S-Adenosylmethionine Rescues Cognitive Deficits in the rTg4510 Animal Model by Stabilizing Protein Phosphatase 2A and Reducing Phosphorylated Tau.

BACKGROUND: Alterations in the methionine cycle and abnormal tau phosphorylation are implicated in many neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia. rTg4510 mice express mutant human P301L tau and are a model of tau hyperphosphorylation. The cognitive deficit seen in these animals correlates with a burden of hyperphosphorylated tau and is a model to test therapies aimed at lowering phosphorylated tau. OBJECTIVE: This study aimed to increase protein phosphatase 2A activity through supplementation of S-adenosylmethionine and analyze the effect on spatial memory and tau in treated animals. METHODS: 6-month-old rTg4510 mice were treated with 100 mg/kg S-adenosylmethionine by oral gavage for 3 weeks. Spatial recognition memory was tested in the Y-maze. Alterations to phosphorylated tau and protein phosphatase 2A were explored using immunohistochemistry, western blot, and enzyme-linked immunosorbent assays. RESULTS: Treatment with S-adenosylmethionine increased the Y-maze novel arm exploration time and increased both the expression and activity of protein phosphatase 2A. Furthermore, treatment reduced the number of AT8 positive neurons and reduced the expression of phosphorylated tau (Ser202/Thr205). S-adenosylmethionine contributes to multiple pathways in neuronal homeostasis and neurodegeneration. CONCLUSION: This study shows that supplementation with S-adenosylmethionine stabilizes the heterotrimeric form of PP2A resulting in an increase the enzymatic activity, a reduced level of pathological tau, and improved cognition.

Characterising the brain metalloproteome in Down syndrome patients with concomitant Alzheimer's pathology.

Down syndrome (DS) is a common intellectual disability, with an incidence of 1 in 700 and is caused by trisomy 21. People with DS develop Alzheimer's disease (AD)-like neuropathology by the age of 40. As metal ion dyshomeostasis (particularly zinc, iron and copper) is one of the characteristics of AD and is believed to be involved in the pathogenesis of disease, we reasoned that it may also be altered in DS. Thus, we used inductively coupled plasma mass spectrometry to examine metal levels in post-mortem brain tissue from DS individuals with concomitant AD pathology. Size exclusion-ICPMS was also utilised to characterise the metalloproteome in these cases. We report here for the first time that iron levels were higher in a number of regions in the DS brain, including the hippocampus (40%), frontal cortex (100%) and temporal cortex (34%), compared to controls. Zinc and copper were also elevated (both 29%) in the DS frontal cortex, but zinc was decreased (23%) in the DS temporal cortex. Other elements were also examined, a number of which also showed disease-specific changes. The metalloproteomic profile in the DS brain was also different to that in the controls. These data suggest that metals and metal:protein interactions are dysregulated in the DS brain which, given the known role of metals in neurodegeneration and AD, is likely to contribute to the pathogenesis of disease. Interrogation of the underlying cellular mechanisms and consequences of this failure in metal ion homeostasis, and the specific contributions of the individual DS and AD phenotypes to these changes, should be explored.

Fibrillar α-synuclein toxicity depends on functional lysosomes.

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

Zn-DTSM, A Zinc Ionophore with Therapeutic Potential for Acrodermatitis Enteropathica?

Acrodermatitis enteropathica (AE) is a rare disease characterised by a failure in intestinal zinc absorption, which results in a host of symptoms that can ultimately lead to death if left untreated. Current clinical treatment involves life-long high-dose zinc supplements, which can introduce complications for overall nutrient balance in the body. Previous studies have therefore explored the pharmacological treatment of AE utilising metal ionophore/transport compounds in an animal model of the disease (conditional knockout (KO) of the zinc transporter, Zip4), with the perspective of finding an alternative to zinc supplementation. In this study we have assessed the utility of a different class of zinc ionophore compound (zinc diethyl bis(N4-methylthiosemicarbazone), Zn-DTSM; Collaborative Medicinal Development, Sausalito, CA, USA) to the one we have previously described (clioquinol), to determine whether it is effective at preventing the stereotypical weight loss present in the animal model of disease. We first utilised an in vitro assay to assess the ionophore capacity of the compound, and then assessed the effect of the compound in three in vivo animal studies (in 1.5-month-old mice at 30 mg/kg/day, and in 5-month old mice at 3 mg/kg/day and 30 mg/kg/day). Our data demonstrate that Zn-DTSM has a pronounced effect on preventing weight loss when administered daily at 30 mg/kg/day; this was apparent in the absence of any added exogenous zinc. This compound had little overall effect on zinc content in various tissues that were assessed, although further characterisation is required to more fully explore the cellular changes underlying the physiological benefit of this compound. These data suggest that Zn-DTSM, or similar compounds, should be further explored as potential therapeutic options for the long-term treatment of AE.

Spatial memory deficits in a mouse model of late-onset Alzheimer's disease are caused by zinc supplementation and correlate with amyloid-beta levels.

Much of the research in Alzheimer's disease (AD) that uses mouse models focuses on the early-onset form of the disease, which accounts for less than 5% of cases. In contrast, this study used a late-onset AD model to examine the interaction between increased dietary zinc (Zn) and the apolipoprotein E (ApoE) gene. ApoE ε4 is overrepresented in late-onset AD and enhances Zn binding to amyloid-ß (Aß). This study sought to determine if elevated dietary Zn would impair spatial memory in CRND8 mice (CRND8), as well as mice who carry both the mutated human amyloid precursor protein (APP) and ApoE ε4 genes (CRND8/E4). Mice were provided with either lab tap water or water enhanced with 10 ppm Zn (ZnCO3) for 4 months. At 6 months of age, spatial memory was measured by the Barnes maze. CRND8 mice exhibited significant memory deficits compared to WT mice, as shown by an increased latency to reach the escape box. For the CRND8/E4, but not the CRND8 mice, those given Zn water made significantly more errors than those on lab water. During the probe trial for the WT group, those on Zn water spent significantly less time in the target quadrant than those on lab water. These data suggest that increased dietary Zn can significantly impair spatial memory in CRND8/E4. WT mice given Zn water were also impaired on the 24-h probe trial when compared to lab water WTs. Within the CRND8/E4 group only, levels of soluble Aß were significantly correlated with average primary latencies. Within the Zn-treated CRND8/E4 group, there was a significant correlation between insoluble Aß and average primary errors. Levels of the zinc transporter 3, ZnT3, were negatively correlated with soluble Aß (p < 0.01). These findings are particularly relevant because increased intake of dietary supplements, such as Zn, are common in the elderly-a population already at risk for AD. Given the effects observed in the CRND8/E4 mice, ApoE status should be taken into consideration when evaluating the efficacy of therapies targeting metals.

Iron accumulation confers neurotoxicity to a vulnerable population of nigral neurons: implications for Parkinson's disease.

BACKGROUND: The substantia nigra (SN) midbrain nucleus is constitutively iron rich. Iron levels elevate further with age, and pathologically in Parkinson's disease (PD). Iron accumulation in PD SN involves dysfunction of ceruloplasmin (CP), which normally promotes iron export. We previously showed that ceruloplasmin knockout (CP KO) mice exhibit Parkinsonian neurodegeneration (~30% nigral loss) by 6 months, which is prevented by iron chelation. Here, we explored whether known iron-stressors of the SN (1) aging and (2) MPTP, would exaggerate the lesion severity of CP KO mice. FINDINGS: We show that while 5 month old CP KO mice exhibited nigral iron elevation and loss of SN neurons, surprisingly, aging CP KO mice to 14 months did not exacerbate iron elevation or SN neuronal loss. Unlike young mice, iron chelation therapy in CP KO mice between 9-14 months did not rescue neuronal loss. MPTP exaggerated iron elevation in young CP KO mice but did not increase cell death when compared to WTs. CONCLUSIONS: We conclude that there may exist a proportion of substantia nigra neurons that depend on CP for protection against iron neurotoxicity and could be protected by iron-based therapeutics. Death of the remaining neurons in Parkinson's disease is likely caused by parallel disease mechanisms, which may call for additional therapeutic options.

Clioquinol synergistically augments rescue by zinc supplementation in a mouse model of acrodermatitis enteropathica.

BACKGROUND: Zinc deficiency due to poor nutrition or genetic mutations in zinc transporters is a global health problem and approaches to providing effective dietary zinc supplementation while avoiding potential toxic side effects are needed. METHODS/PRINCIPAL FINDINGS: Conditional knockout of the intestinal zinc transporter Zip4 (Slc39a4) in mice creates a model of the lethal human genetic disease acrodermatitis enteropathica (AE). This knockout leads to acute zinc deficiency resulting in rapid weight loss, disrupted intestine integrity and eventually lethality, and therefore provides a model system in which to examine novel approaches to zinc supplementation. We examined the efficacy of dietary clioquinol (CQ), a well characterized zinc chelator/ionophore, in rescuing the Zip4 (intest KO) phenotype. By 8 days after initiation of the knockout neither dietary CQ nor zinc supplementation in the drinking water was found to be effective at improving this phenotype. In contrast, dietary CQ in conjunction with zinc supplementation was highly effective. Dietary CQ with zinc supplementation rapidly restored intestine stem cell division and differentiation of secretory and the absorptive cells. These changes were accompanied by rapid growth and dramatically increased longevity in the majority of mice, as well as the apparent restoration of the homeostasis of several essential metals in the liver. CONCLUSIONS: These studies suggest that oral CQ (or other 8-hydroxyquinolines) coupled with zinc supplementation could provide a facile approach toward treating zinc deficiency in humans by stimulating stem cell proliferation and differentiation of intestinal epithelial cells.

Altered expression of ZnT10 in Alzheimer's disease brain.

There is an increasing body of evidence suggesting that metal homeostasis is dysregulated in the pathology of Alzheimer's disease (AD). Although expression levels of several transporters belonging the SLC30 family, which comprises predominantly zinc transporters, have been studied in the AD brain, SLC30A10 (ZnT10) has not been studied in this context. To determine if dysregulated expression of ZnT10, which may transport both Zn and Mn, could be a factor that contributes to AD, we investigated if there were differences in ZnT10 mRNA levels in specimens of frontal cortex from AD patients and controls and also if brain tissue from the APP/PS1 transgenic (Tg) mouse model showed abnormal levels of ZnT10 mRNA expression. Our results show that ZnT10 is significantly (P<0.01) decreased in the frontal cortex in AD. Furthermore, we observed a significant decrease in ZnT10 mRNA levels in the APP/PS1-Tg mice compared with wild-type controls (P<0.01). Our results suggest that this dysregulation in ZnT10 could further contribute to disease progression.

Copper modulation as a therapy for Alzheimer's disease?

The role of metals in the pathophysiology of Alzheimer's disease (AD) has gained considerable support in recent years, with both in vitro and in vivo data demonstrating that a mis-metabolism of metal ions, such as copper and zinc, may affect various cellular cascades that ultimately leads to the development and/or potentiation of AD. In this paper, we will provide an overview of the preclinical and clinical literature that specifically relates to attempts to affect the AD cascade by the modulation of brain copper levels. We will also detail our own novel animal data, where we treated APP/PS1 (7-8 months old) mice with either high copper (20 ppm in the drinking water), high cholesterol (2% supplement in the food) or a combination of both and then assessed ß-amyloid (Aß) burden (soluble and insoluble Aß), APP levels and behavioural performance in the Morris water maze. These data support an interaction between copper/cholesterol and both Aß and APP and further highlight the potential role of metal ion dyshomeostasis in AD.

Three-dimensional elemental bio-imaging of Fe, Zn, Cu, Mn and P in a 6-hydroxydopamine lesioned mouse brain.

Three dimensional maps of iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and phosphorous (P) in a 6-hydroxydopamine (6-OHDA) lesioned mouse brain were constructed employing a novel quantitative laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method known as elemental bio-imaging. The 3D maps were produced by ablating serial consecutive sections taken from the same animal. Each section was quantified against tissue standards resulting in a three dimensional map that represents the variation of trace element concentrations of the mouse brain in the area surrounding the substantia nigra (SN). Damage caused by the needle or the toxin did not alter the distribution of Zn, and Cu but significantly altered Fe in and around the SN and both Mn and Fe around the needle track. A 20% increase in nigral Fe concentration was observed within the lesioned hemisphere. This technique clearly shows the natural heterogeneous distributions of these elements throughout the brain and the perturbations that occur following trauma or intoxication. The method may applied to three-dimensional modelling of trace elements in a wide range of tissue samples.

Behavioural phenotype of APPC100.V717F transgenic mice over-expressing a mutant Abeta-bearing fragment is associated with reduced NMDA receptor density.

The aim of this study was to characterize APPC100.V717F transgenic (TgC100.V717F) mice which over-express a mutant C100 fragment of the amyloid precursor protein. The mice were compared to TgC100 wild type mice (TgC100.WT) and non-transgenic controls at 4-9 and 16-22 months of age. TgC100.V717F mice showed behavioural hyperactivity, particularly at a younger age, as shown by increased numbers of elevated plus maze arm entries and Y-maze arm entries, enhanced baseline locomotor activity in the open field, and enhanced amphetamine-induced hyperlocomotion. This hyperactivity was less pronounced in TgC100.WT which only displayed significant differences to non-transgenic controls at a younger age for the number of Y-maze arm entries and baseline locomotor activity in the open field. In addition, TgC100.V717F mice, but not TgC100.WT, demonstrated cognitive deficits, as shown by reduced spontaneous alternation in the Y-maze and markedly reduced retention in a passive avoidance test. At an older age, TgC100.V717F mice showed enhanced startle and increased immobility time in the forced swim test. In the TgC100.V717F mice, but not TgC100.WT, the behavioural changes were paralleled by a significant reduction in the expression of hippocampal NMDA receptor subunits types 1 and 2A. Concomitantly, we detected axonal disruption and apoptosis in the hippocampus of TgC100.V717F mice. In conclusion, these data demonstrate that the mutant C100 fragment is an effector of biochemical and both cognitive and non-cognitive behaviours. These transgenic mice may be a model for the psychotic features associated with early Alzheimer's disease.

beta-Amyloid as a molecular therapeutic target in Alzheimer's disease.

Alzheimer's disease is the most common form of dementia, primarily affecting individuals during or after their sixth decade of life. Despite decades of research, there are still no effective disease-modifying drugs available to treat this neurodegenerative disorder. Current FDA-approved medications primarily offer symptomatic relief and are based upon known neurotransmitter deficits. There are, however, many drugs in preclinical and clinical development which target other aspects of AD pathogenesis. Principal among these are drugs which modulate beta-amyloid, a protein that is believed to be central to the cascade which leads to the development of Alzheimer's disease. This article will outline the metabolism of beta-amyloid and review a number of different strategies, including pitfalls and future directions of such methods that are directed towards the modulation of this protein. It will become clear that beta-amyloid represents a potent molecular target for pharmacological manipulation to perhaps prevent the onset and progression of Alzheimer's disease.