Andrew Crofton

Targeted proteomics for quantification of histone acetylation in Alzheimer's disease

The epigenetic remodeling of chromatin histone proteins by acetylation has been the subject of recent investigations searching for biomarkers indicative of late onset cognitive loss. Histone acetylations affect the regulation of gene transcription, and the loss of learning induced deacetylation at specific histone sites may represent biomarkers for memory loss and Alzheimers disease (AD). Selected‐reaction‐monitoring (SRM) has recently been advanced to quantitate peptides and proteins in complex biological systems. In this paper, we provide evidence that SRM‐based targeted proteomics can reliably quantify specific histone acetylations in both AD and control brain by identifying the patterns of H3 K18/K23 acetylations Results of targeted proteomics assays have been validated by Western blot (WB) analysis. As compared with LC‐MS/MS‐TMT (tandem‐mass‐tagging) and WB methods, the targeted proteomics method has shown higher throughput, and therefore promised to be more suitable for clinical applications. With this methodology, we find that histone acetylation is significantly lower in AD temporal lobe than found in aged controls. Targeted proteomics warrants increased application for studying epigenetics of neurodegenerative diseases.

Effect of cerebral amyloid angiopathy on brain iron, copper, and zinc in Alzheimer's disease.

Cerebral amyloid angiopathy (CAA) is a vascular lesion associated with Alzheimers disease (AD) present in up to 95% of AD patients and produces MRI-detectable microbleeds in many of these patients. It is possible that CAA-related microbleeding is a source of pathological iron in the AD brain. Because the homeostasis of copper, iron, and zinc are so intimately linked, we determined whether CAA contributes to changes in the brain levels of these metals. We obtained brain tissue from AD patients with severe CAA to compare to AD patients without evidence of vascular amyloid-β. Patients with severe CAA had significantly higher non-heme iron levels. Histologically, iron was deposited in the walls of large CAA-affected vessels. Zinc levels were significantly elevated in grey matter in both the CAA and non-CAA AD tissue, but no vascular staining was noted in CAA cases. Copper levels were decreased in both CAA and non-CAA AD tissues and copper was found to be prominently deposited on the vasculature in CAA. Together, these findings demonstrate that CAA is a significant variable affecting transition metals in AD.

Altered Serum Iron and Copper Homeostasis Predicts Cognitive Decline in Mild Cognitive Impairment

Alzheimers disease (AD) brain is marked by severe neuronal death which has been partly attributed to increased oxidative stress. The pathophysiology accounting for this free radical injury is not well-delineated at this point, but one hypothesis is that a derangement in transition metal metabolism contributes to the process. We tested the hypothesis that peripheral derangement of transition metal metabolism is present early in the dementing process. We analyzed non-heme iron and copper levels in serum from subjects with normal cognition, mild cognitive impairment, and early stage senile dementia and followed these subjects over 5 years. An increase in the ratio of serum copper to non-heme iron levels predicted which subjects with mild cognitive impairment would progress to dementia versus those that would remain cognitively stable. This increase did not correlate with changes in expression of iron regulatory protein 2 or selected downstream targets in peripheral lymphocytes. A cDNA-based microarray (IronChip) containing genes relevant to iron and copper metabolism was used to assess transition metal metabolism in circulating lymphocytes from cognitively normal and demented subjects. No gene was identified as being dysregulated more than 2-fold, and verification using quantitative RT-PCR demonstrated no significant changes in expression for ALAS2, FOS, and CTR1. The increased ratio of serum copper to serum iron prior to dementia has potential as a biomarker for cognitive decline and mirrors other changes in serum previously reported by others, but iron and copper metabolism pathways appear to be broadly unaffected in peripheral blood in AD.

Assessing candidate serum biomarkers for Alzheimer's disease: a longitudinal study.

Because of the growing impact of late onset cognitive loss, considerable effort has been directed toward the development of improved diagnostic techniques for Alzheimers disease (AD) that may pave the way for earlier (and more effective) therapeutic efforts. Serum-based biomarkers are the least expensive and invasive modality for screening and routine monitoring. We systematically reviewed the literature to assemble a list of serum biomarkers relevant to AD. In parallel, we conducted a proteomic LC-MS/MS analysis of serum collected from neurologically normal subjects and subjects with mild cognitive impairment (MCI) and early AD (n = 6 in all). Complement C3 and alpha-2-macroglobulin were identified from both the literature review and our proteomic screen for further validation. For these two candidates, ELISA was performed on serum collected from a small independent cohort of subjects for longitudinal analysis. Serum was serially collected from neurologically normal subjects (n = 5) and subjects with MCI who were subsequently followed for a period of two years (n = 5) and regrouped into stable MCI and progressive MCI or AD (n = 6). The ability of each marker to predict which subjects with MCI would progress to dementia and which would remain cognitively stable was assessed. Patients with probable cerebral amyloid angiopathy were also identified (n = 3). This preliminary analysis tested the most-promising serum protein biomarkers for AD and we concluded that none are yet ready for use in the clinical diagnosis and management of dementia. However, a more thorough assessment in longitudinal studies with higher statistical power is warranted.

A Shift in Microglial β-Amyloid Binding in Alzheimer's Disease Is Associated with Cerebral Amyloid Angiopathy

Alzheimers disease (AD) and cerebral amyloid angiopathy (CAA) are two common pathologies associated with β‐amyloid (Aβ) accumulation and inflammation in the brain; neither is well understood. The objective of this study was to evaluate human post‐mortem brains from AD subjects with purely parenchymal pathology, and those with concomitant CAA (and age‐matched controls) for differential expression of microglia‐associated Aβ ligands thought to mediate Aβ clearance and the association of these receptors with complement activation. Homogenates of brain parenchyma and enriched microvessel fractions from occipital cortex were probed for levels of C3b, membrane attack complex (MAC), CD11b and α‐2‐macroglobulin and immunoprecipitation was used to immunoprecipitate (IP) CD11b complexed with C3b and Aβ. Both C3b and MAC were significantly increased in CAA compared to AD‐only and controls and IP showed significantly increased CD11b/C3b complexes with Aβ in AD/CAA subjects. Confocal microscopy was used to visualize these interactions. MAC was remarkably associated with CAA‐affected blood vessels compared to AD‐only and control vessels. These findings are consistent with an Aβ clearance mechanism via microglial CD11b that delivers Aβ and C3b to blood vessels in AD/CAA, which leads to Aβ deposition and propagation of complement to the cytolytic MAC, possibly leading to vascular fragility.

Formulation and characterization of a plasma sterilized, pharmaceutical grade chitosan powder.

Chitosan has great potential as a pharmaceutical excipient. In this study, chitosan flake was micronized using cryo-ball and cryo-jet milling and subsequently sterilized with nitrogen plasma. Micronized chitosan was characterized by laser diffraction, scanning electron microscopy (SEM), conductometric titration, viscometry, loss on drying, FTIR, and limulus amebocyte lysate (LAL) assays. Cryo-jet milling produced mean particle size of 16.05μm, 44% smaller than cryo-ball milling. Cryomilled chitosan demonstrated increased hygroscopicity, but reduced molecular weight and degree of deacetylation (DD). SEM imaging showed highly irregular shapes. FTIR showed changes consistent with reduced DD and an unexplained shift at 1100cm(-1). Plasma treated chitosan was sterile with <2.5EU/g after low-pressure plasma and <1.3EU/g after atmospheric pressure plasma treatment. Plasma treatment decreased the reduced viscosity of chitosan flake and powder, with a greater effect on powder. In conclusion, pharmaceutical grade, sterile chitosan powder was produced with cryo-jet milling and plasma sterilization.