Enhanced catecholamine transporter binding in the locus coeruleus of patients with early Parkinson disease

We retrospectively reviewed clinical and imaging data of 94 subjects with idiopathic PD in whom FP-CIT SPECT was performed at the "Ospedale Maggiore Policlinico" in Milano within five years of the onset of motor symptoms. Fifteen healthy subjects (healthy controls, HC) were prospectively enrolled for comparisons of FP-CIT binding. At the time of SPECT, HC did not suffer from any disease and were not taking any medications. Clinical inclusion criteria for subjects with PD were: (a) diagnosis according to the UK Parkinson Disease Brain Bank criteria; (b) absence of any signs indicative for atypical parkinsonism (e.g. gaze abnormalities, autonomic dysfunction, significant psychiatric disturbances, etc.) over a follow-up period of at least three years after symptoms onset; (c) Hoehn and Yahr (H&Y) stage 1 or 2 in drugs-off state (i.e. after overnight withdrawal of specific drugs for PD; no patients were taking long-acting dopaminergic drugs) at the time of SPECT; (d) positive clinical improvement at Unified Parkinson Disease Rating Scale (UPDRS) after L-Dopa intake (i.e. > 30% from drug-off state) at some point during the three years of follow up; (e) a normal Magnetic Resonance Imaging (MRI) (no sign of white matter lesion or atrophy). Finally, given a putative role of LC and noradrenaline in cognition and mood (including depression) we excluded from this study patients with a positive score at UPDRS part I.

A quantitative profile of each patient' motor impairment was obtained from clinical assessment performed before SPECT by means of the UPDRS motor part (part III). L-Dopa daily dose and L-Dopa Equivalent Daily Doses (LEDDs) were also recorded, with the latter expressed as follows: 100 mg levodopa = 1.5 mg pramipexole = 6 mg ropinirole. None of the subjects (both PD and HC) were taking or stated to have ever been treated with antipsychotics or drugs known to affect the noradrenergic system (e.g., noradrenaline reuptake inhibitors). Drug naïve patients at the time of SPECT were not included in the study. The Ethics Committee of the Department of Human Physiology approved the study and all subjects gave informed consent.

Intravenous administration of 110-140 MBq of FP-CIT (DaTSCAN, GE-Healthcare, UK) was performed 30-40 minutes after thyroid blockade (10-15 mg of Lugol oral solution) in the control subjects and in patients after overnight withdrawal of dopaminergic therapy [15]. Brain SPECT was performed 3 hours later by means of a dedicated triple detector gamma-camera (Prism 3000, Philips, Eindhoven, the Netherlands) equipped with low-energy ultra-high resolution fan beam collimators (4 subsets of acquisitions, matrix size 128x128, radius of rotation 12.9-13.9 cm, continuous rotation, angular sampling: 3 degree, duration: 28 minutes). Brain sections were reconstructed with an iterative algorithm (OSEM, 4 iterations and 15 subsets) and then processed by 3D filtering (Butterworth, 5^th order, cut-off 0.31 pixel^-1) and attenuation correction (Chang method, factor 0.12).

Unless otherwise stated, data are reported as median and range. Normality of data distribution was tested by means of Shapiro-Wilks test. Chi-Square was used to test gender distribution among groups. Demographic data were compared by means of Wilcoxon two-group test. The Spearman correlation coefficient was calculated to investigate statistical dependence among average binding values, demographic and clinical variables.

Statistical analyses were performed with the JMP statistical package, version 8.0 (SAS Institute, Inc., Cary, NC, USA).

The present study provides in vivo evidence of higher baseline catecholamine transporter binding in the LC region in a large and homogeneous cohort of subjects with early PD. Our findings are consistent with an up-regulation of noradrenaline reuptake in the LC area, which is well compatible with enhanced noradrenaline release [19, 20].

Our results are derived from the analysis of the binding of FP-CIT, a ¹²³I-labeled cocaine derivative with high affinity for dopamine (DAT; K_D = 2nM) and a lesser affinity towards noradrenaline transporter (NET; K_D = 140 nM) [14]. Despite the higher affinity of FP-CIT for DATs, it is unlikely that the higher binding observed in the LC area is due to an enhanced dopaminergic, rather than noradrenergic, transporter for two main reasons: (i) in LC, DAT represent a minor and inconsistent component of the midbrain-derived dopaminergic terminals which degenerates in PD, along with other dopaminergic projections, [4] and (ii) in the LC a major NET component is synthesized in the cell body of pigmented neurons and exposed on their membrane to be transferred toward axonal terminals, [20] with a less consistent NET component localized on terminal projections arising from more caudal noradrenergic cell groups [21].

In PD, reduced dopamine release from nigro-striatal projections results in loss and adaptive down-regulation of DAT binding sites in the striatal region [22]. In line with this notion, and in agreement with studies in de novo and early PD, where 40 to 60% of nigral dopaminergic neurons are lost, [15, 23] we found a significantly reduced FP-CIT binding in the caudate and putamen of PD patients. In contrast to the striatal compartment, analysis of FP-CIT labeling in the upper brainstem revealed significantly increased binding in a pontine area adjacent to the floor of the fourth ventricle and extending into the midbrain to the level of the inferior colliculi. This area corresponds topographically to the LC coordinates identified by other studies including those employing neuromelanin-sensitive MRI methods [6, 17, 24, 25]. In addition, the LC is the sole structure in the posterior rostral pons housing monoamine transporters [1], thus further supporting our claim of anatomical targeting of the LC.

Only two prior studies with PET have specifically investigated the LC in PD patients. A first, [24] reported a reduced ¹⁸F-dopa intake in patients with advanced PD when compared to patients at an early stage of the disease. Because ¹⁸F-dopa intake is more specifically related to dopaminergic neurotransmission, this study does not provide information on noradrenergic functioning of LC. In a second study, [6] PD subjects with depression showed a reduced binding of [¹¹C]RTI-32, a marker of both DAT and NET, when compared to non-depressed patients. Interestingly, the noradrenergic activity of early non-depressed PD patients was within normal range in most patients and enhanced in few of them. In line with these findings, and having enrolled a larger and more selected cohort of subjects, we were able to reveal a significantly higher LC activity at an early stage of PD for the first time.

An acute effect of drugs on FP-CIT binding values appears unlikely since SPECT was performed after overnight withdrawal of anti-parkinsonian drugs. In addition, in animal studies, systemic administration of D₂/D₃ receptor agonists, such as pramipexole or apomorphine, showed little or no effect on the firing rate of LC-NA neurons [26]. Finally, a persistent treatment with dopaminergic drugs will eventually down-regulate, rather than up-regulate, the surface expression of DAT and NET through internalization of the transporters [27, 28]. Accordingly, the average FP-CIT binding values in the LC remained enhanced when data were L-Dopa weighted for equivalent daily dose and L-Dopa daily dose.

Enhanced noradrenergic binding, and possibly activity, in PD might be considered at odds with neuropathological findings, where frank neuronal degeneration has been recognized within LC, based on detection of specific cellular markers. Indeed, morphologic hallmarks of sporadic PD (Lewy bodies and dystrophic neurites containing pathologic α-synuclein) may appear initially in the lower brainstem [2].

However, Lewy pathology can correlate poorly with neuronal loss in specific areas, thus its validity in predicting neuronal disintegration is questionable [29]. In fact, noradrenergic neurons in the LC are relatively preserved in early PD and do not exhibit the same intracellular changes as in the substantia nigra [30].

Accordingly, neuromelanin-sensitive imaging methods in vivo, [25] as well as anatomopathological studies suggested that the loss of NA neurons in PD may be confined to the larger, pigmented cells localized in the caudal part of the nucleus, whereas small unpigmented cells are increased in number, as if derived from shrinkage of larger neurons [31].

However, available information on the LC, so far derived from anatomopathological studies in subjects with PD, is poorly comparable with our findings. In particular, the limited number of PD subjects investigated and the lack of clinical information (e.g. disease duration and the presence of depression or cognitive impairment) of patients in anatomopathological studies prevent a direct comparison between these studies and our results [31, 32].

Based on anatomical and histochemical data, along with neuropharmacological evidence, higher activity of the LC in PD may suggest: (i) in the striatum, noradrenaline plays a compensatory role cross targeting dopaminergic receptors (synaptic action); while (ii) in the substantia nigra, noradrenaline has a neuroprotective bolstering dopaminergic cells (extra-synaptic paracrine action).

As for the compensatory role, there is no absolute specificity for catecholaminergic substrate-receptor interactions, implying that one catecholamine can cross-talk with the pharmacologically defined receptors or transporters belonging to other catecholamines. Indeed, noradrenaline binds to pharmacologically defined dopaminergic receptors [11, 33, 34]. Therefore, enhanced noradrenaline release may be able to partially compensate a dopaminergic innervation loss due to degeneration of the substantia nigra.

With reference to a putative neuroprotective activity, noradrenaline suppresses pro-inflammatory and elevates anti-inflammatory molecules [35] and has the ability to scavenge superoxide and reactive oxygen species, which are thought to contribute to cellular damage and dopaminergic cell death [36]. Furthermore, the tottering mouse, which has noradrenergic hyperinnervation and increased levels of noradrenaline throughout the forebrain, appears to be protected from MPTP toxicity [37] while MPTP-induced damage to nigrostriatal dopaminergic neurons was potentiated by pretreatment with DSP-4, a selective LC neurotoxin [38]. Therefore, we speculate that enhanced LC-NA may be regarded as an endogenous paracrine agent promoting dopaminergic neuron survival [39, 40]. This hypothesis would predict that degeneration of LC noradrenergic neurons in later stages of the disease might accelerate degeneration of substantia nigra dopaminergic neurons. The negative correlation between FP-CIT binding in the striatum and LC area is consistent with the above considerations of LC-NA compensatory and protective activity.