Anniina Snellman

Multifunctional Liposomes Reduce Brain β-Amyloid Burden and Ameliorate Memory Impairment in Alzheimer's Disease Mouse Models

Alzheimers disease is characterized by the accumulation and deposition of plaques of β-amyloid (Aβ) peptide in the brain. Given its pivotal role, new therapies targeting Aβ are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aβ assemblies and evaluated their efficiency in reducing the Aβ burden in Alzheimers disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood–brain barrier targeting and with phosphatidic acid for Aβ binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aβ assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aβ1–42 (−33%), assessed by ELISA, and the number and total area of plaques (−34%) detected histologically. Also, brain Aβ oligomers were reduced (−70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [11C]Pittsburgh compound B (PIB). The reduction of brain Aβ was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aβ aggregates and promote peptide removal across the blood–brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimers disease.

Longitudinal Amyloid Imaging in Mouse Brain with 11C-PIB: Comparison of APP23, Tg2576, and APPswe-PS1dE9 Mouse Models of Alzheimer Disease

Follow-up of β-amyloid (Aβ) deposition in transgenic mouse models of Alzheimer disease (AD) would be a valuable translational tool in the preclinical evaluation of potential antiamyloid therapies. This study aimed to evaluate the ability of the clinically used PET tracer 11C-Pittsburgh compound B (11C-PIB) to detect changes over time in Aβ deposition in the brains of living mice representing the APP23, Tg2576, and APPswe-PS1dE9 transgenic mouse models of AD. Methods: Mice from each transgenic strain were imaged with 60-min dynamic PET scans at 7−9, 12, 15, and 18−22 mo of age. Regional 11C-PIB retention was quantitated as distribution volume ratios using Logan graphical analysis with cerebellar reference input, as radioactivity uptake ratios between the frontal cortex (FC) and the cerebellum (CB) during the 60-min scan, and as bound-to-free ratios in the late washout phase (40−60 min). Ex vivo autoradiography experiments were performed after the final imaging session to validate 11C-PIB binding to Aβ deposits. Additionally, the presence of Aβ deposits was evaluated in vitro using staining with thioflavin-S and Aβ1–40, Aβ1–16, and AβN3(pE) immunohistochemistry. Results: Neocortical 11C-PIB retention was markedly increased in old APP23 mice with large thioflavin-S–positive Aβ deposits. At 12 mo, the Logan distribution volume ratio for the FC was 1.03 and 0.93 (n = 2), increasing to 1.38 ± 0.03 (n = 3) and 1.34 (n = 1) at 18 and 21 mo of age, respectively. An increase was also observed in bound-to-free ratios for the FC between young (7- to 12-mo-old) and old (15- to 22-mo-old) APP23 mice. Binding of 11C-PIB to Aβ-rich cortical regions was also evident in ex vivo autoradiograms of APP23 brain sections. In contrast, no increases in 11C-PIB retention were observed in aging Tg2576 or APPswe-PS1dE9 mice in vivo, although in the latter, extensive Aβ deposition was already observed at 9 mo of age with immunohistochemistry. Conclusion: The results suggest that 11C-PIB binding to Aβ deposits in transgenic mouse brain is highly dependent on the AD model and the structure of its Aβ plaques. Longitudinal in vivo 11C-PIB uptake studies are possible in APP23 mice.

Synthesis and evaluation of a [18F]-curcumin derivate for β-amyloid plaque imaging

INTRODUCTION Curcumin is a neuroprotective compound that inhibits the formation of amyloid oligomers and fibrils and binds to β-amyloid plaques in Alzheimers disease (AD). We aimed to synthesize an (18)F-labeled curcumin derivate ([(18)F]4) and to characterize its positron emission tomography (PET) tracer-binding properties to β-amyloid plaques in a transgenic APP23 mouse model of AD. METHODS We utilized facile one-pot synthesis of [(18)F]4 using nucleophilic (18)F-fluorination and click chemistry. Binding of [(18)F]4 to β-amyloid plaques in the transgenic APP23 mouse brain cryosections was studied in vitro using heterologous competitive binding against PIB. [(18)F]4 uptake was studied ex vivo in rodents and in vivo using PET/computed tomography of transgenic APP23 and wild-type control mice. RESULTS The radiochemical yield of [(18)F]4 was 21 ± 11%, the specific activity exceeded 1TBq/μmol, and the radiochemical purity exceeded 99.3% at the end of synthesis. In vitro studies of [(18)F]4 with the transgenic APP23 mouse revealed high β-amyloid plaque binding. In vivo and ex vivo studies demonstrated that [(18)F]4 has fast clearance from the blood, moderate metabolism but low blood-brain barrier (BBB) penetration. CONCLUSIONS [(18)F]4 was synthesized in high yield and excellent quality. In vitro studies, metabolite profile, and fast clearance from the blood indicated a promising tracer for Aβ imaging. However, [(18)F]4 has low in vivo BBB penetration and thus further studies are needed to reveal the reason for this and to possibly overcome this issue.

EX VIVO TRACING OF NMDA AND GABA-A RECEPTORS IN RAT BRAIN AFTER TRAUMATIC BRAIN INJURY USING 18F-GE-179 AND 18F-GE-194 AUTORADIOGRAPHY

In vivo imaging of N-methyl-d-aspartate (NMDA) glutamate receptor and γ-aminobutyric acid (GABA)-A receptor during progression of brain pathology is challenging because of the lack of imaging tracers with high affinity and specificity. Methods: We monitored changes in NMDA receptor and GABA-A receptor in a clinically relevant model of traumatic brain injury (TBI) induced by lateral fluid percussion in adult rats, using 2 new ligands for PET: 18F-GE-179 for the open/active state of the NMDA receptor ion channel and 18F-GE-194 for GABA-A receptor. Ex vivo brain autoradiography of radioligands was performed at subacute (5–6 d) and chronic (40–42 d) time points after TBI. Results: At 5–6 d after TBI, 18F-GE-179 binding was higher in the cortical lesion area, in the lesion core, and in the hippocampus than in the corresponding contralateral regions; this increase was probably related to increased permeability of the blood–brain barrier. At 40–42 d after TBI, 18F-GE-179 binding was significantly higher in the medial cortex, in the corpus callosum, and in the thalamus than in the corresponding contralateral regions. Five to 6 days after TBI, 18F-GE-194 binding was significantly higher in the lesion core and significantly lower in the ipsilateral thalamus. By 40–42 d after TBI, the reduction in 18F-GE-194 binding extended to the cortical lesion, including the perilesional cortex around the lesion core. The reduction in thalamic binding was more extensive at 40–42 d than at 5–6 d after TBI, suggesting a progressive decrease in thalamic GABA-A receptor density. Immunohistochemistry against GABA-A α1 subunit revealed a similar decrease to 18F-GE-194 binding, particularly during the chronic phase. Conclusion: Our data support the validity of novel 18F-GE-179 and 18F-GE-194 radioligands for the detection of changes in active NMDA receptor and GABA-A receptor in the injured brain. These tools are useful for follow-up evaluation of secondary postinjury pathologies.

Applicability of [11C]PIB micro-PET imaging for in vivo follow-up of anti-amyloid treatment effects in APP23 mouse model

In this study, we evaluated the anti-amyloid effect of functionalized nanoliposomes (mApoE-PA-LIP) in a mouse model of Alzheimers disease with use of positron emission tomography and β-amyloid (Aβ)-targeted tracer [11C]Pittsburgh compound B ([11C]PIB). APP23 mice were injected with mApoE-PA-LIP or saline (3 times per week for 3 weeks) and [11C]PIB imaging was performed at baseline, after the treatment and after 3 months follow-up period, accompanied by Aβ immunohistochemistry and ELISA. After the treatment, [11C]PIB binding ratios between mApoE-PA-LIP and saline groups were equivalent in all analyzed brain regions; however, in the saline group, binding ratios increased from the baseline, whereas no increase was detected in the mApoE-PA-LIP group. During the additional follow-up, [11C]PIB binding increased significantly from baseline in both groups, and binding ratios correlated with the immunohistochemically defined Aβ load. This study further supports the use of [11C]PIB positron emission tomography imaging as a biomarker of Aβ deposition in APP23 mice and highlights the benefits of noninvasive follow-up, that is, using baseline data for animal stratification and normalization of treatment effects to baseline values, for future anti-amyloid treatment studies.

Neuroinflammation appears early and then plateaus in a mouse model of Alzheimer's disease shown by PET imaging

Neuroinflammation has been associated with various neurologic diseases, including Alzheimer disease (AD). In AD, the translocator protein 18 kDa (TSPO) is overexpressed in the activated microglia that surround the β-amyloid plaques. In the current longitudinal study using a mouse model of AD, we evaluated the association between β-amyloid deposition and neuroinflammation in AD. Methods: To monitor the longitudinal changes in β-amyloid deposition and neuroinflammation, we used in vivo PET imaging and ex vivo autoradiography with Pittsburgh compound B (11C-PIB) and a TSPO tracer, flutriciclamide (18F-GE-180), in the APP23 mouse model of AD. We also applied immunohistochemistry to study β-amyloid and activated microglia in the mouse brain tissue. Results: From 17 to 26 mo of age, the mice showed robust increased binding of 11C-PIB with aging in the frontal cortex, parietotemporal cortex, hippocampus, and thalamus whereas the increase in 18F-GE-180 binding with aging was minimal in areas of early amyloidosis such as the frontal cortex and hippocampus. A clear positive correlation between β-amyloid deposition and neuroinflammation was detected with 11C-PIB and 18F-GE-180 only in the parietotemporal cortex and thalamus. Conclusion: The neuroinflammation increase detected with 18F-GE-180 is less than the increase in amyloidosis detected with 11C-PIB. Furthermore, binding of 18F-GE-180 plateaus at an earlier stage of pathogenesis whereas amyloidosis continues to increase. We suggest that TSPO can be a good marker for early pathogenesis detection but not for tracking long-term disease progression.

(18)F-labeling syntheses and preclinical evaluation of functionalized nanoliposomes for Alzheimer's disease.

The aim of the present study was to synthesize functionalized (18)F-labeled NLs ((18)F-NLs) and evaluate their biological behavior in mouse models of Alzheimers disease (AD) using positron emission tomography (PET) and ex vivo brain autoradiography. (18)F-fluorine was introduced to (18)F-NLs either by using a core forming (18)F-lipid or by encapsulating a (18)F-tracer, (18)F-treg-curcumin inside the NLs. Phosphatidic acid (PA) and curcumin derivative (Curc) functionalized (18)F-NLs with or without additional mApoE functionalization were produced using thin film hydration. The biodistribution and β-amyloid plaque-binding ability of (18)F-NLs were studied in wild type mice and AD mouse models using in vivo PET imaging and ex vivo brain autoradiography at 60min after (18)F-NL injection. Functionalized (18)F-NLs were successfully synthesized. The preclinical evaluation in mice showed that the functional group affected the biodistribution of (18)F-NLs. Further functionalization with mApoE increased the brain-to-blood ratio of (18)F-NLs but the overall brain uptake remained low with all functionalized (18)F-NLs. The liposomal encapsulation of (18)F-treg-curcumin was not successful and preclinical results of encapsulated (18)F-treg-curcumin and plain (18)F-treg-curcumin were identical. Although the studied functionalized (18)F-NLs were not suitable for PET imaging as such, the synthesis techniques introduced in this study can be utilized to modify the biological behavior of (18)F-labeled NLs.

19F/18F exchange synthesis for a novel [18F]S1P3-radiopharmaceutical

(19)F/(18)F isotope exchange is a useful method to label drug molecules containing (19)F-fluorine with (18)F without modifying the drug molecule itself. Sphingosine-1-phosphate (S1P) is an important cellular mediator that functions by signaling through cell surface receptors. S1P is involved in several cell responses and may be related to many central nervous system disorders, including neural malfunction in Alzheimers disease. In this study, [(18)F]1-benzyl-N-(3,4-difluorobenzyl)-2-isopropyl-6-(2-methoxyethoxy)-1H-indole-3-carboxamide, a novel (18)F-labeled positron emission tomography tracer for the S1P3 receptor, was successfully synthesized using the (19)F/(18)F isotope exchange reaction. Parameters of the reaction kinetics were studied, and correlations between the initial (18)F-activity, the amount of precursor, radiochemical yield and specific activity (SA) were determined. Contrary to expectations, high initial (18)F-activity decreased the radiochemical yield, and only a minor increase of SA occurred. This is most probably due to the complexity of the molecule and the subsequent susceptibility to radiolytic bond disruption. On the basis of the present results, a convenient condition for the (19)F/(18)F exchange reaction is the use of 2 µmol precursor with 20 GBq of (18)F-activity. This afforded a radiochemical yield of ~10% with an SA of 0.3 GBq/µmol. Results from this study are of interest for new tracer development where high initial (18)F-activity and (19)F/(18)F isotope exchange is used.

6-[18F]Fluoro-l-DOPA Uptake in the Rat Pancreas is Dependent on the Tracer Metabolism

Purpose6-[18F]fluoro-l-3,4-dihydroxyphenyl alanine ([18F]FDOPA) positron emission tomography (PET) is a diagnostic tool which can detect malignancies of the pancreas. We aimed to study whether the manipulation of the [18F]FDOPA metabolic pathway would change the 18F-behavior to provide a biochemical foundation for PET imaging of rat pancreas with [18F]FDOPA.ProceduresInhibitors of aromatic amino acid decarboxylase, catechol-O-methyltransferase, monoamine oxidases A and B, or their combinations on [18F]FDOPA uptake, metabolism, and the regional distribution in the rat pancreas was evaluated using in vivo PET/computed tomography imaging, chromatographic metabolite analyses, and autoradiography.ResultsEnzyme inhibition generally increased the uptake of [18F]FDOPA derived 18F-radioactivity in rat pancreas. Dependent on which enzymatic pathway is blocked (or a combination of pathways), different radiolabeled metabolites in pancreas are responsible for this increase in uptake.ConclusionsAltering the metabolism of [18F]FDOPA by using various enzymatic inhibitors increased the radioactivity uptake and changed the radiometabolic profile in the pancreas allowing better discrimination between pancreas and surrounding tissues of rat. However, these manipulations did not separate islets from the exocrine pancreas. Elucidating the metabolic behavior of [18F]FDOPA provides a biochemical foundation of PET imaging of the rat pancreas.

Long-Term Monoacylglycerol Lipase Inhibitor Treatment Decelerates Pathological Changes in APP/PS1-21 Mice, but Behavioral Improvements Require Early-Stage Treatment Onset—Short Report

The arachidonic acid (AA) pathway produces several essential proinflammatory eicosanoids. However, in many neurodegenerative diseases, e.g. Alzheimer’s disease (AD), this pathway is chronically hyperactivated. In brain, primarily monoacylglycerol lipase (MAGL) hydrolyzes the endocannabinoid 2-arachidonoylglycerol to AA, which is further metabolized to generate many proinflammatory eicosanoids. MAGL inhibition, simultaneously reducing the level of eicosanoids and increasing those of neuroprotective endocannabinoids, has proved efficacious in some AD models, reducing neurotoxic β-amyloid (Aβ) levels and improving memory functions. Here, a MAGL inhibitor, JZL184 was chronically administered (16 mg/kg, i.p., 3 x/wk for 5 mo) for 1 - 1.5 mo and 7 - 8 mo old transgenic (TG) and wild-type (WT) APP/PS1-21 mice modelling cerebral amyloidosis. According to immunohistochemistry, JZL184 significantly increased the expression levels of cannabinoid receptor 1 in older WT and younger TG and WT mice, decreased cannabinoid receptor 2 and oligomeric Aβ in older and younger TG mice and decreased microglia-specific marker Iba1 in younger TG mice, compared to TG mice treated with vehicle only. However, in the Morris Water Maze test, spatial memory functions improved significantly only in younger TG and WT mice, compared to vehicle-treated littermates. These tentative results suggest that chronic, rather long-term MAGL inhibition can decelerate pathological changes in TG APP/PS1-21 mice but it improves memory functions only when administered at an early stage of the pathology