Crafting a PET tracer to detect α-synuclein deposits in the brain has been an uphill struggle. Now, scientists led by Hironobu Endo and Makoto Higuchi at the National Institutes for Quantum Science and Technology in Chiba, Japan, describe a new ligand, C05-05, that is a step in the right direction. In the June 5 Neuron, they report that, in brain tissue from mice, marmosets, and people, the molecule bound α-synuclein fibrils. On PET, C05-05 detected aggregates in multiple system atrophy, dementia with Lewy bodies, and Parkinson’s disease. Tracer brightness in the midbrain correlated with motor symptom severity. Still, C05-05 also bound to neurofibrillary tangles and amyloid plaques, albeit more weakly than to α-synuclein aggregates. The authors are trying to tweak the ligand to make it more selective.

  • A new PET ligand, C05-05, binds α-synuclein fibrils.
  • It lit up the midbrain in PD and DLB; the putamen and cerebellum in MSA.
  • High midbrain C05-05 uptake correlated with worse motor symptoms.

“That C05-05 images α-synuclein in PD demonstrates the target density is high enough to see with PET, a question that has been raised for years,” Robert Mach of the University of Pennsylvania in Philadelphia, told Alzforum.

Francesca Capotosti of AC Immune compared C05-05 to other synuclein tracers for MSA, a disease with dense α-synuclein aggregates. “It's nice to see molecules coming from different chemical structures have a common pattern in what they show on PET,” she said. “This gives confidence to the field that what is imaged corresponds to pathological α-synuclein.”

PD, DLB, and MSA all start with motor symptoms, making them difficult to distinguish, especially at early stages. They can only be definitively diagnosed postmortem, and while detecting α-synuclein aggregates in cerebrospinal fluid and serum is possible, it is not quite up to snuff for diagnostic use (Apr 2023 conference news; Aug 2023 conference news). Measuring the spread of synuclein through a person’s brain using PET imaging could aid in disease staging and diagnosis.

Better Chemistry? Scientists synthesized C05-05 by changing the linker (red) and modifying the benzothiazole ring (left) in PBB3. [Courtesy of Endo et al., Neuron, 2024.]

Scientists have developed α-synuclein PET tracers, but they have limitations (Aug 2023 conference news). One, called F0502B, poorly slipped into the brains of rhesus macaques, while Merck's MK-7337 had off-target binding in monkey cerebella. AC Immune’s ACI-12589 detects α-synuclein aggregates in MSA, but not those in PD, which are much sparser. Capotosti says the company plans to test a more sensitive derivative, dubbed ACI-15916, in a first-in-human trial in PD beginning early next year.

Aiming to develop a better ligand, co-first authors Endo and Maiko Ono modified the tau PET tracer PBB3 because it also bound the β sheets of α-synuclein inclusions (Koga et al., 2017; Perez-Soriano et al., 2017). The scientists swapped a double bond for a triple bond in a hydrocarbon chain linking two aromatic rings in the tracer, then added various functional groups to the rings to create the C05 series of compounds (image above). One, called C05-05, stood out because it tightly bound Lewy bodies and neurites in amygdala tissue from DLB cases. However, it also bound neurofibrillary tangles and amyloid plaques in middle frontal gyrus tissue from a person who had had AD (image below).

Not There Yet. Fluorescence microscopy showed C05-05 bound Lewy bodies and neurites (top left) in DLB brain tissue better than did PBB3 (bottom left), but it also tagged neurofibrillary tangles (arrowheads) and amyloid plaques (arrows) in AD tissue (right). [Courtesy of Endo et al., Neuron, 2024.]

To see how the tracer behaved in vivo, Endo and Ono first used wild-type mice that had been injected with mouse α-synuclein fibrils to seed aggregates throughout the brain. Six months later, the scientists injected an 18F-C05-05 into a vein, then scanned the brains using micro-PET. The highest tracer uptake occurred in the striatum, cortex, and amygdala, areas with abundant synuclein aggregates seen via immunofluorescence (image below). The cerebellum, which had no synuclein inclusions, bound little tracer. The tracer bound off-target in the corpus callosum and the hippocampus, in both α-synuclein and wild-type mice.

Mouse PET. In PET scans (left), 18F-C05-05 was retained in the striata (top) and neocortices (middle) of α-synuclein-seeded mice (left) but not of wild-type mice (right). The cerebellum (bottom) bound no tracer. Autoradiographs and immunofluorescence (right) showed the tracers accumulated in the same areas (arrows) as phosphorylated α-synuclein. The numerals 1 and 2 indicate striatum and corpus callosum, respectively. [Courtesy of Endo et al., Neuron, 2024.]

The scientists next tested the tracer in a nonhuman primate model; that is, marmosets that had α-synuclein fibrils seeded in the brain. 18F-C05-05 accumulated in the caudate nucleus, putamen, and substantia nigra, all places with dense α-synuclein aggregates detected by immunohistochemistry.

What about people? First, the scientists used autoradiography to test tracer binding in amygdala from a DLB donor and in basal ganglia tissue from people who had had MSA or PD. 18F-C05-05 bound all slices and tracked with the level of α-synuclein aggregates, meaning no labeling in cases of mild pathology and intense binding with severe pathology.

Endo and colleagues then tested the tracer in 10 people with PD or DLB and eight controls. The amount of tracer in the brain peaked within minutes after intravenous injection, then fell by half in most areas by 50 minutes. However, in people with PD or DLB, radioactivity remained high in the midbrain, home of the substantia nigra, after two hours (image below). Higher midbrain binding correlated with worse motor impairment, as measured by the revised Unified PD Rating Scale. Capotosti called this correlation remarkable, given such small sample sizes.

18F-C05-05 also lit up the putamen and middle cerebellar peduncle, which connects the cerebellum to the pons, in three people with MSA (image below). These areas are known hotspots for glial cytoplasmic inclusions, the characteristic oligodendrocyte α-synuclein pathology found in MSA (reviewed by Castellani, 1998). The data show that the ligand reacts with α-synuclein aggregates in all three synucleinopathies. “Our work has offered a benchmark for the development of α-synuclein PET tracers by showing that a radiotracer with a dissociation constant of about 1 nM can visualize target pathologies in PD and DLB at moderate and advanced stages,” Higuchi told Alzforum.

Illuminating Synuclein. While 18F-C05-05 bound the choroid plexus (yellow arrows) in healthy controls (top left), it only lit up the midbrain in people with PD or DLB (red arrows, bottom left). The tracer also bound the putamen (red arrows, top right) and middle cerebellar peduncle (red arrows, bottom right) in people with MSA. [Courtesy of Endo et al., Neuron, 2024.]

Other scientists said C05-05 needs more work before it would be suitable for the clinic. Behrooz Yousefi at Philipps University of Marburg, Germany, noted that slow brain uptake and rapid metabolism, at least in mice, make the tracer less than ideal, while Oskar Hansson, Lund University, Sweden, feels the specificity needs to be higher. “I think the current tracer is a great step forward, but the overlap between [tracer uptake in] controls and patients with PD is far too great for it to be a reliable diagnostic marker,” he wrote. 

Capotosti was a bit disappointed by C05-05’s affinity for tangles and plaques, given that more than half of people with PD have these co-pathologies (Irwin et al., 2017). “The lack of selectivity will represent a major liability in further validating the tracer as a diagnostic marker,” she said.

Mach noted that 18F-C05-05 binds α-synuclein near tyrosine 39, a hotspot for phosphorylation or nitration, which he predicted might hinder tracer binding in vivo, but it seems this is not the case for this compound. “We changed our strategy for developing α-synuclein PET ligands to focus on ones that bind near tyrosine 39,” he told Alzforum.

Higuchi and colleagues are making derivatives of C05-05, aiming to find molecules more selective for α-synuclein inclusions.

As for what’s to come for the field, about half a dozen α-synuclein PET tracers in clinical trials are set to read out next year. These include F0502B, Merck’s MK-7337, and AC Immune’s ACI-12589, as well as two tracers, HY215 and M503, that Mach is developing (Kim et al., 2023). “Twenty-twenty-five will be a big year for α-synuclein imaging,” he predicted. —Chelsea Weidman Burke

Comments

  1. I applaud researchers who are developing new potential PET tracers for α-synuclein diseases, because the need is imperative, especially for use in studies determining target engagement of new drugs targeting misfolded α-synuclein. However, it is a challenging task, because the density of misfolded α-synuclein is much lower in these disorders than the densities of misfolded Aβ and tau observed in Alzheimer’s disease. I think the current tracer is a great step forward, but as shown in figure 6, the overlap between controls and patients with PD is far too great for the tracer to be a reliable diagnostic marker. We also need studies imaging patients with other neurodegenerative diseases, such as Alzheimer’s disease and progressive supranuclear palsy, to study how specific the tracer is in vivo in humans.

  2. The in vivo detection of α-synucleinopathies represents a significant unmet need, and it is a highly challenging area of research. Several groups have been developing small molecules that could be utilized as positron emission tomography (PET) tracers for imaging α-synuclein deposits. 18F-C05-05 appears to bind to α-synuclein inclusions in mouse models and in postmortem brains of α-synucleinopathies. The experiments demonstrated the ability of the radiotracer to detect α-synuclein deposits in the midbrains of patients. Fluorescence signal intensities in Lewy bodies and neurites were slightly higher than those in amyloid plaques and neurofibrillary tangles. However, the radioligand exhibited greater affinity for α-synuclein than for Aβ and tau aggregates, with an IC50 of 1.5 versus 12.9 nM (an 8.6-fold difference). Additionally, the authors reported that the radioligand was insensitive to monoamine oxidases.

    This report is a great example of a successful interdisciplinary translational research project that tackles some important challenges in developing α-synuclein tracer. The results show that 18F-C05-05 outperforms other tracers, such as AC Immune's ACI-12589, Ye's F0502B, and Merck's 7337. However, despite these promising findings, there are still some concerns with the tracer's binding affinity, selectivity, lipophilicity, and in vivo stability.

    One notable issue is its tendency to undergo photoisomerization. To prevent this, experiments require the use of UV-cut light. Unfortunately, this limits its usefulness for broader applications in human testing. Another drawback is that when mice were analyzed, it was found that the tracer is rapidly metabolized, with 70 percent of the radiometabolites found in plasma and only 30 percent in the brain within 90 minutes after injection. This fast metabolism makes the tracer less than ideal. Additionally, the tracer shows slow brain uptake kinetics and binds nonspecifically, which are both negative traits for an α-synuclein tracer.

    As a result, finding a suitable α-synuclein tracer, especially for use in Parkinson's disease, remains an ongoing challenge.

  3. After reading this manuscript, I feel that C05-05 indeed performs better in binding to α-syn fibrils than PBB3 and PF-PBBs that were previously developed by the same group. When α-syn fibril concentrations are high enough, this probe indeed visualizes the aggregates in fibril-injected mouse or monkey brain. Nonetheless, C05-05 binding affinity Kd for AD tissue is around 12.9 nM versus 1.5 nM for DLB, indicating the selectivity is not yet good enough for the specific recognition of Lewy bodies, given that the amyloid concentration is approximately 10-fold higher than α-synuclein deposits in patient brains. It will be necessary to explore this probe specificity in tau P301S or 3XTg AD mice and in AD patients to investigate whether it cross-reacts with tau aggregates. The detection of α-synuclein aggregates in the midbrains of PD and DLB patients by PET with F18-C05-05 is encouraging. It is imperative to explore whether F18-C05-05 illuminates amyloid or tau aggregates in AD patients, though FM-PBB3 does not visualize Lewy body in DLB patients.

    The chemical backbone of C05-05 is very similar to our previously reported F0502B. The main difference in the structure is the phenol in F0502B versus methylamino-pyridine in C05-05. We have demonstrated that the hydroxyl phenyl group is critical for the probe to selectively bind to α-synuclein fibrils, which is also validated by our cryo-EM structures. I suspect the amino-pyridine in C05-05 might also cross-react with amyloid or tau fibrils, because this group also shows up in various amyloid and tau probes reported previously. Conceivably, this group is accountable for the close binding affinities of C05-05 for DLB and AD aggregates. 

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References

News Citations

  1. Synuclein Assay Passes the Sniff Test—What of Other Seeds?
  2. Finally, a Diagnostic Marker for Lewy Body Disease?
  3. Spying on α-Synuclein Inclusions: PET Tracers Inch Closer to Success

Paper Citations

  1. . Fluorescence and autoradiographic evaluation of tau PET ligand PBB3 to α-synuclein pathology. Mov Disord. 2017 Jun;32(6):884-892. Epub 2017 Apr 25 PubMed.
  2. . PBB3 imaging in Parkinsonian disorders: Evidence for binding to tau and other proteins. Mov Disord. 2017 Jul;32(7):1016-1024. Epub 2017 Jun 1 PubMed.
  3. . Multiple system atrophy: clues from inclusions. Am J Pathol. 1998 Sep;153(3):671-6. PubMed.
  4. . Neuropathological and genetic correlates of survival and dementia onset in synucleinopathies: a retrospective analysis. Lancet Neurol. 2017 Jan;16(1):55-65. PubMed.
  5. . A Novel Brain PET Radiotracer for Imaging Alpha Synuclein Fibrils in Multiple System Atrophy. J Med Chem. 2023 Sep 14;66(17):12185-12202. Epub 2023 Aug 31 PubMed.

Further Reading

No Available Further Reading

Primary Papers

  1. . Imaging α-synuclein pathologies in animal models and patients with Parkinson's and related diseases. Neuron. 2024 May 27; PubMed.