Imaging dopamine function and microglia in asymptomatic LRRK2 mutation carriers

Neuroinflammation (microglial activation) and subclinical nigrostriatal dysfunction have been reported in subjects at risk of Parkinsonism. Eight non-manifesting carriers (NMCs) of LRRK2 G2019S mutation had 11C-PK11195 and 18F-DOPA PET to assess microglial activation and striatal dopamine system integrity, respectively. Comparisons were made with healthy controls. Five LRRK2-NMCs had subclinical reductions of putaminal 18F-DOPA uptake. Three of them had significantly raised nigral 11C-PK11195 binding bilaterally. These findings indicate that nigrostriatal dysfunction and neuroinflammation occur in LRRK2-NMCs. Studies in larger cohorts with appropriate follow-up are needed to elucidate the significance of neuroinflammation in the premotor phase of LRRK2-PD.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are known to cause inherited Parkinson's disease (PD). LRRK2-associated PD (LRRK2-PD) presents with a low penetrant autosomal-dominant inheritance pattern with a cumulative risk of developing PD ranging from 26 to 80% at the age of 80 years [1,2]. Patients with the G2019S mutation, the most common mutation in LRRK2-PD, are clinically indistinguishable from idiopathic PD and most cases present similar pathological findings with degeneration of dopaminergic neurons in the substantia nigra and occurrence of Lewy-type α-synuclein pathology [3]. Studying non-manifesting carriers (NMCs) of the LRRK2 G2019S mutation provides an opportunity to identify pathophysiological processes occurring in the premotor phase of genetic PD. This is essential to identify biomarkers and therapeutic targets to halt disease progression.
Microglia, the major resident immune cells of the central nervous system, monitors the brain milieu in their resting physiologic state, but when activated by injury, they can express both neuroprotective and cytotoxic phenotypes. It is hypothesized that a pro-inflammatory phenotype, leading to neuronal dysfunction, may drive neurodegeneration [4]. Microglial activation has been observed in idiopathic PD [5], but its role in the pathophysiology of genetic PD is unknown. A recent study using positron emission tomography (PET) imaging demonstrated increased microglial activation in the substantia nigra along with putaminal dopaminergic dysfunction in patients with idiopathic rapid eye movement sleep behavior disorder [6], a condition that may progress over time to a neurodegenerative alpha-synucleinopathy [7].
Morten Gersel Stokholm and Alicia Garrido contributed equally to the manuscript.

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The present study aimed to investigate whether raised levels of microglial activation and alterations of the nigrostriatal dopaminergic function occur as well in LRRK2-NMCs.

Methods
Eight LRRK2-NMCs were recruited from Hospital Clínic de Barcelona between September 2016 and May 2018. Inclusion of LRRK2-NMC was based on the absence of Parkinsonism and availability to perform the study. All NMC had a 60.5-min 11 C-(R)-PK11195 PET scan (ECAT HRRT; CTI/ Siemens, Knoxville, TN, USA) to assess levels of microglial activation (expressing 18-kDa translocator protein) and a 94.5-min 18 F-DOPA PET scan to assess the integrity of the dopaminergic system. Each subject had a T1-weighted MRI (3 T MAGNETOM Skyra, Siemens Healthcare, Germany) performed for co-registration of PET images. PET findings were compared with those of 29 healthy controls ( 11 C-PK11195 PET n = 20, 18 F-DOPA PET n = 9). Control PET scans were acquired for two former projects using the exact same scanner and scan protocols [6,8]. All participants were examined to exclude Parkinsonism. Assessments with the MDS-UPDRS part III, and Mini-Mental State Examination were also performed. All the assessments of this study were performed at the Department of Nuclear Medicine and PET Centre, Aarhus University Hospital.
Volumes of interest (VOI) sampled included the substantia nigra ( 11 C-PK11195 PET only) plus putamen and caudate nucleus for both PET tracers. Image analysis was performed as previously described [6]. Briefly, parametric images of 11 C-PK11195 binding potentials (BP ND ) were generated using a simplified reference tissue model with an individual tissue non-specific input function extracted with a supervised cluster-analysis approach. Similarity between the 11 C-PK11195 reference input function extracted from LRRK2-NMCs and controls was confirmed with a repeated measurement analysis (χ 2 test, p > 0.05). In those VOI's where 11 C-PK11195 binding is lower than that of the selected reference tissue voxels, this model computes a negative BP ND . 18 F-DOPA influx constant (Ki) images were generated using the Patlak graphical approach with the occipital lobe as the non-specific uptake reference region.
Group differences in 11 C-PK11195 BP nd and 18 F-DOPA Ki were interrogated with unpaired Student's t test (p < 0.05), normal distribution of data was checked with normal probability plots and D'Agostino-Pearson normality test. For descriptive purposes, individually raised regional 11 C-PK11195 binding and reduced regional 18 F-DOPA uptake was defined as a statistically significant deviation from the controls mean value (left and right side averaged) of two or more standard deviations (z score ≥ 2 and z score ≤ − 2). Statistical analysis and graphical presentations were performed in Stata IC 14.2 (StataCorp LP, TX, USA) and GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).

Discussion
This PET imaging study performed in LRRK2-NMCs found reduced putaminal 18 F-DOPA uptake in five out of eight LRRK2-NMCs as previously reported in subjects without manifest PD carrying different subtypes of LRRK2 mutations [9]. Three carriers in our cohort had bilaterally raised substantia nigra 11 C-PK11195 binding, indicating microglial activation, together with reductions in putaminal 18 F-DOPA uptake (unilaterally reduced in two subjects and bilaterally in one). To our knowledge, this is the first report of activated microglia in LRRK2-NMC.
While not in all cases, a reduced putaminal 18 F-DOPA uptake without increased ipsilateral nigral 11 C-PK11195 binding was observed (subject 4 and 5; Fig. 1), suggesting that in LRRK2-NMC, dysfunction of putaminal dopaminergic axonal terminals may antedate microglial activation in nigral cell bodies, presumed to be related with neurodegeneration [4]. A similar pattern (normal range substantia nigra 11 C-PK11195 binding and reduced ipsilateral putaminal 18 F-DOPA uptake) has been reported previously in occasional subjects with prodromal PD [6] and manifest idiopathic PD patients [5]. The significance of microglial activation in the pathophysiology of PD is yet to be established. The findings in the current study, together with those found in a previous one examining prodromal PD [6] support the concept that similar alterations occur in the premotor phase in both genetic and idiopathic forms of the disease. Microglial cells are able to express either a neuroprotective or a cytotoxic phenotype [4]. Which of these is occurring in the examined LRRK2-NMC can only be determined with PET tracers binding specifically to each microglia phenotype; however, this kind of in vivo tracer is currently not available.
There are limitations to this study to be considered. First, the small number of cases examined may preclude the generalization of the results. Second, the LRRK2-NMC included in this study are younger than the controls which may account for a selection bias. Considering the age-related and low and penetrance of LRRK2 G20109S mutation [1,2], it is possible that we have studied subjects who will never develop PD or at a very early phase of the disease when biochemical and pathological events have not yet started [10]. However, the alteration of 18 F-DOPA PET in five LRRK2-NMC indicates that in most of LRRK-NMC in this cohort, pathological changes are already occurring. Given that age is the greatest risk factor for developing LRRK2-PD, we investigated whether PET findings were more likely to occur in the eldest LRRK2-NMC. We found that age did not correlate with PET alterations in the studied cohort, suggesting that other factors may influence 18 F-DOPA dysfunction and microglial activation (individual data including age, not shown to avoid subject identification). Third, as previously    F-DOPA Ki values, which are two or more standard deviations below the average mean of controls (z score ≤ − 2). Red colour indicates significantly raised 11 C-PK11195 BP ND two or more standard deviations above the average mean of controls (z score ≥ 2). Observations from each individual carrier are marked with numbers from one to eight, which corresponds to their number in Table 1, individual analysis reported in animal models [11], levels of microglial activation in PD may be influenced by the presence of pathological α-synuclein inclusions. In LRRK2 G2019S PD, a pleiotropic neuropathology has been reported, including subjects without Lewy-type α-synuclein pathology [3,12]. This pathological variability may possibly influence the heterogeneity of the results. Finally, 11 C-PK11195 may not only bind to the 18-kDa translocator protein located on mitochondria inside the activated microglial cells but also on astrocytes; however, observations show that this only accounts for minor degree of 11 C-PK11195 signal [13]. This is the first study performed in LRRK2-NMCs showing dopamine dysfunction in five out of eight subjects and concomitant nigral microglial activation in three of them. These results suggest that PET imaging with 18 F-DOPA and 11 C-PK11195 could help identify NMCs with ongoing nigrostriatal pathology and show that at least in some cases, neuroinflammation could play a role in the pathophysiology of early phases of LRRK2-PD. The contribution of the observed in vivo microglial response and its clinical relevance at individual level still needs to be elucidated. Prospective studies using similar PET tracers in a larger study population with appropriate clinical follow-up may clarify these issues.
Author contributions Study design: NP, ET, MGS, and AG. Data acquisition: MGS, AG, MS, PP, KS, MJM, and NP. All authors contributed to data analyses and the writing of the manuscript. We confirm that all authors have contributed to this work, and have read and approved the manuscript.
Funding The Independent Research Fund Denmark. Ethical approval The local Ethics Committee at both centres, Aarhus and Barcelona, approved the study and all participants gave written informed consent according to the Declaration of Helsinki before study enrollment.

Conflicts of interest
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