Denis Guilloteau

In vivo exploration of neurodegenerative diseases by positron emission tomography (PET) imaging has matured over the last 20 years, using dedicated radiopharmaceuticals targeting cellular metabolism, neurotransmission, neuroinflammation, or abnormal protein aggregates (beta-amyloid and intracellular microtubule inclusions containing hyperphosphorylated tau). The ability of PET to characterize biological processes at the cellular and molecular levels enables early detection and identification of molecular mechanisms associated with disease progression, by providing accurate, reliable, and longitudinally reproducible quantitative biomarkers. Thus, PET imaging has become a relevant imaging method for monitoring response to therapy, approved as an outcome measure in bioclinical trials. The aim of this paper is to review and discuss the current inputs of PET in the assessment of therapeutic effectiveness in neurodegenerative diseases connected by common pathophysiological mechanisms, including Parkinson’s disease, Huntington’s disease, dementia, amyotrophic lateral sclerosis, multiple sclerosis, and also in psychiatric disorders. We also discuss opportunities for PET imaging to drive more personalized neuroprotective and therapeutic strategies, taking into account individual variability, within the growing framework of precision medicine.

Microglia, as cellular mediators of neuroinflammation, are implicated in the pathogenesis of a wide range of neurodegenerative diseases. Positron emission tomography (PET) imaging of microglia has matured over the last 20 years, through the development of radiopharmaceuticals targeting several molecular biomarkers of microglial activation and, among these, mainly the translocator protein-18 kDa (TSPO). Nevertheless, current limitations of TSPO as a PET microglial biomarker exist, such as low brain density, even in a neurodegenerative setting, expression by other cells than the microglia (astrocytes, peripheral macrophages in the case of blood brain barrier breakdown), genetic polymorphism, inducing a variation for most of TSPO PET radiopharmaceuticals’ binding affinity, or similar expression in activated microglia regardless of its polarization (pro- or anti-inflammatory state), and these limitations narrow its potential interest. We overview alternative molecular targets, for which dedicated radiopharmaceuticals have been proposed, including receptors (purinergic receptors P2X7, cannabinoid receptors, α7 and α4β2 nicotinic acetylcholine receptors, adenosine 2A receptor, folate receptor β) and enzymes (cyclooxygenase, nitric oxide synthase, matrix metalloproteinase, β-glucuronidase, and enzymes of the kynurenine pathway), with a particular focus on their respective contribution for the understanding of microglial involvement in neurodegenerative diseases. We discuss opportunities for these potential molecular targets for PET imaging regarding their selectivity for microglia expression and polarization, in relation to the mechanisms by which microglia actively participate in both toxic and neuroprotective actions in brain diseases, and then take into account current clinicians’ expectations.

In vivo exploration of activated microglia in neurodegenerative diseases is achievable by Positron Emission Tomography (PET) imaging, using dedicated radiopharmaceuticals targeting the translocator protein-18 kDa (TSPO). In this review, we emphasized the major advances made over the last 20 years, thanks to TSPO PET imaging, to define the pathophysiological implication of microglia activation and neuroinflammation in neurodegenerative diseases, including Parkinson’s disease, Huntington’s disease, dementia, amyotrophic lateral sclerosis, multiple sclerosis, and also in psychiatric disorders. The extent and upregulation of TSPO as a molecular biomarker of activated microglia in the human brain is now widely documented in these pathologies, but its significance, and especially its protective or deleterious action regarding the disease’s stage, remains under debate. Thus, we exposed new and plausible suggestions to enhance the contribution of TSPO PET imaging for biomedical research by exploring microglia’s role and interactions with other cells in brain parenchyma. Multiplex approaches, associating TSPO PET radiopharmaceuticals with other biomarkers (PET imaging of cellular metabolism, neurotransmission or abnormal protein aggregates, but also other imaging modalities, and peripheral cytokine levels measurement and/or metabolomics analysis) was considered. Finally, the actual clinical impact of TSPO PET imaging as a routine biomarker of neuroinflammation was put into perspective regarding the current development of diagnostic and therapeutic strategies for neurodegenerative diseases.

Introduction: DOTANOC is a largely used somatostatin analogue in NETs investigations. In NODAGANOC, the DOTA is replaced by NODAGA, a chelator derivative from NOTA which is the ‘gold standard’ for Ga³⁺ and Cu²⁺ chelation because it forms more stable complexes than DOTA.[1] The aim of our study is to evaluate the affinity of NODAGANOC for somatostatin receptors (sst) and to develop a ⁶⁸Ga and ⁶⁴Cu labelled NODAGANOC for a future use in PET imaging. Material and methods: The affinity of NODAGANOC was evaluated (n=3) in AR4-2J rat pancreatic cell line which express mRNA of sst_1,2,3,5.[2] IC₅₀ values for the binding to living AR4-2J cells were determined from competition experiments between NODAGANOC (Concentrations from 1.7×10⁻¹² to 10⁻⁶M) and 15.10⁻⁹M ⁶⁸Ga-DOTANOC. Radiolabelling: ⁶⁸Ga-NODAGANOC was prepared instantly at RT by mixing the fraction of eluate containing >90% (613±54MBq) of the available generator activity with 35 nmol of peptide and 800 μl of 1M ammonium acetate buffer (pH 4.4) without further purification of the final product. ⁶⁴Cu-NODAGANOC was prepared after incubation 15 min at RT of 10 nmol of conjugate with 50 μl of ⁶⁴CuCl₂ (67MBq) in ammonium acetate buffer (0.1 M, pH 8.54)[3]. The radiochemical purities (RCP) were determined by RP-HPLC. The stabilities of the labelled compounds were evaluated by HPLC at RT and in human plasma for 2 h. Results: The competition curve was analyzed with GraphPad Prism Software, the IC50 of NODAGANOC is 1.2±0.5 nM. The labelled products were obtained with high radiochemical purities (>95% for ⁶⁸Ga-NODAGANOC and 100% for ⁶⁴Cu-NODAGANOC) without purification of the final product. In all cases, RCP remains >90% for 2 h. Conclusion: The nanomolar affinity for living cells AR4-2J makes NODAGANOC a potential interesting vector for targeting of pathologies overexpressing the sst. The radiolabelling described methods can be performed without costly equipment and allows to ⁶⁸Ga and ⁶⁴Cu-NODAGANOC with high radiochemical purities without further purification. References [1] E. W. Price and C. Orvig, “Matching chelators to radiometals for radiopharmaceuticals,” Chem. Soc. Rev., vol. 43, no. 1, pp. 260–290, Dec. 2013. [2] E. Hofsli, L. Thommesen, K. Nørsett, S. Falkmer, U. Syversen, A. Sandvik, and A. Laegreid, “Expression of chromogranin A and somatostatin receptors in pancreatic AR42J cells,” Mol. Cell. Endocrinol., vol. 194, no. 1–2, pp. 165–173, Aug. 2002. [3] J. Oxboel, M. Brandt-Larsen, C. Schjoeth-Eskesen, R. Myschetzky, H. H. El-Ali, J. Madsen, and A. Kjaer, “Comparison of two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2 and 64Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in human xenograft tumors,” Nucl. Med. Biol., vol. 41, no. 3, pp. 259–267, Mar. 2014.

Introduction: Subjective cognitive decline (SCD) could indicate preclinical Alzheimer's disease, but the existing literature is confounded by heterogeneous approaches to studying SCD. We assessed the differential cognitive, affective, and neuroimaging correlates of two aspects of SCD: reporting high cognitive difficulties on a self‐rated questionnaire versus consulting at a memory clinic.Methods: We compared 28 patients from a memory clinic with isolated SCD, 35 community‐recruited elders with similarly high levels of self‐reported cognitive difficulties, and 35 community‐recruited controls with low self‐reported cognitive difficulties.Results: Increased anxiety and amyloid β deposition were observed in both groups with high self‐reported difficulties, whereas subclinical depression and (hippocampal) atrophy were specifically associated with medical help seeking. Cognitive tests showed no group differences.Discussion: These results further validate the concept of SCD in both community‐ and clinic‐based groups. Yet, recruitment methods influence associated biomarkers and affective symptomatology, highlighting the heterogeneous nature of SCD depending on study characteristics.

Subarachnoid hemorrhage (SAH) can lead to delayed cerebral ischemia, which increases the rate of morbidity and mortality. The detection of microglial activation may serve as a biomarker for the identification of patients at risk of this deleterious consequence. We assessed this hypothesis in a rat model of SAH in which the exploration of neuroinflammation related to microglial activation was correlated with the degree of bleeding. We used the rat filament model and evaluated (at 48 hours postsurgery) the intensity of neuroinflammation using positron emission tomography (PET) imaging with the 18-kDa translocator protein (TSPO) tracer [¹⁸F]DPA-714, quantitative autoradiography with [³H]PK-11195, and SAH grade by postmortem brain picture. High SAH grades were strongly and positively correlated with in vivo PET imaging of TSPO in the cortex and striatum. In addition, a positive correlation was found in the cortex in TSPO, with densities determined by imaging and autoradiographic approaches. Qualitative immunofluorescence studies indicated that overexpression of TSPO was linked to astrocytic/microglial activation. In this model, PET imaging of TSPO using [¹⁸F]DPA-714 appeared to be a relevant index of the degree of bleeding, indicating that this imaging method could be used in human patients to improve the management of patients with SAH.

In quantitative PET measurements, the analysis of radiometabolites in plasma is essential for determining the exact arterial input function. Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT) and it is therefore important to investigate their radiometabolism. We have chosen to explore the radiometabolic profile of [¹¹C]MADAM, one of these radioligands widely used for in vivo PET-SERT studies. The metabolism of [¹¹C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the radiometabolic rate of the radioligand [¹¹C]MADAM in vitro and in vivo was examined by radio-HPLC. RLM and HLM incubations were carried out at two different carrier concentrations of 1 and 10 μM. Urine samples after perfusion of [¹¹C]MADAM/MADAM in rats were also analysed by radio-HPLC. Analysis by UHPLC/Q-ToF-MS identified the metabolites produced in vitro to be results of N-demethylation, S-oxidation and benzylic hydroxylation. The presence of carrier greatly affected the radiometabolism rate of [¹¹C]MADAM in both RLM/HLM experiments and in vivo rat studies. The good concordance between the results predicted by RLM and HLM experiments and the in vivo data obtained in rat studies indicate that the kinetics of the radiometabolism of the radioligand [¹¹C]MADAM is dose-dependent. This issue needs to be addressed when the diarylsulfide class of compounds are used in PET quantifications of SERT.