Up‐regulation of β‐amyloidogenesis in neuron‐like human cells by both 24‐ and 27‐hydroxycholesterol: protective effect of N‐acetyl‐cysteine

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27‐hydroxycholesterol (27‐OH) and 24‐hydroxycholesterol (24‐OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK‐N‐BE human neuroblastoma cells with patho‐physiologically relevant amounts of 27‐OH and 24‐OH showed that both oxysterols induce a net synthesis of Aβ_1‐42 by up‐regulating expression levels of amyloid precursor protein and β‐secretase, as well as the β‐secretase activity. Interestingly, cell pretreatment with N‐acetyl‐cysteine (NAC) fully prevented the enhancement of β‐amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β‐amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols‐induced Aβ toxic peptide accumulation in the brain.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

Cholesterol metabolism is associated with soluble amyloid precursor protein production in Alzheimer's disease

Disturbances of the cholesterol metabolism are associated with Alzheimer's disease (AD) risk and related cerebral pathology. Experimental studies found changing levels of cholesterol and its metabolites 24S‐hydroxycholesterol (24S‐OHC) and 27‐hydroxycholesterol (27‐OHC) to contribute to amyloidogenesis by increasing the production of soluble amyloid precursor protein (sAPP). The aim of this study was to evaluate the relationship between the CSF and circulating cholesterol 24S‐OHC and 27‐OHC, and the sAPP production as measured by CSF concentrations of sAPP forms in humans. The plasma and the CSF concentrations of cholesterol, 24S‐OHC and 27‐OHC, and the CSF concentrations of sAPPα, sAPPβ, and Aß1‐42 were assessed in subjects with AD and controls with normal cognition. In multivariate regression tests including age, gender, albumin ratio, and apolipoprotein E (APOE)ε4 status CSF cholesterol, 24S‐OHC, and 27‐OHC independently predicted the concentrations of sAPPα and sAPPβ. The associations remained significant when analyses were separately performed in the AD group. Furthermore, plasma 27‐OHC concentrations were associated with the CSF sAPP levels. The results suggest that high CSF concentrations of cholesterol, 24S‐OHC, and 27‐OHC are associated with increased production of both sAPP forms in AD.     

Non‐motor parkinsonian pathology in aging A53T α‐Synuclein mice is associated with progressive synucleinopathy and altered enzymatic function

Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α–synuclein levels in substantia nigra pars compacta (SNc). Excess α‐synuclein spurs Lewy‐like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate‐limiting enzyme, tyrosine hydroxylase (TH). A loss of nigral dopaminergic neurons induces PD movement problems, but before those abnormalities occur, behaviors such as olfactory loss, anxiety, and constipation often manifest. Identifying mouse models with early PD behavioral changes could provide a model in which to test emerging therapeutic compounds. To this end, we evaluated mice expressing A53T mutant human (A53T) α–synuclein for behavior and α–synuclein pathology in olfactory bulb, adrenal gland, and gut. Aging A53T mice exhibited olfactory loss and anxiety that paralleled olfactory and adrenal α‐synuclein aggregation. PP2A activity was also diminished in olfactory and adrenal tissues harboring insoluble α‐synuclein. Low adrenal PP2A activity co‐occurred with TH hyperactivity, making this the first study to link adrenal synucleinopathy to anxiety and catecholamine dysregulation. Aggregated A53T α–synuclein recombinant protein also had impaired stimulatory effects on soluble recombinant PP2A. Collectively, the data identify an excellent model in which to screen compounds for their ability to block the spread of α‐synuclein pathology associated with pre‐motor stages of PD.

     

Relation between TLR4/NF‐κB signaling pathway activation by 27‐hydroxycholesterol and 4‐hydroxynonenal,and atherosclerotic plaque instability

It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll‐like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low‐density lipoproteins, the oxysterol 27‐hydroxycholesterol (27‐OH) and the aldehyde 4‐hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high‐risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27‐OH and HNE were found to enhance cell release of IL‐8, IL‐1β, and TNF‐α and to upregulate matrix metalloproteinase‐9 (MMP‐9) via TLR4/NF‐κB‐dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP‐9 upregulation, thus enhancing the release of this matrix‐degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP‐9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF‐κB downstream signaling.     

Monoclonal antibodies selective for α‐synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α‐synuclein transgenic mice with the disease‐causing A30P mutation

Inclusions of intraneuronal alpha‐synuclein (α‐synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α‐synuclein is a central feature of the disease pathogenesis. Among the different α‐synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α‐synuclein oligomers were generated. These antibodies, which do not bind amyloid‐beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α‐synuclein transgenic mice than linear epitope antibodies. An oligomer‐selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α‐synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α‐synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer‐selective α‐synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.     

Measurements of auto‐antibodies to α‐synuclein in the serum and cerebral spinal fluids of patients with Parkinson's disease

Biomarkers for α‐synuclein are needed for diagnosis and prognosis in Parkinson's disease (PD ). Endogenous auto‐antibodies to α‐synuclein could serve as biomarkers for underlying synucleinopathy, but previous assessments of auto‐antibodies have shown variability and inconsistent clinical correlations. We hypothesized that auto‐antibodies to α‐synuclein could be diagnostic for PD and explain its clinical heterogeneity. To test this hypothesis, we developed an enzyme‐linked immunosorbent assay for measuring α‐synuclein auto‐antibodies in human samples. We evaluated 69 serum samples (16 healthy controls (HC ) and 53 PD patients) and 145 CSF samples (52 HC and 93 PD patients) from our Institution. Both serum and CSF were available for 24 participants. Males had higher auto‐antibody levels than females in both fluids. CSF auto‐antibody levels were significantly higher in PD patients as compared with HC , whereas serum levels were not significantly different. CSF auto‐antibody levels did not associate with amyloid‐β_1–42, total tau, or phosphorylated tau. CSF auto‐antibody levels correlated with performance on the Montreal Cognitive Assessment, even when controlled for CSF amyloidβ_1–42. CSF hemoglobin levels, as a proxy for contamination of CSF by blood during lumbar puncture, did not influence these observations. Using recombinant α‐synuclein with N‐ and C‐terminal truncations, we found that CSF auto‐antibodies target amino acids 100 through 120 of α‐synuclein. We conclude that endogenous CSF auto‐antibodies are significantly higher in PD patients as compared with HC , suggesting that they could indicate the presence of underlying synucleinopathy. These auto‐antibodies associate with poor cognition, independently of CSF amyloidβ_1–42, and target a select C‐terminal region of α‐synuclein.

Read the Editorial Highlight for this article on page 433 .

     

β‐Methylphenylalanine exerts neuroprotective effects in a Parkinson's disease model by protecting against tyrosine hydroxylase depletion

We evaluated the neuroprotective effects of β‐methylphenylalanine in an experimental model of rotenone‐induced Parkinson's disease (PD) in SH‐SY5Y cells and rats. Cells were pre‐treated with rotenone (2.5 µg/mL) for 24 hours followed by β‐methylphenylalanine (1, 10 and 100 mg/L) for 72 hours. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), mitochondrial fragmentation, apoptosis, and mRNA and protein levels of tyrosine hydroxylase were determined. In a rat model of PD, dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels, bradykinesia and tyrosine hydroxylase expression were determined. In rotenone–pre‐treated cells, β‐methylphenylalanine significantly increased cell viability and MMP, whereas ROS levels, apoptosis and fragmented mitochondria were reduced. β‐Methylphenylalanine significantly increased the mRNA and protein levels of tyrosine hydroxylase in SH‐SY5Y cells. In the rotenone‐induced rat model of PD, oral administration of β‐methylphenylalanine recovered DA and DOPAC levels and bradykinesia. β‐Methylphenylalanine significantly increased the protein expression of tyrosine hydroxylase in the striatum and substantia nigra of rats. In addition, in silico molecular docking confirmed binding between tyrosine hydroxylase and β‐methylphenylalanine. Our experimental results show neuroprotective effects of β‐methylphenylalanine via the recovery of mitochondrial damage and protection against the depletion of tyrosine hydroxylase. We propose that β‐methylphenylalanine may be useful in the treatment of PD.     

Alpha‐Synuclein as a Pathological Link Between Chronic Traumatic Brain Injury and Parkinson's Disease

The long‐term consequences of traumatic brain injury (TBI) are closely associated with the development of histopathological deficits. Notably, TBI may predispose long‐term survivors to age‐related neurodegenerative diseases, such as Parkinson's disease (PD), which is characterized by a gradual degeneration of the nigrostriatal dopaminergic neurons. However, preclinical studies on the pathophysiological changes in substantia nigra (SN) after chronic TBI are lacking. In the present in vivo study, we examined the pathological link between PD‐associated dopaminergic neuronal loss and chronic TBI. Sixty days post‐TBI, rats were euthanized and brain tissues harvested. Immunostaining was performed using tyrosine hydroxylase (TH), an enzyme required for the synthesis of dopamine in neurons, α‐synuclein, a presynaptic protein that plays a role in synaptic vesicle recycling, and major histocompatibility complex II (MHCII), a protein found in antigen presenting cells such as inflammatory microglia cells, all key players in PD pathology. Unbiased stereology analyses revealed significant decrease of TH‐positive expression in the surviving dopaminergic neurons of the SN pars compacta (SNpc) relative to sham control. In parallel, increased α‐synuclein accumulation was detected in the ipsilateral SN compared to the contralateral SN in TBI animals or sham control. In addition, exacerbation of MHCII+ cells was recognized in the SN and cerebral peduncle ipsilateral to injury relative to contralateral side and sham control. These results suggest α‐synuclein as a pathological link between chronic effects of TBI and PD symptoms as evidenced by significant overexpression and abnormal accumulation of α‐synuclein in inflammation‐infiltrated SN of rats exposed to chronic TBI. J. Cell. Physiol. 230: 1024–1032, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols

Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000–80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1–21% and 2–20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.     

α‐Synuclein sequesters arachidonic acid to modulate SNARE‐mediated exocytosis

α‐Synuclein is a synaptic modulatory protein implicated in the pathogenesis of Parkinson disease. The precise functions of this small cytosolic protein are still under investigation. α‐Synuclein has been proposed to regulate soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, α‐synuclein fails to interact with SNARE proteins in conventional protein‐binding assays, thus suggesting an indirect mode of action. As the structural and functional properties of both α‐synuclein and the SNARE proteins can be modified by arachidonic acid, a common lipid regulator, we analysed this possible tripartite link in detail. Here, we show that the ability of arachidonic acid to stimulate SNARE complex formation and exocytosis can be controlled by α‐synuclein, both in vitro and in vivo. α‐Synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of α‐synuclein in the modulation of neurotransmission.     

Characterizing the inhibition of α‐synuclein oligomerization by a pharmacological chaperone that prevents prion formation by the protein PrP

Aggregation of the disordered protein α‐synuclein into amyloid fibrils is a central feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease. Small, pre‐fibrillar oligomers of misfolded α‐synuclein are thought to be the key toxic entities, and α‐synuclein misfolding can propagate in a prion‐like way. We explored whether a compound with anti‐prion activity that can bind to unfolded parts of the protein PrP, the cyclic tetrapyrrole Fe‐TMPyP, was also active against α‐synuclein aggregation. Observing the initial stages of aggregation via fluorescence cross‐correlation spectroscopy, we found that Fe‐TMPyP inhibited small oligomer formation in a dose‐dependent manner. Fe‐TMPyP also inhibited the formation of mature amyloid fibrils in vitro, as detected by thioflavin T fluorescence. Isothermal titration calorimetry indicated Fe‐TMPyP bound to monomeric α‐synuclein with a stoichiometry of 2, and two‐dimensional heteronuclear single quantum coherence NMR spectra revealed significant interactions between Fe‐TMPyP and the C‐terminus of the protein. These results suggest commonalities among aggregation mechanisms for α‐synuclein and the prion protein may exist that can be exploited as therapeutic targets.     

α‐Synuclein stimulation of monoamine oxidase‐B and legumain protease mediates the pathology of Parkinson's disease

Dopaminergic neurodegeneration in Parkinson's disease (PD) is associated with abnormal dopamine metabolism by MAO‐B (monoamine oxidase‐B) and intracellular α‐Synuclein (α‐Syn) aggregates, called the Lewy body. However, the molecular relationship between α‐Syn and MAO‐B remains unclear. Here, we show that α‐Syn directly binds to MAO‐B and stimulates its enzymatic activity, which triggers AEP (asparagine endopeptidase; legumain) activation and subsequent α‐Syn cleavage at N103, leading to dopaminergic neurodegeneration. Interestingly, the dopamine metabolite, DOPAL, strongly activates AEP, and the N103 fragment of α‐Syn binds and activates MAO‐B. Accordingly, overexpression of AEP in SNCA transgenic mice elicits α‐Syn N103 cleavage and accelerates PD pathogenesis, and inhibition of MAO‐B by Rasagiline diminishes α‐Syn‐mediated PD pathology and motor dysfunction. Moreover, virally mediated expression of α‐Syn N103 induces PD pathogenesis in wild‐type, but not MAO‐B‐null mice. Our findings thus support that AEP‐mediated cleavage of α‐Syn at N103 is required for the association and activation of MAO‐B, mediating PD pathogenesis.     

Poster Session BP07: Neurodegenerative Diseases. The role of α‐synuclein in cell division,differentiation and survival

Presynaptic terminals contain an abundant 140‐amino acid phosphoprotein, dubbed α‐synuclein, which is accumulated in Lewy bodies typically observed in neurons in neurodegenerative diseases, such as Parkinson's disease. In this study, the role of α‐synuclein in regulating cycle, differentiation, and survival of neuronal cells was studied using a rat dopaminergic cell line ZN27D. To delineate specific effects of α‐synuclein the same cell line was engineered to express human α‐synuclein and a vector‐transfected cell line RK27 was used as a second control. All three cell lines showed significant proliferation even in serum‐free medium, and complete inhibition of cell division and differentiation could be achieved in the ZN27D cells only when both dibutyryl cAMP (dbcAMP) and retinoic acid were present. In contrast, the α‐synuclein expressing cells could be differentiated in the presence of only dbcAMP. Dose dependence of MPP+(1‐methyl‐4‐phenylpyridinium iodide)‐mediated caspase3 activation was studied in undifferentiated ZN27D cells. At 200 μm MPP+ a significant cleavage of the caspase3 substrate PARP was observed and it was reversed in the presence of α‐synuclein. MPP+ also inhibited aminophospholipid translocase (APTL), a P‐type ATPase that is responsible for inner plasma membrane localization of phophotidylserine in healthy cells. The role of α‐synuclein in regulating cell cycle, differentiation, APTL activity and cell death is being investigated further in the dopaminergic ZN27D cell line.     

Long‐lasting pathological consequences of overexpression‐induced α‐synuclein spreading in the rat brain

Increased expression of α‐synuclein can initiate its long‐distance brain transfer, representing a potential mechanism for pathology spreading in age‐related synucleinopathies, such as Parkinson's disease. In this study, the effects of overexpression‐induced α‐synuclein transfer were assessed over a 1‐year period after injection of viral vectors carrying human α‐synuclein DNA into the rat vagus nerve. This treatment causes targeted overexpression within neurons in the dorsal medulla oblongata and subsequent diffusion of the exogenous protein toward more rostral brain regions. Protein advancement and accumulation in pontine, midbrain, and forebrain areas were contingent upon continuous overexpression, because death of transduced medullary neurons resulted in cessation of spreading. Lack of sustained spreading did not prevent the development of long‐lasting pathological changes. Particularly remarkable were findings in the locus coeruleus, a pontine nucleus with direct connections to the dorsal medulla oblongata and greatly affected by overexpression‐induced transfer in this model. Data revealed progressive degeneration of catecholaminergic neurons that proceeded long beyond the time of spreading cessation. Neuronal pathology in the locus coeruleus was accompanied by pronounced microglial activation and, at later times, astrocytosis. Interestingly, microglial activation was also featured in another region reached by α‐synuclein transfer, the central amygdala, even in the absence of frank neurodegeneration. Thus, overexpression‐induced spreading, even if temporary, causes long‐lasting pathological consequences in brain regions distant from the site of overexpression but anatomically connected to it. Neurodegeneration may be a consequence of severe protein burden, whereas even a milder α‐synuclein accumulation in tissues affected by protein transfer could induce sustained microglial activation.     

Endonuclease G mediates α‐synuclein cytotoxicity during Parkinson's disease

Malfunctioning of the protein α‐synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson‐diseased patients, while EndoG depletion largely reduces α‐synuclein‐induced cell death in human neuroblastoma cells. Xenogenic expression of human α‐synuclein in yeast cells triggers mitochondria‐nuclear translocation of EndoG and EndoG‐mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α‐synuclein‐driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α‐synuclein‐expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α‐synuclein cytotoxicity.