Quantitative proteomics of tau and Aß in detergent fractions from Alzheimer's disease brains.

The two hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aß drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aß mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aß species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aß42 at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT181 (~22%) and pT217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non-filamentous form (N-terminal intact tau) co-localized with Aß in the sarkosyl-insoluble pellets along with tau filaments (N-truncated MTBR tau). Our results suggest a model that Aß and tau interact forming globular aggregates, from which filamentous tau and Aß emerge. These characterizations contribute towards unravelling the sequence of events which lead to end-stage AD changes.

Vascular remodeling in sheep implanted with endovascular neural interface.

Objective.The aim of this work was to assess vascular remodeling after the placement of an endovascular neural interface (ENI) in the superior sagittal sinus (SSS) of sheep. We also assessed the efficacy of neural recording using an ENI.Approach.The study used histological analysis to assess the composition of the foreign body response. Micro-CT images were analyzed to assess the profiles of the foreign body response and create a model of a blood vessel. Computational fluid dynamic modeling was performed on a reconstructed blood vessel to evaluate the blood flow within the vessel. Recording of brain activity in sheep was used to evaluate efficacy of neural recordings.Main results.Histological analysis showed accumulated extracellular matrix material in and around the implanted ENI. The extracellular matrix contained numerous macrophages, foreign body giant cells, and new vascular channels lined by endothelium. Image analysis of CT slices demonstrated an uneven narrowing of the SSS lumen proportional to the stent material within the blood vessel. However, the foreign body response did not occlude blood flow. The ENI was able to record epileptiform spiking activity with distinct spike morphologies.Significance. This is the first study to show high-resolution tissue profiles, the histological response to an implanted ENI and blood flow dynamic modeling based on blood vessels implanted with an ENI. The results from this study can be used to guide surgical planning and future ENI designs; stent oversizing parameters to blood vessel diameter should be considered to minimize detrimental vascular remodeling.

Rapid and persistent decrease in brain tissue oxygenation in ovine gram-negative sepsis.

The changes in brain perfusion and oxygenation in critical illness, which are thought to contribute to brain dysfunction, are unclear due to the lack of methods to measure these variables. We have developed a technique to chronically measure cerebral tissue perfusion and oxygen tension in unanesthetized sheep. Using this technique, we have determined the changes in cerebral perfusion and Po2 during the development of ovine sepsis. In adult Merino ewes, fiber-optic probes were implanted in the brain, renal cortex, and renal medulla to measure tissue perfusion, oxygen tension (Po2), and temperature, and flow probes were implanted on the pulmonary and renal arteries. Conscious sheep were infused with live Escherichia coli for 24 h, which induced hyperdynamic sepsis; mean arterial pressure decreased (from 85.2 ± 5.6 to 71.5 ± 8.7 mmHg), while cardiac output (from 4.12 ± 0.70 to 6.15 ± 1.26 L/min) and total peripheral conductance (from 48.9 ± 8.5 to 86.8 ± 11.5 mL/min/mmHg) increased (n = 8, all P < 0.001) and arterial Po2 decreased (from 104 ± 8 to 83 ± 10 mmHg; P < 0.01). Cerebral perfusion tended to decrease acutely, although this did not reach significance, but there was a significant and sustained decrease in cerebral tissue Po2 (from 32.2 ± 10.1 to 18.8 ± 11.7 mmHg) after 3 h and to 22.8 ± 5.2 mmHg after 24 h of sepsis (P < 0.02). Sepsis induced large reductions in both renal medullary perfusion and Po2 but had no effect in the renal cortex. In ovine sepsis, there is an early decrease in cerebral Po2 that is maintained for 24 h despite minimal changes in cerebral perfusion. Cerebral hypoxia may be one of the factors causing sepsis-induced malaise and lethargy.

Citrullination of Amyloid-ß Peptides in Alzheimer's Disease.

Protein citrullination (deimination of arginine residue) is a well-known biomarker of inflammation. Elevated protein citrullination has been shown to colocalize with extracellular amyloid plaques in postmortem AD patient brains. Amyloid-ß (Aß) peptides which aggregate and accumulate in the plaques of Alzheimer's disease (AD) have sequential N-terminal truncations and multiple post-translational modifications (PTM) such as isomerization, pyroglutamate formation, phosphorylation, nitration, and dityrosine cross-linking. However, no conclusive biochemical evidence exists whether citrullinated Aß is present in AD brains. In this study, using high-resolution mass spectrometry, we have identified citrullination of Aß in sporadic and familial AD brains by characterizing the tandem mass spectra of endogenous N-truncated citrullinated Aß peptides. Our quantitative estimations demonstrate that ∼ 35% of pyroglutamate3-Aß pool was citrullinated in plaques in the sporadic AD temporal cortex and ∼ 22% in the detergent-insoluble frontal cortex fractions. Similarly, hypercitrullinated pyroglutamate3-Aß (∼ 30%) was observed in both the detergent-soluble as well as insoluble Aß pool in familial AD cases. Our results indicate that a common mechanism for citrullination of Aß exists in both the sporadic and familial AD. We establish that citrullination of Aß is a remarkably common PTM, closely associated with pyroglutamate3-Aß formation and its accumulation in AD. This may have implications for Aß toxicity, autoantigenicity of Aß, and may be relevant for the design of diagnostic assays and therapeutic targeting.

Quantification of N-terminal amyloid-ß isoforms reveals isomers are the most abundant form of the amyloid-ß peptide in sporadic Alzheimer's disease.

Plaques that characterize Alzheimer's disease accumulate over 20 years as a result of decreased clearance of amyloid-ß peptides. Such long-lived peptides are subjected to multiple post-translational modifications, in particular isomerization. Using liquid chromatography ion mobility separations mass spectrometry, we characterized the most common isomerized amyloid-ß peptides present in the temporal cortex of sporadic Alzheimer's disease brains. Quantitative assessment of amyloid-ß N-terminus revealed that > 80% of aspartates (Asp-1 and Asp-7) in the N-terminus was isomerized, making isomerization the most dominant post-translational modification of amyloid-ß in Alzheimer's disease brain. Total amyloid-ß1-15 was ∼85% isomerized at Asp-1 and/or Asp-7 residues, with only 15% unmodified amyloid-ß1-15 left in Alzheimer's disease. While amyloid-ß4-15 the next most abundant N-terminus found in Alzheimer's disease brain, was only ∼50% isomerized at Asp-7 in Alzheimer's disease. Further investigations into different biochemically defined amyloid-ß-pools indicated a distinct pattern of accumulation of extensively isomerized amyloid-ß in the insoluble fibrillar plaque and membrane-associated pools, while the extent of isomerization was lower in peripheral membrane/vesicular and soluble pools. This pattern correlated with the accumulation of aggregation-prone amyloid-ß42 in Alzheimer's disease brains. Isomerization significantly alters the structure of the amyloid-ß peptide, which not only has implications for its degradation, but also for oligomer assembly, and the binding of therapeutic antibodies that directly target the N-terminus, where these modifications are located.

Reversal of the Pathophysiological Responses to Gram-Negative Sepsis by Megadose Vitamin C.

OBJECTIVES: Oxidative stress appears to initiate organ failure in sepsis, justifying treatment with antioxidants such as vitamin C at megadoses. We have therefore investigated the safety and efficacy of megadose sodium ascorbate in sepsis. DESIGN: Interventional study. SETTING: Research Institute. SUBJECTS: Adult Merino ewes. INTERVENTIONS: Sheep were instrumented with pulmonary and renal artery flow-probes, and laser-Doppler and oxygen-sensing probes in the kidney. Conscious sheep received an infusion of live Escherichia coli for 31 hours. At 23.5 hours of sepsis, sheep received fluid resuscitation (30 mL/kg, Hartmann solution) and were randomized to IV sodium ascorbate (0.5 g/kg over 0.5 hr + 0.5 g/kg/hr for 6.5 hr; n = 5) or vehicle (n = 5). Norepinephrine was titrated to restore mean arterial pressure to baseline values (~80 mm Hg). MEASUREMENTS AND MAIN RESULTS: Sepsis-induced fever (41.4 ± 0.2°C; mean ± se), tachycardia (141 ± 2 beats/min), and a marked deterioration in clinical condition in all cases. Mean arterial pressure (86 ± 1 to 67 ± 2 mm Hg), arterial Po2 (102.1 ± 3.3 to 80.5 ± 3.4 mm Hg), and renal medullary tissue Po2 (41 ± 5 to 24 ± 2 mm Hg) decreased, and plasma creatinine doubled (71 ± 2 to 144 ± 15 µmol/L) (all p < 0.01). Direct observation indicated that in all animals, sodium ascorbate dramatically improved the clinical state, from malaise and lethargy to a responsive, alert state within 3 hours. Body temperature (39.3 ± 0.3°C), heart rate (99.7 ± 3 beats/min), and plasma creatinine (32.6 ± 5.8 µmol/L) all decreased. Arterial (96.5 ± 2.5 mm Hg) and renal medullary Po2 (48 ± 5 mm Hg) increased. The norepinephrine dose was decreased, to zero in four of five sheep, whereas mean arterial pressure increased (to 83 ± 2 mm Hg). We confirmed these physiologic findings in a coronavirus disease 2019 patient with shock by compassionate use of 60 g of sodium ascorbate over 7 hours. CONCLUSIONS: IV megadose sodium ascorbate reversed the pathophysiological and behavioral responses to Gram-negative sepsis without adverse side effects. Clinical studies are required to determine if such a dose has similar benefits in septic patients.

Plant poisoning leads to alpha-synucleinopathy and neuromelanopathy in kangaroos.

The pathogenesis of synucleinopathies, common neuropathological lesions normally associated with some human neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, remains poorly understood. In animals, ingestion of the tryptamine-alkaloid-rich phalaris pastures plants causes a disorder called Phalaris staggers, a neurological syndrome reported in kangaroos. The aim of the study was to characterise the clinical and neuropathological changes associated with spontaneous cases of Phalaris staggers in kangaroos. Gross, histological, ultrastructural and Immunohistochemical studies were performed to demonstrate neuronal accumulation of neuromelanin and aggregated α-synuclein. ELISA and mass spectrometry were used to detect serum-borne α-synuclein and tryptamine alkaloids respectively. We report that neurons in the central and enteric nervous systems of affected kangaroos display extensive accumulation of neuromelanin in the perikaryon without affecting neuronal morphology. Ultrastructural studies confirmed the typical structure of neuromelanin. While we demonstrated strong staining of α-synuclein, restricted to neurons, intracytoplasmic Lewy bodies inclusions were not observed. α-synuclein aggregates levels were shown to be lower in sera of the affected kangaroos compared to unaffected herd mate kangaroos. Finally, mass spectrometry failed to detect the alkaloid toxins in the sera derived from the affected kangaroos. Our preliminary findings warrant further investigation of Phalaris staggers in kangaroos, potentially a valuable large animal model for environmentally-acquired toxic synucleinopathy.

Iron, Copper, and Zinc Concentration in Aß Plaques in the APP/PS1 Mouse Model of Alzheimer's Disease Correlates with Metal Levels in the Surrounding Neuropil.

The metal ions of iron, copper, and zinc have long been associated with the aggregation of ß-amyloid (Aß) plaques in Alzheimer's disease; an interaction that has been suggested to promote increased oxidative stress and neuronal dysfunction. We examined plaque metal load in the hippocampus of APP/PS1 mice using X-ray fluorescence microscopy to assess how the anatomical location of Aß plaques was influenced by the metal content of surrounding tissue. Immunohistochemical staining of Aß plaques colocalized with areas of increased X-ray scattering power in unstained tissue sections, allowing direct X-ray based-assessment of plaque metal levels in sections subjected to minimal chemical fixation. We identified and mapped 48 individual plaques in four subregions of the hippocampus from four biological replicates. Iron, Cu, and Zn areal concentrations (ng cm-2) were increased in plaques compared to the surrounding neuropil. However, this elevation in metal load reflected the local metal makeup of the surrounding neuropil, where different brain regions are enriched for different metal ions. After correcting for tissue density, only Zn levels remained elevated in plaques. This study suggests that the in vivo binding of Zn to plaques is not simply due to increased protein deposition.

Direct in vivo imaging of ferrous iron dyshomeostasis in ageing Caenorhabditis elegans.

Iron is essential for eukaryotic biochemistry. Systematic trafficking and storage is required to maintain supply of iron while preventing it from catalysing unwanted reactions, particularly the generation of oxidising reactive species. Iron dyshomeostasis has been implicated in major age-associated diseases including cancers, neurodegeneration and heart disease. Here, we employ population-level X-ray fluorescence imaging and native-metalloproteomic analysis to determine that altered iron coordination and distribution is a pathological imperative of ageing in the nematode, Caenorhabditis elegans. Our approach provides a method to simultaneously study iron metabolism across different scales of biological organisation, from populations to cells. Here we report how and where iron homeostasis is lost during C. elegans ageing, and its relationship to the age-related elevation of damaging reactive oxygen species. We find that wild types utilise ferritin to sustain longevity, buffering against exogenous iron and showing rapid ageing if ferritin is ablated. After reproduction, escape of iron from safe-storage in ferritin raised cellular Fe2+ load in the ageing C. elegans, and increased generation of reactive species. These findings support the hypothesis that iron-mediated processes drive senescence. We propose that loss of iron homeostasis may be a fundamental and inescapable consequence of ageing that could represent a critical target for therapeutic strategies to improve health outcomes in ageing.

Assessing THK523 selectivity for tau deposits in Alzheimer's disease and non-Alzheimer's disease tauopathies.

INTRODUCTION: The introduction of tau imaging agents such as (18)F-THK523 offers new hope for the in vivo assessment of tau deposition in tauopathies such as Alzheimer's disease (AD), where preliminary (18)F-THK523-PET studies have demonstrated significantly higher cortical retention of (18)F-THK523 in AD compared to age-matched healthy individuals. In addition to AD, tau imaging with PET may also be of value in assessing non-AD tauopathies, such as corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and Pick's disease (PiD). METHODS: To further investigate the ability of THK523 to recognize tau lesions, we undertook immunohistochemical and fluorescence studies in serial brain sections taken from individuals with AD (n = 3), CBD (n = 2), PSP (n = 1), PiD (n = 2) and Parkinson's disease (PD; n = 2). In addition to the neuropathological analysis, one PSP patient had undergone a (18)F-THK523 PET scan 5 months before death. RESULTS: Although THK523 labelled tau-containing lesions such as neurofibrillary tangles and neuropil threads in the hippocampus and frontal regions of AD brains, it failed to label tau-containing lesions in non-AD tauopathies. Furthermore, though THK523 faintly labelled dense-cored amyloid-ß plaques in the AD frontal cortex, it failed to label α-synuclein-containing Lewy bodies in PD brain sections. CONCLUSION: The results of this study suggest that (18)F-THK523 selectively binds to paired helical filament tau in AD brains but does not bind to tau lesions in non-AD tauopathies, or to α-synuclein in PD brains.

Morphological changes in the ovine carotid artery wall induced by cold storage.

Blood vessels obtained from cadavers and amputated limbs stored at 4°C (i.e., cold stored) potentially represent an economical and readily sourced alternative to autologous vessels and synthetic prostheses for vascular reconstructive surgery. However, cold-stored vessels would need to have a reduced antigenicity and an antithrombogenic autologous endothelial cell (EC) lining before they could function as patent vascular allografts. The aim of this study was to determine the effect of cold storage for 1-16 weeks on the morphology of the ovine carotid artery wall. Ovine carotid arteries (n = 6) were rinsed and flushed with 0.9% saline, cut into segments, wrapped in 0.9% saline-soaked gauze, and stored at 4°C for 1, 2, 4, 8, or 16 weeks. Following storage, the segments were sampled and the samples fixed and sectioned for light microscopic, immunohistochemical, or transmission electron microscopic examination. After 1 and 2 weeks the ECs were karyolitic or contained pyknotic nuclei. After 4 weeks the EC layer was depleted, the subendothelial matrix exposed, and the number of smooth muscle cells (SMCs) and fibroblasts reduced. The 8- and 16-week samples demonstrated complete loss of the EC lining and only occasional remnants of SMCs or fibroblasts. The subendothelial basement membrane appeared to undergo degradative changes as early as 1 week following cold storage. At each time point examined, the subendothelial connective tissue stroma, the internal elastic lamina (IEL), and the collagenous and elastic extracellular framework were retained. These results demonstrate that the ovine carotid artery wall progressively loses its cells but retains its extracellular components during cold storage for up to 16 weeks. They suggest that cold-stored vessels may function as allografts with a reduced antigenicity for vascular reconstructive surgery. It is conceivable that seeded autologous ECs may be used to restore the antithrombogenic EC lining prior to graft implantation.

The impact of experimental hypoperfusion on subsequent kidney function.

OBJECTIVE: To investigate the short- and medium-term renal hemodynamic and functional responses to both short and sustained hypoperfusion. SUBJECTS: Eleven Merinos ewes. SETTING: Animal laboratory of the University Physiology Institute. DESIGN: Prospective observational study. INTERVENTIONS: Studies were performed in conscious sheep after unilateral nephrectomy with a vascular occluder and flow probe implanted on the remaining renal artery. In five sheep, renal blood flow (RBF) was reduced by 25, 50 and 75%, respectively, by acute vascular occlusion for 30 min at weekly intervals. In another six sheep, RBF was reduced by 80% for 2 h. MEASUREMENTS AND RESULTS: After 25, 50 or 75% renal hypoperfusion for 30 min, there was no associated extended loss of renal function. During 2 h of 80% hypoperfusion, urine output decreased from 80 to 17 ml, and creatinine clearance from 32 to 3 ml/min, whereas plasma creatinine increased from 103 to 132 mumol/l, and fractional excretion of sodium and urea increased. Release of occlusion induced brief hyperemia before all measured variables returned to normal within 8 h and remained normal for the following 72 h. At autopsy, the kidneys were histopathologically normal. CONCLUSIONS: Various degrees of renal hypoperfusion for 30 min did not induce prolonged changes in renal function or blood flow. Even with sustained severe hypoperfusion, there was rapid recovery to baseline function and flow. Unlike total ischemia, severe hypoperfusion alone is insufficient to induce subsequent persistent AKI.

Tissue engineering a functional aortic heart valve: an appraisal.

Valvular heart disease is an important cause of morbidity and mortality, and currently available substitutes for failing hearts have serious limitations. A new promising alternative that may overcome these shortcomings is provided by the relatively new field of tissue engineering (TE). TE techniques involve the growth of autologous cells on a 3D matrix that can be a biodegradable polymer scaffold, or an acellular tissue matrix. These approaches provide the potential to create living matrix valve structures with an ability to grow, repair and remodel within the recipient. This article provides an appraisal of artificial heart valves and an overview of developments in TE that includes the current limitations and challenges for creating a fully functional valve. Biomaterials and stem cell technologies are now providing the potential for new avenues of research and if combined with advances in the rapid prototyping of biomaterials, the engineering of personalized, fully functional, and autologous tissue valve replacements, may become a clinical alternative.