Identification of a novel tetrameric structure for human apolipoprotein-D.

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded ß-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-ß burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

ABCA8 stimulates sphingomyelin production in oligodendrocytes.

Members of the ABCA (ATP-binding cassette subfamily A) family are characterized by their ability to transport lipids across cellular membranes and regulate lipid homoeostasis in the brain and peripheral tissues. ABCA8 is a little-known member of this subfamily that was originally cloned from human brain libraries and has no known function. In an effort to elucidate the role of ABCA8 in the brain we first undertook a comprehensive analysis of its expression in the human brain. ABCA8 was differentially expressed in multiple regions of adult human brains with significantly higher expression in oligodendrocyte-enriched white matter regions compared with grey matter cortical regions. We then assessed the impact of ABCA8 on sphingomyelin production in oligodendrocyte and showed that ABCA8 was able to significantly stimulate both sphingomyelin synthase 1 expression and sphingomyelin production. Furthermore, ABCA8 expression in the prefrontal cortex across the human life span correlated strongly with age-associated myelination, and the myelinating gene p25α was significantly up-regulated with ABCA8. The present study represents the first extensive expression and functional study of ABCA8 in the human brain and the results strongly suggest that ABCA8 regulates lipid metabolism in oligodendrocytes and potentially plays a role in myelin formation and maintenance.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease.

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.

Multiple pathways of lipid dysregulation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease characterized by the degeneration of motor neurons and loss of various muscular functions. Dyslipidaemia is prevalent in amyotrophic lateral sclerosis with aberrant changes mainly in cholesterol ester and triglyceride. Despite this, little is known about global lipid changes in amyotrophic lateral sclerosis or in relation to disease progression. The present study incorporated a longitudinal lipidomic analysis of amyotrophic lateral sclerosis serum with a comparison with healthy controls using advanced liquid chromatography-mass spectrometry. The results established that diglyceride, the precursor of triglyceride, was enriched the most, while ceramide was depleted the most in amyotrophic lateral sclerosis compared with controls, with the diglyceride species (18:1/18:1) correlating significantly to neurofilament light levels. The prenol lipid CoQ8 was also decreased in amyotrophic lateral sclerosis and correlated to neurofilament light levels. Most interestingly, the phospholipid phosphatidylethanolamine and its three derivatives decreased with disease progression, in contrast to changes with normal ageing. Unsaturated lipids that are prone to lipid peroxidation were elevated with disease progression with increases in the formation of toxic lipid products. Furthermore, in vitro studies revealed that phosphatidylethanolamine synthesis modulated TARDBP expression in SH-SY5Y neuronal cells. Finally, diglyceride, cholesterol ester and ceramide were identified as potential lipid biomarkers for amyotrophic lateral sclerosis diagnosis and monitoring disease progression. In summary, this study represents a longitudinal lipidomics analysis of amyotrophic lateral sclerosis serum and has provided new insights into multiple pathways of lipid dysregulation in amyotrophic lateral sclerosis.

ATP-binding cassette transporter expression is widely dysregulated in frontotemporal dementia with TDP-43 inclusions.

The human brain is highly enriched in lipids and increasing evidence indicates that dysregulation of lipids in the brain is associated with neurodegeneration. ATP-binding cassette subfamily A (ABCA) transporters control the movement of lipids across cellular membranes and are implicated in a number of neurodegenerative diseases. However, very little is known about the role of ABCA transporters in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), which is a common form of younger-onset dementia. We therefore undertook a comprehensive analysis of the expression of ABCA transporters (ABCA1-13) in five key brain regions (amygdala, inferior temporal cortex, superior frontal cortex, cerebellum and parietal cortex) in FTLD-TDP and controls. We found that the expression of ABCA2, ABCA3, ABCA4, ABCA7, ABCA9, ABCA10 and ABCA13 was significantly altered in FTLD-TDP in a region-specific manner. In addition, the expression of ABCA transporters correlated specifically to different neural markers and TARDBP. These results suggest substantial dysregulation of ABCA transporters and lipid metabolism in FTLD-TDP and these changes are associated with neuroinflammation.

Increased unsaturated lipids underlie lipid peroxidation in synucleinopathy brain.

Lipid peroxidation is a process of oxidative degradation of cellular lipids that is increasingly recognized as an important factor in the pathogenesis of neurodegenerative diseases. We were therefore interested in the manifestation of lipid peroxidation in synucleinopathies, a group of neurodegenerative diseases characterized by the central pathology of α-synuclein aggregates, including Parkinson's disease, multiple system atrophy, dementia with Lewy bodies and Alzheimer's disease with Lewy bodies. We assessed lipid peroxidation products, lipid aldehydes, in the amygdala, a common disease-affected region in synucleinopathies, and in the visual cortex, a disease-unaffected region. We found that the levels of lipid aldehydes were significantly increased in the amygdala, but not in the visual cortex. We hypothesized that these increases are due to increases in the abundance of unsaturated lipids, since lipid aldehydes are formed from unsaturated lipids. We undertook a comprehensive analysis of membrane lipids using liquid chromatography-mass spectrometry and found that unsaturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin were specifically elevated in the amygdala and correlated with increases in lipid aldehydes. Furthermore, unsaturated phosphatidylethanolamine levels were associated with soluble α-synuclein. Put together, these results suggest that manifestation of lipid peroxidation is prevalent in synucleinopathies and is likely to be due to increases in unsaturated membrane lipids. Our findings underscore the importance of lipid peroxidation in α-synuclein pathology and in membrane structure maintenance.

Increased VLCFA-lipids and ELOVL4 underlie neurodegeneration in frontotemporal dementia.

Rare, yet biologically critical, lipids that contain very long chain fatty acids (VLCFA-lipids) are synthesized in the brain by the enzyme ELOVL4. High levels of VLCFA-lipids are toxic to cells and excess VLCFA-lipids are actively removed by ABCD1 in an ATP-dependent manner. Virtually nothing is known about the impact of VLCFA-lipids in neurodegenerative diseases. Here, we investigated the possible role of VLCFA-lipids in frontotemporal dementia (FTD), which is a leading cause of younger-onset dementia. Using quantitative discovery lipidomics, we identified three VLCFA-lipid species that were significantly increased in FTD brain compared to controls, with strong correlations with ELOVL4. Increases in ELOVL4 expression correlated with significant decreases in the membrane-bound synaptophysin in FTD brain. Furthermore, increases in ABCD1 expression correlated with increases in VLCFA-lipids. We uncovered a new pathomechanism that is pertinent to understanding the pathogenesis of FTD.

Pathological manifestation of human endogenous retrovirus K in frontotemporal dementia.

BACKGROUND: Behavioral variant frontotemporal dementia (bvFTD) is a common form of younger-onset dementia with a proportion of cases overlapping pathologically and genetically with amyotrophic lateral sclerosis (ALS). Previous studies have identified that the human endogenous retrovirus K (HERV-K) is elevated in ALS serum and is associated with ALS TDP-43 pathology. In contrast, little is known about HERV-K changes in bvFTD. Here, we investigated the possible role of HERV-K in bvFTD. METHODS: We measured the HERV-K env gene in sporadic bvFTD (N=63), sporadic ALS (N=89) and control (N=21) serum by ddPCR. We also analyzed HERV-K env, by qPCR, and the HERV-K reverse transcriptase protein, by confocal immunofluorescence microscopy, in the disease-affected superior frontal cortex of bvFTD with TDP-43 pathology. RESULTS: Here, we show that HERV-K env levels are significantly elevated (P=3.5×10-6) in bvFTD compared to control serum, differentiating cases with an AUC value of 0.867. HERV-K env levels are also specifically elevated in the superior frontal cortex of bvFTD with TDP-43 pathology, with the HERV-K reverse transcriptase protein and TDP-43 deposit localized to the neuronal cytoplasm. Furthermore, in a neuronal cell line overexpression of TDP-43 induces HERV-K env transcription. CONCLUSIONS: These results suggest that manifestation of HERV-K is associated with bvFTD TDP-43 pathology. Analysis of HERV-K in bvFTD may provide insight into an unrecognized but targetable perturbed pathology.

Uncovering pathophysiological changes in frontotemporal dementia using serum lipids.

Blood serum is enriched in lipids and has provided a platform to understand the pathogenesis of a number of human diseases with improved diagnosis and development of biomarkers. Understanding lipid changes in neurodegenerative diseases is particularly important because of the fact that lipids make up >50% of brain tissues. Frontotemporal dementia (FTD) is a common cause of early onset dementia, characterized by brain atrophy in the frontal and temporal regions, concomitant loss of lipids and dyslipidemia. However, little is known about the link between dyslipidemia and FTD pathophysiology. Here, we utilized an innovative approach - lipidomics based on mass spectrometry - to investigate three key aspects of FTD pathophysiology - mitochondrial dysfunction, inflammation, and oxidative stress. We analyzed the lipids that are intrinsically linked to neurodegeneration in serum collected from FTD patients and controls. We found that cardiolipin, acylcarnitine, lysophosphatidylcholine, platelet-activating factor, o-acyl-ω-hydroxy fatty acid and acrolein were specifically altered in FTD with strong correlation between the lipids, signifying pathophysiological changes in FTD. The lipid changes were verified by measurement of the common disease markers (e.g. ATP, cytokine, calcium) using conventional assays. When put together, these results support the use of lipidomics technology to detect pathophysiological changes in FTD.

Apolipoprotein D Upregulation in Alzheimer's Disease but Not Frontotemporal Dementia.

Frontotemporal dementia (FTD) and Alzheimer's disease (AD) are the two common forms of dementia. FTD syndromes are characterized by lobar atrophy (frontotemporal lobar degeneration or FTLD) and the presence of either cellular TDP43 (FTLD-TDP), tau (FTLD-tau), or FUS aggregates, while extracellular ß-amyloid plaques and hyperphosphorylated tau tangles develop in AD. Oxidative stress can induce these pathological modifications in disease models, and is thought to play a role in these syndromes. Apolipoprotein D (apoD) is a glial-expressed lipocalin known to protect against oxidative stress, with increased levels in AD, supporting a protective role. The expression of apoD has not been studied in FTLD. This study assesses apoD expression in FTLD-TDP and FTLD-tau in comparison to AD and controls. It also analyzes the effect of apoD on TARDBP (TDP43 gene) and ß-amyloid precursor protein (APP). The expression of apoD was analyzed by Western blotting in FTLD-TDP, FTLD-tau, AD, and control post-mortem brain tissue. An apoD-overexpressing cell model was used to study the impact of increased apoD on APP and TARDBP expression. We confirm that apoD expression was increased in AD but surprisingly it was not affected in either of the two main pathological forms of FTLD. Under oxidative stress conditions, apoD had no effect on TDP43 expression but it did decrease APP expression. This suggests that apoD does not act as a neuroprotective factor in FTLD in the same way as in AD. This could contribute to the more rapid degeneration observed in FTLD.

Deletion of Alzheimer's Disease Risk Gene ABCA7 Alters White Adipose Tissue Development and Leptin Levels.

ATP-binding cassette A7 (ABCA7) is a genetic risk factor for late-onset Alzheimer's disease (AD). It belongs to a group of transporter genes that specializes in regulating lipid transport in the periphery as well as in the brain. ABCA7 has been implicated in a number of roles relating to AD pathology, including phagocytic clearance of amyloid-ß peptides. We have discovered that deletion of ABCA7 in mouse causes a dramatic reduction in white adipose tissue (WAT) in female mice. WAT is important in AD context because it is the primary producer of leptin, which is a hormone that is known to modulate AD neuropathology. WAT in male Abca7-/- mice was not altered. The pathological link between ABCA7 and WAT that impacts on AD is unknown. Our transcription analysis revealed that lipin-1 expression was significantly upregulated in female Abca7-/- mice, indicating that ABCA7 affects WAT development. The circulating leptin level was significantly reduced in female Abca7-/- mice without any change in WAT leptin mRNA or protein expression, indicating that ABCA7 does not affect leptin production, but alters the circulating leptin level indirectly by affecting WAT development. Insulin is a key hormone that regulates WAT development, i.e., adipogenesis, and it was significantly reduced in female Abca7-/- mice. These data when put together suggest that ABCA7 plays a role in regulating WAT development and consequently circulating leptin levels, which are known to modulate AD neuropathology.

Increased apolipoprotein D dimer formation in Alzheimer's disease hippocampus is associated with lipid conjugated diene levels.

Previous studies indicate that apolipoprotein D (apoD) may have a lipid antioxidant function in the brain. We have shown that apoD can reduce free radical-generating lipid hydroperoxides to inert lipid hydroxides in a reaction that involves conversion of surface exposed apoD methione-93 (Met93) residue to Met93-sulfoxide (Met93-SO). One consequence of this reaction is the formation of a stable dimerized form of apoD. As cerebral lipid peroxidation is associated with Alzheimer's disease (AD), in the present study we aimed to assess the possible presence of apoD dimers in postmortem hippocampal and cerebellar tissues derived from a cohort of pathologically defined cases ranging from control to late stage AD. Both soluble and insoluble (requiring guanidine HCl extraction) fractions of tissue homogenates were analyzed for apoD and its dimerized form. We also assessed amyloid-ß levels by ELISA and levels of lipid peroxidation by lipid conjugated diene and F2-isoprostane analysis. Our studies reveal a significant association between soluble apoD levels and AD Braak stage whereas apoD dimer formation appears to increase predominantly in the advanced stages of disease. The formation of apoD dimers is closely correlated to lipid conjugated diene levels and occurs in the hippocampus but not in the cerebellum. These results are consistent with the hypothesis that apoD acts as a lipid antioxidant in the brain.

Molecular dynamics analysis of apolipoprotein-D-lipid hydroperoxide interactions: mechanism for selective oxidation of Met-93.

BACKGROUND: Recent studies suggest reduction of radical-propagating fatty acid hydroperoxides to inert hydroxides by interaction with apolipoprotein-D (apoD) Met93 may represent an antioxidant function for apoD. The nature and structural consequences of this selective interaction are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Herein we used molecular dynamics (MD) analysis to address these issues. Long-timescale simulations of apoD suggest lipid molecules are bound flexibly, with the molecules free to explore multiple conformations in a binding site at the entrance to the classical lipocalin ligand-binding pocket. Models of 5s- 12s- and 15s-hydroperoxyeicosatetraenoic acids were created and the lipids found to wrap around Met93 thus providing a plausible mechanism by which eicosatetraenoic acids bearing hydroperoxides on different carbon atoms can interact with Met93 to yield Met93 sulfoxide (Met93SO). Simulations of glycosylated apoD indicated that a second solvent exposed Met at position 49 was shielded by a triantennerary N-glycan attached to Asn45 thereby precluding lipid interactions. MD simulations of apoD showed B-factors of the loop containing Met93SO were higher in the oxidized protein, indicating increased flexibility that is predicted to destabilize the protein and promote self-association. CONCLUSIONS/SIGNIFICANCE: These studies provide novel insights into the mechanisms that may contribute to the antioxidant function of apoD and the structural consequences that result if Met93SO is not redox-cycled back to its native state.

Cognitive phenotyping of amyloid precursor protein transgenic J20 mice.

Transgenic mice that express familial Alzheimer's disease mutant forms of the human amyloid precursor protein (hAPP) have proved to be invaluable in determining the impact that the neurotoxic amyloid-ß peptide has in vivo. In addition to the propensity to accumulate cerebral amyloid plaques, a crucial characteristic of hAPP mouse models is their cognitive impairments. To date the most widely used test for analyzing cognitive impairment in hAPP mice is the Morris water maze (MWM) which, due to the fact that mice are not "natural" swimmers, may not always be the ideal paradigm to investigate cognitive behaviours. Furthermore, not all cognitive impairments have been replicated across research laboratories. In the current study, we characterised the cognitive abilities of the J20 transgenic mouse line (expressing the Swedish 670/671(KM->NL) and Indiana (717(V->F)hAPP mutations) and non-transgenic mice. Mice were assessed in the cheeseboard task (i.e., a 'dry version' of the MWM) and a variety of other cognitive paradigms to test fear conditioning, object recognition and short-term memory to broaden the understanding of the cognitive deficits in J20 mice. hAPP transgenic mice perform normally in tasks for fear conditioning, short-term object recognition and short-term memory of context familiarity. However, they were profoundly impaired in their spatial reference memory capabilities in the cheeseboard task. The cheeseboard task has potential to replace the MWM task in situations where the MWM is not suitable for particular mouse models.

Role of Abca7 in mouse behaviours relevant to neurodegenerative diseases.

ATP-binding cassette transporters of the subfamily A (ABCA) are responsible for the translocation of lipids including cholesterol, which is crucial for neurological function. Recent studies suggest that the ABC transporter ABCA7 may play a role in the development of brain disorders such as schizophrenia and Alzheimer's disease. However, Abca7's role in cognition and other behaviours has not been investigated. Therefore, we characterised homozygous Abca7 knockout mice in a battery of tests for baseline behaviours (i.e. physical exam, baseline locomotion and anxiety) and behaviours relevant to schizophrenia (i.e. prepulse inhibition and locomotor response to psychotropic drugs) and Alzheimer's disease (i.e. cognitive domains). Knockout mice had normal motor functions and sensory abilities and performed the same as wild type-like animals in anxiety tasks. Short-term spatial memory and fear-associated learning was also intact in Abca7 knockout mice. However, male knockout mice exhibited significantly impaired novel object recognition memory. Task acquisition was unaffected in the cheeseboard task. Female mice exhibited impaired spatial reference memory. This phenomenon was more pronounced in female Abca7 null mice. Acoustic startle response, sensorimotor gating and baseline locomotion was unaltered in Abca7 knockout mice. Female knockouts showed a moderately increased motor response to MK-801 than control mice. In conclusion, Abca7 appears to play only a minor role in behavioural domains with a subtle sex-specific impact on particular cognitive domains.

Increased ATP-binding cassette transporter A1 expression in Alzheimer's disease hippocampal neurons.

ATP-binding cassette transporter A1 (ABCA1) reduces amyloid-beta burden in transgenic mouse models of Alzheimer's disease (AD). Associations between ABCA1 polymorphisms and AD risk are also established. Little is known regarding the regulation of ABCA1 expression in the brain and how this may be affected by AD. In the present study we assessed ABCA1 mRNA and protein expression in the hippocampus of AD cases compared to controls. ABCA1 was clearly expressed in hippocampal neurons and expression was increased two- to three-fold in AD cases. The increased hippocampal ABCA1 expression was associated with increased APOE and PUMA gene expression, implying an association with neuronal stress. Consistent with this, treatment of SK-N-SH neurons with amyloid-beta peptide resulted in a 48% loss in survival and a significant upregulation of ABCA1, APOE, and PUMA gene expression. Studies in young (2 month) and old (12 month) transgenic mice expressing a familial AD form of human amyloid-beta protein precursor and presenilin-1 revealed a significant age-dependent upregulation of hippocampal Abca1 compared to wild-type control mice. However, hippocampal Apoe and Puma gene expression were not correlated with increased Abca1 expression in mice. Our data indicate that ABCA1 is upregulated in AD hippocampal neurons potentially via an amyloid-beta-mediated pathway.