We have several ongoing studies including:
1). In Vivo metabolism of amyloid-beta (Aβ) in Alzheimer’s disease (AD)
We have pioneered a novel stable isotope labeling kinetics (SILK) technique that measures Aβ metabolism in humans. To measure the production and clearance of Aβ in AD, we developed a method to measure human CNS Aβ production and clearance, and compared Aβ42 and Aβ40 production and clearance rates in individuals with symptomatic AD and cognitively normal persons to determine if either or both is altered in AD. This initial study demonstrates the average clearance rate of Aβ42 was slower for AD individuals compared with cognitively normal controls (5.3%/hr vs. 7.6%/hr, p=0.03), as was the average clearance rate of Aβ40 (5.2%/hr for AD individuals vs. 7.0%/hr for controls; p=0.01). (Figure 1 from Mawuenyega et al., 2011 Science; below).
Aβ kinetics in the CNS of twelve AD participants (red triangles) and twelve controls (blue circles). The amount of labeled Aβ42 and Aβ40 was measured and compared between groups to measure production and clearance rates of both Aβ species. (A) The average normalized labeled Aβ42 time course. (B) Aβ42 clearance rate during the clearance phase (hours 24–36). (C) Normalized labeled Aβ40 time course. (D) Aβ40 clearance in AD compared to controls. (E) The average fractional synthesis rates of Aβ42 and Aβ40 in AD participants and cognitively normal controls. (F) The average fractional clearance rates of Aβ42 and Aβ40.
In follow up studies using the SILK method, we demonstrate a highly significant correlation between increasing age and slowed Aβ turnover rates (2.5-fold longer half-life over five decades of age). In addition, we found independent effects on Aβ42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble Aβ42 and a 10-fold higher Aβ42 reversible exchange rate. (Figure 1 from Patterson et al., 2015, Annals of Neurology; below).
Amyloid-beta (Aβ)38, Aβ40, and Aβ42 turnover rates slow with increased age. (A) Stable isotope labeling kinetics time-course profiles of Aβ38 (left), Aβ40 (middle), and Aβ42 (right) from 51 amyloid-negative subjects from the present sporadic Alzheimer’s disease cohort are summarized along with 12 amyloid-negative subjects who were previously reported on (Potter et al., 2013). Results are averaged across three age groups spanning decade ranges: black = age 30s to 50s, n = 9; blue = age 60s, n = 25; red = age 70s to 80s, n = 29. Error bars represent 95% confidence intervals (CIs). Solid lines represent average model fits to the data for each age group. (B) Turnover rates of all Aβ isoforms are highly negatively correlated with increased age. Results from older amyloid-negative (blue circles) and amyloid-positive (red triangles) are shown with 12 younger amyloid-negative participants (green asterisks). Linear fit with 95% CIs are shown for age versus FTR of Aβ. FTR = fractional turnover rate.
2.) Longitudinal changes that occur in Autosomal Dominant Alzheimer Disease (ADAD):
Increased in vivo amyloid-β42 production, exchange, and loss in presenilin mutation carriers. We are investigating the changes that occur in ADAD including structural changes by MRI, pathological changes by PET-PIB, functional changes by Clinical Dementia Rating and neuropsychometric testing, and pathophysiological changes in CSF biomarkers and CNS protein production and clearance rates. We found that CNS Aβ42 to Aβ40 production rates were 24% higher in mutation carriers compared to noncarriers, and this was independent of fibrillar amyloid deposits quantified by PET PIB imaging. Furthermore, the fractional turnover rate of soluble Aβ42 relative to Aβ40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aβ42 into plaques, leading to reduced recovery of Aβ42 in cerebrospinal fluid (CSF). These findings support the hypothesis that Aβ42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aβ42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aβ42 concentrations in the CSF. (Figure 1 from Potter et al., 2013, Science and Translational Medicine; below).
PET images and isotopic enrichment time course profiles of CSF Aβ peptides. (A) Composite PET images showing [11C]Pittsburgh compound B binding in participants who are non-carriers of PSEN mutations (left column), and PSEN mutation carriers who lack (PIB−, middle column) or have (PIB+, right column) evidence of amyloidosis. (B, C) Average Aβ isotopic kinetic time course profiles in CSF showing the Aβ42:40, Aβ38:40, and Aβ42:38 isotopic enrichment ratios (B, middle panel: Aβ38:40, blue circles, Aβ42:38 green squares, and Aβ42:40 red triangles) and as enrichment ratios normalized to plasma leucine plateau enrichments (C, lower panel: Aβ38 blue circles, Aβ40 green squares, Aβ42 red triangles). Aβ38 and Aβ40 present similar labeling profiles in all subject groups, whereas Aβ42 kinetics deviate from Aβ38 and Aβ40 only in mutation carriers, as evident in the Aβ42:40 and Aβ42:38 ratio profiles.
3.) In vivo strategies to assess disease-modifying therapies for Alzheimer disease
Pharmacodynamic response of proposed disease-modifying therapies for Alzheimer disease are tested by directly measuring the production, clearance and steady-state levels of the targeted proteins, including amyloid-beta. These studies quantitate targeted activity of therapeutics and provide evidence that these compounds may be effective in humans. (Figure 4 from Dobrowolska et al. 2014, Journal of Neuroscience; below)
BACE inhibitor dose dependently decreased sAPPβ and Aβ in non-human primate (NHP) CSF. Effects of a BACE inhibitor on stable isotope labeling kinetics (SILK) relative values, ELISA absolute concentrations, and concentrations of newly generated Aβ (A–C), sAPPβ (D–F), and sAPPα (G–I) in CSF of NHP. A, D, G, SILK MFL Aβ and sAPPβ decreases dose dependently with BACE inhibitor, and MFL sAPPα indicated no measurable difference among vehicle and drug groups (measured by LC-MS). B, E, H, Concentrations of Aβ and sAPPβ decreased dose dependently and absolute concentrations of sAPPα increased dose dependently with a BACE inhibitor (measured by ELISA). C, F, I, Newly generated Aβ and sAPPβ decreased dose dependently and newly generated sAPPα increased dose dependently with a BACE inhibitor (measured as product of LC-MS labeling and ELISA absolute concentrations at each time point). Error bars indicate SD.
4.) CNS derived proteomics and measurements
We are currently investigating multiple other CNS derived proteins and are developing methods to measure hundreds of protein metabolism profiles in humans using highly sensitive nano-flow mass spectrometry and in vivo labeling techniques. Advanced bio-informatics, cutting edge mass spectrometry, and in vivo and in vitro labeling experiments are used for highly quantitative analysis of proteins.
Articles about laboratory projects: