Kristina Fischer

A point mutation in the dynein heavy chain gene leads to striatal atrophy and compromises neurite outgrowth of striatal neurons

The molecular motor dynein and its associated regulatory subunit dynactin have been implicated in several neurodegenerative conditions of the basal ganglia, such as Huntingtons disease (HD) and Perry syndrome, an atypical Parkinson-like disease. This pathogenic role has been largely postulated from the existence of mutations in the dynactin subunit p150(Glued). However, dynactin is also able to act independently of dynein, and there is currently no direct evidence linking dynein to basal ganglia degeneration. To provide such evidence, we used here a mouse strain carrying a point mutation in the dynein heavy chain gene that impairs retrograde axonal transport. These mice exhibited motor and behavioural abnormalities including hindlimb clasping, early muscle weakness, incoordination and hyperactivity. In vivo brain imaging using magnetic resonance imaging showed striatal atrophy and lateral ventricle enlargement. In the striatum, altered dopamine signalling, decreased dopamine D1 and D2 receptor binding in positron emission tomography SCAN and prominent astrocytosis were observed, although there was no neuronal loss either in the striatum or substantia nigra. In vitro, dynein mutant striatal neurons displayed strongly impaired neuritic morphology. Altogether, these findings provide a direct genetic evidence for the requirement of dynein for the morphology and function of striatal neurons. Our study supports a role for dynein dysfunction in the pathogenesis of neurodegenerative disorders of the basal ganglia, such as Perry syndrome and HD.

Transgenic overexpression of the alpha-synuclein interacting protein synphilin-1 leads to behavioral and neuropathological alterations in mice

Synphilin-1 has been identified as an interacting protein of alpha-synuclein, Parkin, and LRRK2, proteins which are mutated in familial forms of Parkinson’s disease (PD). Subsequently, synphilin-1 has also been shown to be an intrinsic component of Lewy bodies in sporadic PD. In order to elucidate the role of synphilin-1 in the pathogenesis of PD, we generated transgenic mice overexpressing wild-type and mutant (R621C) synphilin-1 driven by a mouse prion protein promoter. Transgenic expression of both wild-type and the R621C variant synphilin-1 resulted in increased dopamine levels of the nigrostriatal system in 3-month-old mice. Furthermore, we found pathological ubiquitin-positive inclusions in cerebellar sections and dark-cell degeneration of Purkinje cells. Both transgenic mouse lines showed significant reduction of motor skill learning and motor performance. These findings suggest a pathological role of overexpressed synphilin-1 in vivo and will help to further elucidate the mechanisms of protein aggregation and neuronal cell death.

In vivo quantification of dopamine transporters in mice with unilateral 6-OHDA lesions using [11C]methylphenidate and PET.

UNLABELLED Quantification of the binding of [11C]methylphenidate to the dopamine transporter (DAT) using positron emission tomography (PET) is often used to evaluate the integrity of dopaminergic neurons in the striatal regions of the brain. Over the past decade, many genetically engineered mouse models of human disease have been developed and have become particularly useful for the study of disease onset and progression over time. Quantitative imaging of small structures such as the mouse brain is especially challenging. Thus, the aims of this study were (1) to evaluate the accuracy of quantifying DAT binding using in vivo PET and (2) to examine the impact of different methodologies. METHODS Eight mice were scanned with [11C]methylphenidate under true or transient equilibrium conditions using a bolus and constant infusion protocol or a bolus injection protocol to evaluate the accuracy of the Logan graphical approach for [11C]methylphenidate imaging in mice. Displacement with unlabeled methylphenidate (0.1, 3 and 10 mg/kg) was used to verify specific binding. In a second experiment, 30 mice were lesioned by injection of 6-hydroxydopamine (6-OHDA) at doses of 0, 2 or 4 μg (n=10) into the right striatum to assess the dose-dependent correlation between the PET signal and dopaminergic degeneration. In addition, we performed test-retest experiments and used ex vivo autoradiography (AR) to validate the effect of partial volume on the accuracy of the [11C]methylphenidate PET quantification in the mouse striatum. RESULTS The binding potentials (BPND) calculated from the Logan graphical analysis under transient equilibrium conditions (1.03±0.1) were in excellent agreement with those calculated at true equilibrium (1.07±0.1). Displacement of specific binding with 0.1, 3 and 10mg/kg methylphenidate resulted in 38%, 77% and 81% transporter occupancy in the striatum. Intra-striatal injections of 6-OHDA caused a dose-dependent decrease in the specific binding of [11C]methylphenidate to the DAT in the striatum. The BPND was reduced by 49% and 61% after injection with 2 and 4 μg of 6-OHDA, respectively. The test-retest reproducibility was 6% in the healthy striatum and 27% in the lesioned striatum. In addition, only a small (15%) difference was found between the [11C]methylphenidate DVR-1 values determined by PET and AR on the healthy side, and no differences were observed on the lesioned side. CONCLUSION The present work demonstrates for the first time that [11C]methylphenidate PET is useful for the quantification of striatal dopamine transporters at the dopaminergic nerve terminals in the mouse striatum; therefore, this marker may be used as a biomarker in genetically engineered mouse models of neurodegenerative disorders. However, only changes resulting in greater than 10% differences in BPND values can reliably be detected in vivo.

In Vivo Dopamine Transporter Imaging in a Unilateral 6-Hydroxydopamine Rat Model of Parkinson Disease Using 11C-Methylphenidate PET

Dopamine transporter (DAT) function is altered by many neurodegenerative diseases. For instance, in Parkinson disease DAT density has been shown to decrease in early disease and to play a role in the occurrence of motor complications. DAT is thus an important imaging target with potential therapeutic relevance in humans and animal models of disease. The PET DAT marker 11C-methylphenidate is commonly used to quantify DAT function. Here we investigate the characteristics of the 11C-methylphenidate–derived quantification of DAT in rodents using the 6-hydroxydopamine Parkinson disease rat model. Methods: Seven unilaterally 6-hydroxydopamine–lesioned rats (dopaminergic denervation [DD] range, 36%–94%) were injected with 3.7 MBq/100 g of body weight and tracer masses ranging from 93.8 to 0.0041 μg/100 g of body weight. We evaluated the maximum available transporter density and the in vivo (apparent) ligand-transporter dissociation constant (Bmax and Kdapp, respectively) with an in vivo Scatchard method using several modeling approaches and estimated the transporter occupancy as a function of the amount of tracer injected and tracer specific activity (SA). Results: Strong evidence of different nonspecific binding in the striatal region, compared with the reference region, leading to bias in the estimate of DD severity was found. One percent transporter occupancy was reached with 0.14 μg of tracer/100 g of body weight, corresponding to an SA of 5.7 kBq/pmol for the given radioactivity dose, and 10% occupancy was reached at 1.5 μg of tracer/100 g of body weight, corresponding to an SA of 0.57 kBq/pmol. The 6-hydroxydopamine lesion affected Bmax (control, 402 ± 94 pmol/mL; lesioned, 117 ± 120 pmol/mL; P = 0.003) but not Kdapp (control, 331 ± 63 pmol/mL; lesioned, 362 ± 119 pmol/mL; P = 0.63). Conclusion: Although DAT imaging can be performed at a relatively high mass of 11C-methylphenidate (low SA), the additional nonspecific binding found in the striatum can introduce a DD severity–dependent bias in the estimate of tissue-derived binding potential and care must be taken in comparing 11C-methylphenidate–derived assessment of DD with that obtained using other dopaminergic tracers.

Imaging DA release in a rat model of L-DOPA-induced dyskinesias: A longitudinal in vivo PET investigation of the antidyskinetic effect of MDMA

In the context of Parkinsons disease, motor symptoms result from the degeneration of nigrostriatal neurons. Dopamine (DA) replacement using l-3,4-dihydroxyphenylalanine (L-DOPA) has been the treatment of choice in the early stages of the disease. However, with disease progression, patients suffer from motor complications, which have been suggested to arise from DA released from serotonergic terminals according to the false neurotransmitter hypothesis. The synthetic amphetamine derivative (±) 3,4-methylenedioxymethamphetamine (MDMA) has been shown to significantly inhibit dyskinesia in humans and in animal models of PD. In this study, we examined the effect of MDMA on L-DOPA-induced DA release by using [(11)C]raclopride kinetic modeling to assess alterations in DA neurotransmission in a rat model of L-DOPA-induced dyskinesia (LID) in a longitudinal in vivo PET study. Rats were submitted to 6-OHDA lesions, and the lesions were confirmed to be sufficiently severe based on the performance during stepping tests and [(11)C]methylphenidate PET scans. The rats underwent two [(11)C]raclopride PET sessions before (baseline) and after two weeks of chronic L-DOPA treatment (priming). L-DOPA priming led to strong abnormal involuntary movements (AIMs). In group 1, L-DOPA priming reduced L-DOPA-induced DA release in the lesioned striatum with no effect on the healthy side, while the concomitant administration of L-DOPA and MDMA (group 2) increased the DA levels in the lesioned and healthy striatum. In addition, behavioral analysis, which was performed two weeks after the second PET session, confirmed the antidyskinetic effect of MDMA. Our data show that L-DOPA-induced DA release is attenuated in the Parkinsonian striatum after chronic L-DOPA pretreatment and that the antidyskinetic mechanism of MDMA does not depend primarily on dopaminergic neurotransmission.

Simulation-based optimisation of the PET data processing for Partial Saturation Approach protocols

Positron emission tomography (PET) with [(11)C]Raclopride is an important tool for studying dopamine D2 receptor expression in vivo. [(11)C]Raclopride PET binding experiments conducted using the Partial Saturation Approach (PSA) allow the estimation of receptor density (B(avail)) and the in vivo affinity appK(D). The PSA is a simple, single injection, single scan experimental protocol that does not require blood sampling, making it ideal for use in longitudinal studies. In this work, we generated a complete Monte Carlo simulated PET study involving two groups of scans, in between which a biological phenomenon was inferred (a 30% decrease of B(avail)), and used it in order to design an optimal data processing chain for the parameter estimation from PSA data. The impact of spatial smoothing, noise removal and image resolution recovery technique on the statistical detection was investigated in depth. We found that image resolution recovery using iterative deconvolution of the image with the system point spread function associated with temporal data denoising greatly improves the accuracy and the statistical reliability of detecting the imposed phenomenon. Before optimisation, the inferred B(avail) variation between the two groups was underestimated by 42% and detected in 66% of cases, while a false decrease of appK(D) by 13% was detected in more than 11% of cases. After optimisation, the calculated B(avail) variation was underestimated by only 3.7% and detected in 89% of cases, while a false slight increase of appK(D) by 3.7% was detected in only 2% of cases. We found during this investigation that it was essential to adjust a factor that accounts for difference in magnitude between the non-displaceable ligand concentrations measured in the target and in the reference regions, for different data processing pathways as this ratio was affected by different image resolutions.

In Vivo Evaluation of [11C]DASB for Quantitative SERT Imaging in Rats and Mice

Serotonin, or 5-hydroxytryptamine (5-HT), plays a key role in the central nervous system and is involved in many essential neurologic processes such as mood, social behavior, and sleep. The serotonin transporter ligand 11C-3-amino-4(2-dimethylaminomethyl-phenylsufanyl)-benzonitrile (11C-DASB) has been used to determine nondisplaceable binding potential (BPND), which is defined as the quotient of the available receptor density (Bavail) and the apparent equilibrium dissociation rate constant (1/appKD) under in vivo conditions. Because of the increasing number of animal models of human diseases, there is a pressing need to evaluate the applicability of 11C-DASB to rats and mice. Here, we assessed the feasibility of using 11C-DASB for quantification of serotonin transporter (SERT) density and affinity in vivo in rats and mice. Methods: Rats and mice underwent 4 PET scans with increasing doses of the unlabeled ligand to calculate Bavail and appKD using the multiple-ligand concentration transporter assay. An additional PET scan was performed to calculate test–retest reproducibility and reliability. BPND was calculated using the simplified reference tissue model, and the results for different reference regions were compared. Results: Displaceable binding of 11C-DASB was found in all brain regions of both rats and mice, with the highest binding being in the thalamus and the lowest in the cerebellum. In rats, displaceable binding was largely reduced in the cerebellar cortex, which in mice was spatially indistinguishable from cerebellar white matter. Use of the cerebellum with fully saturated binding sites as the reference region did not lead to reliable results. Test–retest reproducibility in the thalamus was more than 90% in both mice and rats. In rats, Bavail, appKD, and ED50 were 3.9 ± 0.4 pmol/mL, 2.2 ± 0.4 nM, and 12.0 ± 2.6 nmol/kg, respectively, whereas analysis of the mouse measurements resulted in inaccurate fits due to the high injected tracer mass. Conclusion: Our data showed that in rats, 11C-DASB can be used to quantify SERT density with good reproducibility. BPND agreed with the distribution of SERT in the rat brain. It remains difficult to estimate quantitative parameters accurately from mouse measurements because of the high injected tracer mass and underestimation of the binding parameters due to high displaceable binding in the reference region.

A data driven method for estimation of Bavail and appKD using a single injection protocol with [11C]raclopride in the mouse

PURPOSE The partial saturation approach (PSA) is a simple, single injection experimental protocol that will estimate both B(avail) and appK(D) without the use of blood sampling. This makes it ideal for use in longitudinal studies of neurodegenerative diseases in the rodent. The aim of this study was to increase the range and applicability of the PSA by developing a data driven strategy for determining reliable regional estimates of receptor density (B(avail)) and in vivo affinity (1/appK(D)), and validate the strategy using a simulation model. METHODS The data driven method uses a time window guided by the dynamic equilibrium state of the system as opposed to using a static time window. To test the method, simulations of partial saturation experiments were generated and validated against experimental data. The experimental conditions simulated included a range of receptor occupancy levels and three different B(avail) and appK(D) values to mimic diseases states. Also the effect of using a reference region and typical PET noise on the stability and accuracy of the estimates was investigated. RESULTS The investigations showed that the parameter estimates in a simulated healthy mouse, using the data driven method were within 10±30% of the simulated input for the range of occupancy levels simulated. Throughout all experimental conditions simulated, the accuracy and robustness of the estimates using the data driven method were much improved upon the typical method of using a static time window, especially at low receptor occupancy levels. Introducing a reference region caused a bias of approximately 10% over the range of occupancy levels. CONCLUSIONS Based on extensive simulated experimental conditions, it was shown the data driven method provides accurate and precise estimates of B(avail) and appK(D) for a broader range of conditions compared to the original method.

Comparison of different Scatchard approaches for quantitative [11C]raclopride PET imaging in Mice

The increasing use of genetically engineered mice in biomedical research and the latest advances in imaging technologies provide a great potential to study receptor expression and gene function non invasively in mice. [C]raclopride (RAC) is a widely used positron emission tomography (PET) tracer to measure striatal D2 receptor binding. To determine receptor density Bmax and the apparent affinity Kd separately, multiple injections with high and low specific activities (SA) or single injections with low SA have been performed in PET studies of humans, non human primates and rats. We tested the feasibility of three approaches for D2 receptor quantification in mice: the multiple ligand concentration receptor assay (MLCRA), the transient/peak equilibrium approach (PEA) and the partial saturation approach (PSA). Since these methods differ in terms of accuracy and complexity, we aimed to identify the method, which is best suited for quantitative PET analysis in mice. 12 mice underwent a total of 3 scans with decreasing SA. Injected activity was 458±33 MBq/kg and SA ranged from 190 to 1.8 GBq/μmol, corresponding to injectedmasses of 0.02 to 0.5 μg. In sixmice the bolus injection protocol was used and receptor binding parameters were estimated from theequilibriumratio betweenbound (B) to free (F) ligand,whichwas calculated (i) from the tissue input Logangraphical approach, (ii) fromthe ratio method, (iii) at peak equilibrium and (iv) at partial saturation. Receptor occupancy plotted as a function of log10(1/SA). For the PSA an injected mass of 4.5 μg was chosen. In addition, we used the bolus plus constant infusion protocol (BI) to attain true equilibrium between bound and free tracer concentrations, avoiding difficult arterial cannulation in mice. Six mice underwent three 90 min emission scans using a Kbol of 88 min. The tracer concentration was adjusted to 148 MBq/ml. B/F was calculated (i) from the Logan graphical analysis and (ii) from the ratio method. The average D2 receptor density Bmax was 26±4 pmol/ml and the apparent Kd was 13±2 pmol/ml using the bolus plus constant infusion protocol, which was defined as gold standard. If the tracer was injected by bolus, we found lower values for Bmax and Kd when using the Logan graphical analysis (Bmax=15±4; Kd=7±1) and the ratio from last 30 min (Bmax=22±4; Kd=10±2) and higher values when using the PEA (Bmax=46±4; Kd=23±2) and the PSA (Bmax=35±8; Kd=15±6). The receptor occupancy curves showed that an injected tracer mass of 0.07 μg induces approximately 10% receptor occupancy with corresponding SA values of 1400 Ci/mmol. Our data showed that the tracer mass, if higher than 0.07 μg can highly effect binding parameter estimations and has to be taken into account when performing kinetic analysis, specifically in mice. We also demonstrated that in vivo determination of D2 receptor density and affinity using multiple injection protocols and single injection protocols is feasible in mice. However slight overand underestimations were found when comparing the different analysis methods to the bolus plus constant infusion protocol.