP following stroke in an ovine model, warranting future clinical evaluation.
RESUMEN
Heterozygous mutations in the GRN gene and hexanucleotide repeat expansions in C9orf72 are the two most common genetic causes of Frontotemporal Dementia (FTD) with TDP-43 protein inclusions. The triggers for neurodegeneration in FTD with GRN (FTD-GRN) or C9orf72 (FTD-C9orf72) gene abnormalities are unknown, although evidence from mouse and cell culture models suggests that GRN mutations disrupt lysosomal lipid catabolism. To determine how brain lipid metabolism is affected in familial FTD with TDP-43 inclusions, and how this is related to myelin and lysosomal markers, we undertook comprehensive lipidomic analysis, enzyme activity assays, and western blotting on grey and white matter samples from the heavily-affected frontal lobe and less-affected parietal lobe of FTD-GRN cases, FTD-C9orf72 cases, and age-matched neurologically-normal controls. Substantial loss of myelin-enriched sphingolipids (sulfatide, galactosylceramide, sphingomyelin) and myelin proteins was observed in frontal white matter of FTD-GRN cases. A less-pronounced, yet statistically significant, loss of sphingolipids was also observed in FTD-C9orf72. FTD-GRN was distinguished from FTD-C9orf72 and control cases by increased acylcarnitines in frontal grey matter and marked accumulation of cholesterol esters in both frontal and parietal white matter, indicative of myelin break-down. Both FTD-GRN and FTD-C9orf72 cases showed significantly increased lysosomal and phagocytic protein markers, however galactocerebrosidase activity, required for lysosomal catabolism of galactosylceramide and sulfatide, was selectively increased in FTD-GRN. We conclude that both C9orf72 and GRN mutations are associated with disrupted lysosomal homeostasis and white matter lipid loss, but GRN mutations cause a more pronounced disruption to myelin lipid metabolism. Our findings support the hypothesis that hyperactive myelin lipid catabolism is a driver of gliosis and neurodegeneration in FTD-GRN. Since FTD-GRN is associated with white matter hyperintensities by MRI, our data provides important biochemical evidence supporting the use of MRI measures of white matter integrity in the diagnosis and management of FTD.
Asunto(s)
Proteína C9orf72 , Demencia Frontotemporal , Enfermedad de Pick , Progranulinas , Animales , Ratones , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Galactosilceramidas/metabolismo , Metabolismo de los Lípidos/genética , Mutación/genética , Vaina de Mielina/metabolismo , Enfermedad de Pick/metabolismo , Progranulinas/genética , Progranulinas/metabolismo , Sulfoglicoesfingolípidos/metabolismo , HumanosRESUMEN
Background: Assessment of functional impairment following ischaemic stroke is essential to determine outcome and efficacy of intervention in both clinical patients and pre-clinical models. Although paradigms are well described for rodents, comparable methods for large animals, such as sheep, remain limited. This study aimed to develop methods to assess function in an ovine model of ischaemic stroke using composite neurological scoring and gait kinematics from motion capture. Methods: Merino sheep (n = 26) were anaesthetised and subjected to 2 hours middle cerebral artery occlusion. Animals underwent functional assessment at baseline (8-, 5-, and 1-day pre-stroke), and 3 days post-stroke. Neurological scoring was carried out to determine changes in neurological status. Ten infrared cameras measured the trajectories of 42 retro-reflective markers for calculation of gait kinematics. Magnetic resonance imaging (MRI) was performed at 3 days post-stroke to determine infarct volume. Intraclass Correlation Coefficients (ICC's) were used to assess the repeatability of neurological scoring and gait kinematics across baseline trials. The average of all baselines was used to compare changes in neurological scoring and kinematics at 3 days post-stroke. A principal component analysis (PCA) was performed to determine the relationship between neurological score, gait kinematics, and infarct volume post-stroke. Results: Neurological scoring was moderately repeatable across baseline trials (ICC > 0.50) and detected marked impairment post-stroke (p < 0.05). Baseline gait measures showed moderate to good repeatability for the majority of assessed variables (ICC > 0.50). Following stroke, kinematic measures indicative of stroke deficit were detected including an increase in stance and stride duration (p < 0.05). MRI demonstrated infarction involving the cortex and/or thalamus (median 2.7 cm3, IQR 1.4 to 11.9). PCA produced two components, although association between variables was inconclusive. Conclusion: This study developed repeatable methods to assess function in sheep using composite scoring and gait kinematics, allowing for the evaluation of deficit 3 days post-stroke. Despite utility of each method independently, there was poor association observed between gait kinematics, composite scoring, and infarct volume on PCA. This suggests that each of these measures has discreet utility for the assessment of stroke deficit, and that multimodal approaches are necessary to comprehensively characterise functional impairment.
RESUMEN
Dementia poses an ever-growing burden to health care and social services as life expectancies have grown across the world and populations age. The most common forms of dementia are Alzheimer's disease (AD), vascular dementia, frontotemporal dementia (FTD), and Lewy body dementia, which includes Parkinson's disease (PD) dementia and dementia with Lewy bodies (DLB). Genomic studies over the past 3 decades have identified variants in genes regulating lipid transporters and endosomal processes as major risk determinants for AD, with the most significant being inheritance of the ε4 allele of the APOE gene, encoding apolipoprotein E. A recent surge in research on lipid handling and metabolism in glia and neurons has established defective lipid clearance from endolysosomes as a central driver of AD pathogenesis. The most prevalent genetic risk factors for DLB are the APOE ε4 allele, and heterozygous loss of function mutations in the GBA gene, encoding the lysosomal catabolic enzyme glucocerebrosidase; whilst heterozygous mutations in the GRN gene, required for lysosomal catabolism of sphingolipids, are responsible for a significant proportion of FTD cases. Homozygous mutations in the GBA or GRN genes produce the lysosomal storage diseases Gaucher disease and neuronal ceroid lipofuscinosis. Research from mouse and cell culture models, and neuropathological evidence from lysosomal storage diseases, has established that impaired cholesterol or sphingolipid catabolism is sufficient to produce the pathological hallmarks of dementia, indicating that defective lipid catabolism is a common mechanism in the etiology of dementia.
Asunto(s)
Colesterol/metabolismo , Demencia/metabolismo , Lisosomas/metabolismo , Esfingolípidos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Apolipoproteína E4/metabolismo , Apolipoproteínas/metabolismo , Química Encefálica , Demencia/etiología , Demencia/genética , Modelos Animales de Enfermedad , Endosomas/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Inflamación , Metabolismo de los Lípidos , Metabolismo , Ratones , Microglía/metabolismo , Mutación Missense , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Agregación Patológica de Proteínas , Ratas , Proteínas tau/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Cerebral edema and elevated intracranial pressure (ICP) are the leading cause of death in the first week following stroke. Despite this, current treatments are limited and fail to address the underlying mechanisms of swelling, highlighting the need for targeted treatments. When screening promising novel agents, it is essential to use clinically relevant large animal models to increase the likelihood of successful clinical translation. As such, we sought to develop a survival model of transient middle cerebral artery occlusion (tMCAO) in the sheep and subsequently characterize the temporal profile of cerebral edema and elevated ICP following stroke in this novel, clinically relevant model. METHODS: Merino-sheep (27M;31F) were anesthetized and subject to 2 h tMCAO with reperfusion or sham surgery. Following surgery, animals were allowed to recover and returned to their home pens. At preselected times points ranging from 1 to 7 days post-stroke, animals were re-anesthetized, ICP measured for 4 h, followed by imaging with MRI to determine cerebral edema, midline shift and infarct volume (FLAIR, T2 and DWI). Animals were subsequently euthanized and their brain removed for immunohistochemical analysis. Serum and cerebrospinal fluid samples were also collected and analyzed for substance P (SP) using ELISA. RESULTS: Intracranial pressure and MRI scans were normal in sham animals. Following stroke, ICP rose gradually over time and by 5 days was significantly (p < 0.0001) elevated above sham levels. Profound cerebral edema was observed as early as 2 days post-stroke and continued to evolve out to 6 days, resulting in significant midline shift which was most prominent at 5 days post-stroke (p < 0.01), in keeping with increasing ICP. Serum SP levels were significantly elevated (p < 0.01) by 7 days post-tMCAO. CONCLUSION: We have successfully developed a survival model of ovine tMCAO and characterized the temporal profile of ICP. Peak ICP elevation, cerebral edema and midline shift occurred at days 5-6 following stroke, accompanied by an elevation in serum SP. Our findings suggest that novel therapeutic agents screened in this model targeting cerebral edema and elevated ICP would most likely be effective when administered prior to 5 days, or as early as possible following stroke onset.