[18F]Nifene PET/CT Imaging in Mice: Improved Methods and Preliminary Studies of α4β2* Nicotinic Acetylcholinergic Receptors in Transgenic A53T Mouse Model of α-Synucleinopathy and Post-Mortem Human Parkinson's Disease

Abstract

We report [¹⁸F]nifene binding to α4β2* nicotinic acetylcholinergic receptors (nAChRs) in Parkinson's disease (PD). The study used transgenic Hualpha-Syn(A53T) PD mouse model of α-synucleinopathy for PET/CT studies in vivo and autoradiography in vitro. Additionally, postmortem human PD brain sections comprising of anterior cingulate were used in vitro to assess translation to human studies. Because the small size of mice brain poses challenges for PET imaging, improved methods for radiosynthesis of [¹⁸F]nifene and simplified PET/CT procedures in mice were developed by comparing intravenous (IV) and intraperitoneal (IP) administered [¹⁸F]nifene. An optimal PET/CT imaging time of 30-60 min post injection of [¹⁸F]nifene was established to provide thalamus to cerebellum ratio of 2.5 (with IV) and 2 (with IP). Transgenic Hualpha-Syn(A53T) mice brain slices exhibited 20-35% decrease while in vivo a 20-30% decrease of [¹⁸F]nifene was observed. Lewy bodies and α-synuclein aggregates were confirmed in human PD brain sections which lowered the [¹⁸F]nifene binding by more than 50% in anterior cingulate. Thus [¹⁸F]nifene offers a valuable tool for PET imaging studies of PD.

Keywords: Hualpha-Syn(A53T); Lewy bodies; PET/CT imaging; Parkinson’s disease; [18F]nifene; transgenic mice; α-synucleinopathy.

Figure 1

Chemical structures of PET and SPECT radiotracers for α4β2* nAChRs. 1. [¹⁸F]Nifrolidine (PET); 2. [¹⁸F]nifzetidine (PET); 3. [¹⁸F]nifrolene (PET); 4. [¹⁸F]nifene (PET); 5. [¹²³I]niodene (SPECT); 6. [¹⁸F] or [¹²³I]niofene (PET or SPECT).

Figure 2

Improved [¹⁸F]nifene (4) synthesis. N-BOC-nifene (7) was converted to the 2-(trimethylamino)-3-[2-((S)-N-tert-butoxycarbonyl-3-pyrrolinyl)methoxy]pyridine triflate (TMAT) (8) by reacting with dimethylamine and methyl trifluorosulfonate. (A). HPLC chromatogram showing TMAT precursor retention time of approx. 6 min; (B). HPLC chromatogram showing N-BOC-nitronifene (9) precursor with retention time of approx. 9.5 min; (C). N-BOC-[¹⁸F]nifene (10) eluting at approx. 10 min on HPLC which is well separated from TMAT precursor, but close the N-BOC-nitronifene precursor.

Figure 3

PET/CT of intravenous (IV) [¹⁸F]nifene (5.55 MBq in 20 mL saline) administered in wild-type mouse (BALB/c, female 24 g). (A) Coronal, transaxial, and sagittal images of mouse head at 0.5 min after injection showing high levels of [¹⁸F]nifene in the brain; (B) coronal, transaxial, and sagittal images of same mouse head at 47.5 min after injection; (C) PET intensity increased (×1.5) of images in (B) showing [¹⁸F]nifene bound the thalamus and cortical regions; (D) time–activity curve of thalamus and cerebellum of region-of-interest shown on the sagittal slices; (E) ratio of thalamus to cerebellum of time–activity curve showing plateau between 30 to 60 min (dotted line).

Figure 4

PET/CT of intraperitoneal (IP) [¹⁸F]nifene (5.77 MBq in 50 mL saline) administered in wild-type mouse (BALB/c, female 24 g). (A) Coronal, transaxial, and sagittal images of mouse head at 0.5 min after injection showing no [¹⁸F]nifene in the brain; (B) coronal, transaxial, and sagittal images of same mouse head at 47.5 min after injection; (C) PET intensity increased (×1.5) of images in (B) showing [¹⁸F]nifene bound the thalamus and cortical regions; (D) time–activity curve of thalamus and cerebellum of region-of-interest shown on the sagittal slices; (E) ratio of thalamus to cerebellum of time–activity curve showing plateau between 30 to 60 min (dotted line).

Figure 5

PET-MR of [¹⁸F]nifene: (A) Mouse brain MR template showing coronal, sagittal and transaxial slices, with cross hairs placed on the thalamus; (B) [¹⁸F]nifene PET coronal, sagittal, and transaxial slices, 30 min post IP injection (5.50 MBq in 100 mL saline) in wild-type mouse (C57BL/6, male 28 g); (C) [¹⁸F]nifene PET co-registered with mouse MR template (A,B); (D) mouse brain slice ex vivo 30 μm thick; (E) ex vivo autoradiograph of [¹⁸F]nifene of mouse slice (D), after PET experiment in (B), showing regions-of-interest for analysis; (F) comparison of [¹⁸F]nifene in vivo PET and [¹⁸F]nifene ex vivo brain slice autoradiograph (B vs. E) using four brain regions; (G–K) in vitro [¹⁸F]nifene binding in 10 mm sagittal mice brain (G) slices showing [¹⁸F]nifene binding (H; 100%) and effect of 0.01 mM (I; 72%), 0.1 mM (J; 15%), and 1 mM (K; 6%) unlabeled Nifene.

Figure 6

In vitro [¹⁸F]nifene in Hualpha-Syn ((A53T) PD mice model: (A) Non-carrier mouse brain slice 10 μm thick; (B) in vitro autoradiograph of [¹⁸F]nifene of non-carrier mouse brain slice; (C) A53T PD mouse brain slice; 10 μm thick; (D) in vitro autoradiograph of [¹⁸F]nifene of Hualpha-Syn ((A53T) mouse brain slice; (E) comparison of [¹⁸F]nifene in non-carrier mice (n = 3) and Hualpha-Syn ((A53T) PD mice (n = 3) in different brain regions. Inset in (E) shows percent decrease of [¹⁸F]nifene binding in A53T PD mice compared to non-carrier mice (p < 0.05 for frontal cortex, anterior cingulate, striatum, and thalamus; not significant for subiculum, cerebellum, and hippocampus).

Figure 7

Hualpha-Syn ((A53T) PD mice brain PET-MR of [¹⁸F]nifene. (A–C): [¹⁸F]Nifene PET co-registered to mouse MR template non-carrier mice; (D–F): [¹⁸F]Nifene PET co-registered to mouse MR template Hualpha-Syn ((A53T) PD mice; (G): Plot of [¹⁸F]nifene SUV of Hualpha-Syn ((A53T) PD mice versus non-carrier mice in different brain regions (n = 4 WT and n = 4 TG). CP = caudate putamen; SC = superior colliculus; IC = inferior colliculus (p < 0.05 for all regions except brain stem, midbrain, and hippocampus, SC, IC, and cerebellum were not significant).

Figure 8

Comparison of [¹⁸F]nifene in post-mortem human PD versus controls. (A) CN brain section, 10 micron thick consisting of grey matter (GM) anterior cingulate (AC) and white matter (WM) corpus callosum (CC); (B) ubiquitin IHC for LB; (C) [¹⁸F]nifene binding in adjacent section; (D) PD brain section, 10 micron thick consisting of grey matter (GM) anterior cingulate (AC) and white matter (WM) corpus callosum (CC); (E) ubiquitin IHC for LB; inset at 20 mm shows LB and measured diameter of LB in inset was 6 to 9 microns; (F) [¹⁸F]nifene binding in adjacent section showing reduced binding compared to CN brain.

Figure 9

(A) Anti-α-synuclein IHC of PD brain slice; (B) magnified view at 50 μm showing presence of Lewy neurites; (C) Lewy bodies stained for α-synuclein in Lewy bodies see at 20 μm; (D) anti-ubiquitin staining showing presence of Lewy bodies in the PD and absent in the CN brains (p < 0.001). Cortical layers IV–VI had significantly greater amounts compared to outer I–III layers. White matter did not reveal presence of Lewy bodies; (E) comparison of [¹⁸F]nifene in post-mortem human PD versus controls showing decreases in PD GM (p < 0.01).