Stephen H. Pasternak

Four‐pool modeling of proton exchange processes in biological systems in the presence of MRI–paramagnetic chemical exchange saturation transfer (PARACEST) agents

Signal loss due to magnetization transfer (MT) from the macromolecular protons of biological tissues is an important consideration for the in vivo detection of paramagnetic chemical exchange saturation transfer (PARACEST) agents. In this study, a four‐pool model is presented that is based on the modified Bloch equations and incorporates terms for the proton exchange processes that occur in biological systems in the presence of MRI‐PARACEST contrast agents. The effect of the exchangeable proton chemical shift and PARACEST agent concentration are modeled in the presence of macromolecule‐derived MT. Experimental validation of the model was performed at 9.4 Tesla using Eu3+‐DOTAM‐glycine (Gly)‐phenylalanine (Phe) in both aqueous solution and samples containing 10% bovine serum albumin (BSA). The model was then used to measure the agent‐bound‐water chemical shift and the transverse relaxation time of macromolecular protons of a sample of Vero (nonhuman primate) cells labeled with Eu3+‐DOTAM‐Gly‐Phe and a phantom containing mouse brain tissue and 7 mM Eu3+‐DOTAM‐Gly‐Phe. In the brain tissue phantom, a chemical shift map with standard deviation (SD) < 0.7 ppm and a T2 map with SD < 0.6 μs were obtained. The results demonstrate the feasibility of in vivo temperature measurement based on the bound‐water chemical shift of Eu3+‐DOTAM‐Gly‐Phe in combination with this four‐pool model despite the inherent MT effect. Magn Reson Med 60:1197–1206, 2008.

The effects of oxytocin on social cognition and behaviour in frontotemporal dementia

Patients with behavioural variant frontotemporal dementia demonstrate abnormalities in behaviour and social cognition, including deficits in emotion recognition. Recent studies suggest that the neuropeptide oxytocin is an important mediator of social behaviour, enhancing prosocial behaviours and some aspects of emotion recognition across species. The objective of this study was to assess the effects of a single dose of intranasal oxytocin on neuropsychiatric behaviours and emotion processing in patients with behavioural variant frontotemporal dementia. In a double-blind, placebo-controlled, randomized cross-over design, 20 patients with behavioural variant frontotemporal dementia received one dose of 24 IU of intranasal oxytocin or placebo and then completed emotion recognition tasks known to be affected by frontotemporal dementia and by oxytocin. Caregivers completed validated behavioural ratings at 8 h and 1 week following drug administrations. A significant improvement in scores on the Neuropsychiatric Inventory was observed on the evening of oxytocin administration compared with placebo and compared with baseline ratings. Oxytocin was also associated with reduced recognition of angry facial expressions by patients with behavioural variant frontotemporal dementia. Together these findings suggest that oxytocin is a potentially promising, novel symptomatic treatment candidate for patients with behavioural variant frontotemporal dementia and that further study of this neuropeptide in frontotemporal dementia is warranted.

Rapid and Direct Transport of Cell Surface APP to the Lysosome defines a novel selective pathway

BackgroundA central feature of Alzheimers disease is the cleavage of the amyloid precursor protein (APP) to form beta-amyloid peptide (Aβ) by the β-secretase and γ-secretase enzymes. Although this has been shown to occur after endocytosis of APP from the cell surface, the exact compartments of APP processing are not well defined. We have previously demonstrated that APP and γ-secretase proteins and activity are highly enriched in purified rat liver lysosomes. In order to examine the lysosomal distribution and trafficking of APP in cultured cells, we generated constructs containing APP fused to a C-terminal fluorescent protein tag and N-terminal HA-epitope tag. These were co-transfected with a panel of fluorescent-protein tagged compartment markers.ResultsHere we demonstrate using laser-scanning confocal microscopy that although APP is present throughout the endosomal/lysosomal system in transfected Cos7 and neuronal SN56 cell lines as well as in immunostained cultured mouse neurons, it is enriched in the lysosome. We also show that the Swedish and London mutations reduce the amount of APP in the lysosome. Surprisingly, in addition to its expected trafficking from the cell surface to the early and then late endosomes, we find that cell-surface labelled APP is transported rapidly and directly from the cell surface to lysosomes in both Cos7 and SN56 cells. This rapid transit to the lysosome is blocked by the presence of either the London or Swedish mutations.ConclusionsThese results demonstrate the presence of a novel, rapid and specific transport pathway from the cell surface to the lysosomes. This suggests that regulation of lysosomal traffic could regulate APP processing and that the lysosome could play a central role in the pathophysiology of Alzheimers disease.

A paramagnetic chemical exchange-based MRI probe metabolized by cathepsin D: design, synthesis and cellular uptake studies.

Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimers disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE).

Oligomeric aggregates of amyloid β peptide 1-42 activate ERK/MAPK in SH-SY5Y cells via the α7 nicotinic receptor

The production and aggregation of amyloid beta peptides (Abeta) has been linked to the development and progression of Alzheimers disease. It is apparent that the various structural forms of Abeta can affect cell signalling pathways and the activity of neurons differently. In this study, we investigated the effects of oligomeric and fibrillar aggregates of Abeta 1-42 (Abeta42) and non-aggregated peptide upon activation of the ERK/MAPK signalling pathway. In SH-SY5Y cells, acute exposure to oligomeric Abeta42 led to phosphorylation of ERK1/2 at concentrations as low as 1 nM and up to 100 nM. These changes were detected as early as 5 min following exposure to 100 nM oligomeric Abeta42, reaching a maximum level after 10 min. Phosphorylation of ERK1/2 subsequently declined to and remained at basal levels after 30 min to 2h of exposure. Fibrillar aggregates of Abeta42 did not significantly induce phosphorylation of ERK1/2 and non-aggregated Abeta42 did not activate the pathway. The effects of oligomeric Abeta42 to increase ERK phosphorylation above basal levels were inhibited by MLA, a specific antagonist of the alpha7 nAChR. U0126, an inhibitor of MEK, the upstream activator of ERK1/2, completely blocked induction of ERK1/2 phosphorylation. Oligomeric aggregates of Abeta42 are the principal structural form of the peptide that activates ERK/MAPK in SH-SY5Y cells and these effects are mediated by the alpha7 nAChR.

The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid

BackgroundAlzheimer s disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing.ResultsWe have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces Aβ production by more than a third.ConclusionThese data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of Aβ production.

Oxytocin for frontotemporal dementia: A randomized dose-finding study of safety and tolerability

Objective: To determine the safety and tolerability of 3 doses of intranasal oxytocin (Syntocinon; Novartis, Bern, Switzerland) administered to patients with frontotemporal dementia (FTD). Methods: We conducted a randomized, parallel-group, double-blind, placebo-controlled study using a dose-escalation design to test 3 clinically feasible doses of intranasal oxytocin (24, 48, or 72 IU) administered twice daily for 1 week to 23 patients with behavioral variant FTD or semantic dementia (clinicaltrials.gov registration number NCT01386333). Primary outcome measures were safety and tolerability at each dose. Secondary measures explored efficacy across the combined oxytocin vs placebo groups and examined potential dose-related effects. Results: All 3 doses of intranasal oxytocin were safe and well tolerated. Conclusions: A multicenter trial is warranted to determine the therapeutic efficacy of long-term intranasal oxytocin for behavioral symptoms in FTD. Classification of evidence: This study provides Class I evidence that for patients with FTD, intranasal oxytocin is not significantly associated with adverse events or significant changes in the overall neuropsychiatric inventory.

MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures.

Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

The Fibroblast Growth Factor Receptor Substrate 3 Adapter Is a Developmentally Regulated Microtubule-associated Protein Expressed in Migrating and Differentiated Neurons

J. Neurochem. (2009) 112, 924–939.

A dual magnetic resonance imaging/fluorescent contrast agent for Cathepsin-D detection.

Currently there are no approved biomarkers for the pre-symptomatic diagnosis of Alzheimers 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 Alzheimers 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 Alzheimers disease.