Emily House

Human pro-islet amyloid polypeptide (ProIAPP1–48) forms amyloid fibrils and amyloid spherulites in vitro

Deposition of beta sheets of islet amyloid polypeptide (IAPP) in pancreatic tissue is implicated in the aetiology of type 2 diabetes mellitus (T2DM). IAPP is cleaved from its precursor protein, pro-islet amyloid polypeptide (ProIAPP) and incomplete cleavage results in ProIAPP(1-48), which is found co-deposited with IAPP. Cu(II) prevents IAPP from forming amyloid and herein we investigated if it would also prevent ProIAPP(1-48) from forming beta sheets. Excess Cu(II) prevented ProIAPP(1-48) from forming amyloid and additionally reversed the formation of beta sheets in pre-formed fibrils of the peptide. The latter was also true for ProIAPP(1-48) fibrils formed in the presence of Al(III). An unexpected finding was the formation of spherulites of ProIAPP(1-48) which were only observed in preparations which included Al(III). The spherulites were 40-100 microm in diameter and stained positively for Al(III) suggesting a role for this metal in their formation. The abolition by Cu(II) of the propensity of ProIAPP(1-48) to form amyloid may have important implications for the treatment of T2DM. The immediate significance for diabetes of the equally novel observation of spherulites of ProIAPP(1-48) is unknown though, as with spherulites of Abeta(42) in Alzheimers disease, there may be implications for the aetiology of the disease.

Brain Burdens of Aluminum, Iron, and Copper and their Relationships with Amyloid-β Pathology in 60 Human Brains

The deposition in the brain of amyloid-β as beta sheet conformers associated with senile plaques and vasculature is frequently observed in Alzheimer’s disease. While metals, primarily aluminum, iron, zinc, and copper, have been implicated in amyloid-β deposition in vivo, there are few data specifically relating brain metal burden with extent of amyloid pathologies in human brains. Herein brain tissue content of aluminum, iron, and copper are compared with burdens of amyloid-β, as senile plaques and as congophilic amyloid angiopathy, in 60 aged human brains. Significant observations were strong negative correlations between brain copper burden and the degree of severity of both senile plaque and congophilic amyloid angiopathy pathologies with the relationship with the former reaching statistical significance. While we did not have access to the dementia status of the majority of the 60 brain donors, this knowledge for just 4 donors allowed us to speculate that diagnosis of dementia might be predicted by a combination of amyloid pathology and a ratio of the brain burden of copper to the brain burden of aluminum. Taking into account only those donor brains with either senile plaque scores ≥4 and/or congophilic amyloid angiopathy scores ≥12, a Cu:Al ratio of <20 would predict that at least 39 of the 60 donors would have been diagnosed as suffering from dementia. Future research should test the hypothesis that, in individuals with moderate to severe amyloid pathology, low brain copper is a predisposition to developing dementia.

Copper Abolishes the β-Sheet Secondary Structure of Preformed Amyloid Fibrils of Amyloid-β42

The observation of the co-deposition of metals and amyloid-beta(42) (Abeta(42)) in brain tissue in Alzheimers disease prompted myriad investigations into the role played by metals in the precipitation of this peptide. Copper is bound by monomeric Abeta(12) and upon precipitation of the copper-peptide complex thereby prevents Abeta(42) from adopting a beta-sheet secondary structure. Copper is also bound by beta-sheet conformers of Abeta(42), and herein we have investigated how this interaction affects the conformation of the precipitated peptide. Copper significantly reduced the thioflavin T fluorescence of aged, fibrillar Abeta(42) with, for example, a 20-fold excess of the metal resulting in a ca 90% reduction in thioflavin T fluorescence. Transmission electron microscopy showed that copper significantly reduced the quantities of amyloid fibrils while Congo red staining and polarized light demonstrated a copper-induced abolition of apple-green birefringence. Microscopy under cross-polarized light also revealed the first observation of spherulites of Abeta(42). The size and appearance of these amyloid structures were found to be very similar to spherulites identified in Alzheimers disease tissue. The combined results of these complementary methods strongly suggested that copper abolished the beta-sheet secondary structure of pre-formed, aged amyloid fibrils of Abeta(42). Copper may protect against the presence of beta-sheets of Abeta(42) in vivo, and its binding by fibrillar Abeta(42) could have implications for Alzheimers disease therapy.

Iron deficiency in parkinsonism : region-specific iron dysregulation in Parkinson's disease and multiple system atrophy

Alpha synuclein pathology is widespread and found in diverse cell types in multiple system atrophy (MSA) as compared to Parkinsons disease (PD). The reason for this differential distribution is unknown. Regional differences in the distribution of iron are associated with neurodegenerative diseases, and here we characterize the relationship between iron homeostasis proteins and regional concentration, distribution and form of iron in MSA and PD. In PD substantia nigra, tissue iron and expression of the iron export protein ferroportin increased, while the iron storage protein ferritin expression was unchanged. In the basis pontis of MSA cases, increased total iron concentration coupled with a disproportionate increase in ferritin in dysmorphic microglia and a reduction in ferroportin expression. This is supported by isothermal remanent magnetisation evidence consistent with elevated concentrations of ferritin-bound iron in MSA basis pontis. Conventional opinion holds that excess iron is involved in neurodegeneration. Our data support that this may be the case in PD. While region-specific changes in iron are evident in both PD and MSA, the mechanisms of iron dysregulation appear quite distinct, with a failure to export iron from the MSA basis pontis coupling with significant intracellular accumulation of ferritin iron. This pattern also occurs, to a lesser extent, in the MSA putamen. Despite the excess tissue iron, the manner of iron dysregulation in MSA is reminiscent of changes in anemia of chronic disease, and our preliminary data, coupled with the widespread pathology and involvement of multiple cell types, may evidence a deficit in bioavailabile iron.

Spherulites of Amyloid-β42 In Vitro and in Alzheimer's Disease

Several amyloidogenic proteins including insulin, beta-lactoglobulin, and albumin form spherulites in vitro under non-physiological conditions. These micrometer-sized, roughly spherical structures are composed of ordered arrays of amyloid fibrils in radial arrangements which, characteristically, show a typical Maltese cross pattern of light extinction under the polarizing microscope. The physiological significance of amyloid spherulites is unknown though in Alzheimers disease, senile plaques composed primarily of beta sheets of amyloid-beta (Abeta)42 have, very occasionally, been shown to give a Maltese cross pattern of light extinction under crossed polarizers. Herein we describe the first observation of the formation in vitro of spherulites of Abeta42. They were formed under near-physiological conditions in which the beta sheet conformation of pre-formed aggregates of Abeta42 had been abolished following the addition of an excess of copper. Incubation of these preparations at 37 degrees C for up to 9 months resulted in the formation of globular structures, 5-20 microm in diameter, which exhibited a Maltese cross pattern of light extinction typical of spherulites. Near-identical spherulitic structures were also observed in abundance in 30 microm thick sections of Alzheimers disease brain tissue. Synchrotron x-ray fluorescence showed that the location of these spherulites in AD tissue coincided with locally elevated concentrations of tissue copper. The formation in vitro of spherulites of Abeta42 which morphologically appeared analogous to spherulitic structures observed in vivo strongly supports the hypothesis that spherulites and senile plaques in AD tissue are one and the same structures and that their ultimate formation may involve copper.

The aluminium content of breast tissue taken from women with breast cancer.

The aetiology of breast cancer is multifactorial. While there are known genetic predispositions to the disease it is probable that environmental factors are also involved. Recent research has demonstrated a regionally specific distribution of aluminium in breast tissue mastectomies while other work has suggested mechanisms whereby breast tissue aluminium might contribute towards the aetiology of breast cancer. We have looked to develop microwave digestion combined with a new form of graphite furnace atomic absorption spectrometry as a precise, accurate and reproducible method for the measurement of aluminium in breast tissue biopsies. We have used this method to test the thesis that there is a regional distribution of aluminium across the breast in women with breast cancer. Microwave digestion of whole breast tissue samples resulted in clear homogenous digests perfectly suitable for the determination of aluminium by graphite furnace atomic absorption spectrometry. The instrument detection limit for the method was 0.48 μg/L. Method blanks were used to estimate background levels of contamination of 14.80 μg/L. The mean concentration of aluminium across all tissues was 0.39 μg Al/g tissue dry wt. There were no statistically significant regionally specific differences in the content of aluminium. We have developed a robust method for the precise and accurate measurement of aluminium in human breast tissue. There are very few such data currently available in the scientific literature and they will add substantially to our understanding of any putative role of aluminium in breast cancer. While we did not observe any statistically significant differences in aluminium content across the breast it has to be emphasised that herein we measured whole breast tissue and not defatted tissue where such a distribution was previously noted. We are very confident that the method developed herein could now be used to provide accurate and reproducible data on the aluminium content in defatted tissue and oil from such tissues and thereby contribute towards our knowledge on aluminium and any role in breast cancer.

Spherulites in Human Brain Tissue are Composed of Beta Sheets of Amyloid and Resemble Senile Plaques

Recent evidence showed that amyloid-β, Aβ(42), formed spherulites in vitro and, possibly, in vivo in Alzheimers disease brain tissue. We now confirm the presence of spherulites in human brains and that they are composed of β sheets of amyloid. The spherulites were identical in appearance to spherulites of Aβ(42) formed in vitro which suggested that they may too be composed of Aβ. The physiological significance of this finding may be in its support of previous speculation that spherulites in human brain tissue are the 3-dimensional manifestations of what are otherwise identified as senile or neuritic plaques.