title: A dual magnetic resonance imaging/fluorescent contrast agent for Cathepsin-D detection.
abstract: Currently there are no approved biomarkers for the pre-symptomatic diagnosis of Alzheimer's disease (AD). Cathepsin-D (Cat-D) is a lysosomal protease that is present at elevated levels in amyloid plaques and neurons in patients with AD and is also elevated in some cancers. We have developed a magnetic resonance imaging (MRI)/fluorescent contrast agent to detect Cat-D enzymatic activity. The purpose of this study was to investigate the cellular and tissue uptake of this MRI/fluorescent contrast agent. The agent consists of an MRI probe [DOTA-caged metal ion (Gd³⁺ or Tm³⁺)] and a fluorescent probe coupled to a cell-penetrating-peptide sequence by a Cat-D recognition site. The relaxivity of Gd³⁺-DOTA-CAT(cleaved) was measured in 10% heat-treated bovine serum albumin (BSA) phantoms to assess contrast efficacy at magnetic fields ranging from 0.24 mT to 9.4 T. In vitro, Tm³⁺-DOTA-CAT was added to neuronal SN56 cells over-expressing Cat-D and live-cell confocal microscropy was performed at 30 min. Tm³⁺-DOTA-CAT was also intravenously injected into APP/PS1-dE9 Alzheimer's disease mice (n = 9) and controls (n = 8). Cortical and hippocampal uptake was quantified at 30, 60 and 120 min post-injection using confocal microscopy. The liver and kidneys were also evaluated for contrast agent uptake. The relaxivity of Gd³⁺-DOTA-CAT(cleaved) was 3.3 (mM s)⁻¹ in 10% BSA at 9.4 T. In vitro, cells over-expressing Cat-D preferentially took up the contrast agent in a concentration-dependent manner. In vivo, the contrast agent effectively crossed the blood-brain barrier and exhibited a distinct time course of uptake and retention in APP/PS1-dE9 transgenic mice compared with age-matched controls. At clinical and high magnetic field strengths, Gd³⁺-DOTA-CAT produced greater T₁ relaxivity than Gd³⁺-DTPA. Tm³⁺-DOTA-CAT was taken up in a dose-dependent manner in cells over-expressing Cathepsin-D and was shown to transit the blood-brain barrier in vivo. This strategy may be useful for the in vivo detection of enzyme activity and for the diagnosis of Alzheimer's disease.
authors.affiliation: Department of Medical Biophysics, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Ta
authors.forename: Mojmir
authors.initials: M
authors.lastname: Suchy
authors.forename: Joshua H K
authors.initials: JH
authors.lastname: Tam
authors.forename: Alex X
authors.initials: AX
authors.lastname: Li
authors.forename: Francisco S
authors.initials: FS
authors.lastname: Martinez-Santiesteban
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Scholl
authors.forename: Robert H E
authors.initials: RH
authors.lastname: Hudson
authors.forename: Robert
authors.initials: R
authors.lastname: Bartha
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Pasternak
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D002402:Cathepsin D / Q000378:metabolism*; D003287:Contrast Media / Q000138:chemical synthesis; D006571:Heterocyclic Compounds; D008279:Magnetic Resonance Imaging / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D009942:Organometallic Compounds; D010455:Peptides; D013382:Subtraction Technique
chemical_list: D003287:Contrast Media; D006571:Heterocyclic Compounds; D009942:Organometallic Compounds; D010455:Peptides; C050823:gadolinium 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetate; D002402:Cathepsin D