Imaging alpha-synuclein pathology in Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. The clinical manifestations of PD include motor symptoms, such as bradykinesia, resting tremor, rigidity, and nonmotor symptoms, which include disturbances in sleep, gastrointestinal function, and olfaction. PD misdiagnosis rates have been reported to reach approximately 30%, partly owing to the heterogeneity of parkinsonism with non-PD pathologies, and the differential diagnosis of PD from neurodegenerative diseases such as multiple systemic atrophy (MSA) and progressive supranuclear palsy poses another unmet need. These nonmotor symptoms may emerge more than a decade prior to the onset of motor impairments. Pathologically, PD is characterized by the accumulation of Lewy bodies (LBs), which are composed of misfolded alpha-synuclein, and the early loss of dopaminergic neurons. Recent studies have introduced two biomarker-based systems for PD research: the SyNeurGe system and the neuronal alpha-synuclein disease integrated staging system (Höglinger et al., 2024; Simuni et al., 2024). Misfolded alpha-synuclein has been shown to spread between cells, serving as a template for further alpha-synuclein misfolding. Although the precise etiological role of alpha-synuclein in PD has not been fully elucidated, a recent study has revealed heterogeneity in the trajectories of alpha-synuclein pathology in PD and the toxicity of alpha-synuclein especially soluble aggregates (Mastenbroek et al., 2024). Currently, there are several clinical trials targeting alpha-synuclein as disease modifying treatments of PD, either by using active or passive immunotherapy, by preventing alpha-synuclein aggregation, or by disaggregation of existing complexes.

Recent advancements in alpha-synuclein seed amplification assays in cerebrospinal fluid and blood, as well as immunohistochemical techniques for skin biopsy, present promising new avenues for the diagnosis of PD. These methods have demonstrated significant potential in differentiating between PD patients and healthy controls (Okuzumi et al., 2023). Additionally, fluid biomarkers have been employed to assess target engagement in a recent clinical trial of an alpha-synuclein active immunotherapy in PD patients (Eijsvogel et al., 2024). Furthermore, other fluid biomarkers, including cell-free DNA, cell-free RNA, circulating microRNAs, and the blood proteome, hold significant promise for precision medicine. Further methodological development and validation of the novel fluid biomarkers in a large cohort of patients are critical.

Positron emission tomography (PET) is a noninvasive imaging technique that enables visualization and quantification of pathophysiological processes using radioligands. Alpha-synuclein PET imaging allows for early and accurate diagnosis and longitudinal monitoring of LB pathology progression and in vivo staging. Understanding the location and presence of alpha-synuclein will increase our understanding of PD onset and the diversity of these diseases, potentially aiding in more accurate patient diagnosis and classification. Additionally, such PET tracers will enable target engagement evaluation and patient selection and provide surrogate markers in addition to clinical outcomes in clinical trials targeting alpha-synuclein. Currently, several alpha-synuclein PET imaging biomarkers are under development/clinical trials and are positive only in MSA patients but not in PD patients. Targeting alpha-synuclein pathology in PD patients is challenging owing to its primarily intracellular distribution and the significantly lower levels of alpha-synuclein aggregates in the PD brain than in the MSA brain and those of amyloid-β and tau aggregates in the Alzheimer’s disease and primary tauopathy brain. A few alpha-synuclein PET tracers have been developed and evaluated in rodent and nonhuman primate models of PD, such as [¹¹C]MK-7337 (chemical structure not known), benzamide derivatives, diphenylpyrazole derivatives e.g., [¹¹C]MODAG-001, [¹¹C]MODAG-005, benzothiazole-ethenyl-phenol derivative e.g., [¹⁸F]F0502B, 2-styrylbenzothiazole derivatives, e.g., [¹⁸F]MFSB, indolinone/indolinone derivatives, phenothiazine derivatives, e.g., [¹¹C]SIL5, arylpyrazolethiazole derivatives, e.g., [¹¹C]APT-13, chalcone derivatives, disarylbisthiazole compounds, e.g., [¹⁸F]FS3-1, [¹⁸F]FS9, etc., as well as antibody-based tracers. With recent advancements in structure-based techniques, such as cryo-electron microscopy and solid-state nuclear magnetic resonance, the structural characteristics of alpha-synuclein fibrils derived from postmortem tissue from PD, dementia with Lewy bodies (DLB), and MSA patients have been revealed. The binding pocket of a few alpha-synuclein PET tracers to alpha-synuclein fibrils or structurally based designs has already been employed in the tracer development. Xiang et al. (2023) reported the PET tracer [¹⁸F]F0502B, which shows a high binding affinity for alpha-synuclein but not for amyloid-beta or tau fibrils (supported by cryo-electron microscopy findings), with brain permeability and rapid washout. [¹⁸F]F0502B was able to visualize alpha-synuclein deposits in the brains of rodent and nonhuman primate PD models (Xiang et al., 2023). This group further demonstrated the spread of alpha-synuclein from the gut to the brain by PET using [¹⁸F]F0502B in a rodent model in vivo. In addition, optical approaches such as chemiluminescence imaging (e.g., using the probe CyLumi-3) and near-infrared imaging methods have been used to visualize alpha-synuclein in M83 transgenic mouse model of PD in vivo. Compared with PET imaging, optical imaging is cost-efficient and logistically convenient approaches for preclinical investigations in disease animal models. Straumann et al. (2024) demonstrated that the pyrimidoindole derivative THK-565 detected alpha-synuclein and showed greater cerebral fluorescence intensity than nontransgenic littermate mice. However, THK-565 is not specific for alpha-synuclein and binds to amyloid-beta or tau, thus further optimization is needed based on this scaffold.

To date, four studies have reported the imaging of alpha-synuclein in vivo in humans. Matsuoka et al. (2022) reported that PET using [¹⁸F]SPAL‐T‐06 demonstrated increased retentions in the putamen, pons, and cerebellar white matter and peduncles of MSA patients with predominant parkinsonism and MSA patients with predominant cerebellar ataxia, compared with healthy controls. Smith et al. (2023) reported that the alpha-synuclein PET tracer [¹⁸F]ACI-12589 could effectively distinguish MSA from other neurodegenerative diseases. These findings demonstrated that [¹⁸F]ACI-12589 has robust in vitro affinity and specificity for pathological alpha-synuclein in tissue samples from patients with various alpha-synuclein-related disorders, including PD and MSA, as evidenced by autoradiography. In vivo studies have indicated that [¹⁸F]ACI-12589 binds prominently to the cerebellar white matter and middle cerebellar peduncles in MSA patients but shows limited binding in PD patients (Smith et al., 2023). Saw et al. (2024) recently reported that [¹¹C]MODAG-005 exhibits sub-nanomolar binding affinity for recombinant alpha-synuclein fibrils and glial cytoplasmic inclusions in MSA brain tissue, favorable brain penetration, and rapid clearance. In vivo imaging in transgenic rodent models and nonhuman primates, as well as first-in-human studies in MSA patients, revealed marked tracer binding in regions with high alpha-synuclein loads. However, this tracer has shown limited efficacy in detecting LBs and Lewy neurites in PD brain tissue, as well as off-target binding to amyloid-beta and tau (Saw et al., 2024). Endo et al. (2024) developed an alpha-synuclein PET tracer, [¹⁸F]C05‒05, which is derived from the tau tracer [¹¹C]PBB3 and exhibits cross-reactivity with alpha-synuclein. This tracer demonstrated a high binding affinity for alpha-synuclein compared with amyloid-beta and tau fibrils. In vivo, two-photon laser scanning microscopy, PET imaging, and ex vivo autoradiography revealed increased binding of [¹⁸F]C05‒05 in the striatum and cortex of alpha-synuclein-inoculated mice and marmoset models, which correlated with reduced dopaminergic innervation and the distribution of phosphorylated alpha-synuclein. Importantly, increased retention of [¹⁸F]C05-05 was detected in the midbrain of PD patients and DLB patients compared to control cases, correlated with clinical motor symptoms (indicated by MDS-UPDRS part III scores). This provides promising evidence that the alpha-synuclein density in the brains of PD patients is sufficiently high to be visible in vivo by PET (Figure 1). The pattern of [¹⁸F]C05-05 uptake is also different in MSA patients, with increased retention in the putamen and cerebellum regions (Endo et al., 2024). Moreover, a clear difference between the pattern of [¹⁸F]C05-05 (alpha-synuclein) and [¹⁸F]PM-PBB3 (tau) supported the detection specificity of the tracers.

Figure 1.

Detection of alpha-synuclein pathologies in the midbrains of PD and DLB patients by PET with [¹⁸F]C05-05.

(A) Representative parametric images of SUVRs at 100–120 minutes after ¹⁸F-C05-05 injection at midbrain level with deep white matter as reference region (SUVRdwm) (B, C) Group comparison of SUVR values for [¹⁸F]C05-05 and [¹⁸F]PM-PBB3 in the midbrain between HC and PD/DLB patients. (D) Correlation between midbrain SUVR values for ¹⁸F-C05-05 and the MDS-UPDRS part III scores in PD and DLB cases. Reprinted with permission from Endo et al. (2024). DLB: Dementia with Lewy bodies; HC: healthy control; MDS-UPDRS: Movement Disorder Society-Unified Parkinson’s Disease Rating Scale; PD: Parkinson’s disease; SUVR: standardized uptake value ratio; SUVRdwm: SUVR with the deep white matter as the reference region.

Current limitations and future considerations: With the recent promising development of alpha-synuclein tracers, several concerns remain, for further translation as biomarkers for the detection of alpha-synuclein pathology in PD.

Sensitivity and specificity of tracers: While alpha-synuclein is a hallmark of PD, it is also implicated in other alpha-synucleinopathies, such as MSA and DLB. This can lead to false-positive results when the patient has a different neurodegenerative disease other than PD. In addition, the concentration of fibrillar alpha-synuclein aggregates (such as LBs and Lewy neurites) is more than 10 times lower than that in amyloid-beta, and tau in the brain. Given the prevalence of copathologies in the brain of PD, DLB, PD dementia, and related disorders, as well as other forms of parkinsonism associated with tau aggregation, high binding affinity and selectivity of the tracer to alpha-synuclein (over amyloid and tau) are particularly critical. However [¹⁸F]C05-05, the only tracer that so far showed positive midbrain detection in PD patients, showed an affinity of 1.5 nM in the brain from DLB cases compared to 12.9 nM in the brain from AD cases (< 10-fold difference), which still has room for improvement. Moreover, several tracers have shown off-target binding to white matter, and the choroid plexus may complicate assessments, especially in MSA. The tracer [¹¹C]MODAG-005 has demonstrated no off-target binding to monoamine oxidase B (Saw et al., 2024), which needs to be evaluated for other alpha-synuclein tracers as well.

Preclinical evaluation model: A recent study revealed that mouse alpha-synuclein fibrils differ from human alpha-synuclein fibrils, both structurally by cryo-electron microscopy as well as functionally (Sokratian et al., 2024). Thus, an animal model inoculated with patient brain-derived alpha-synuclein preformed fibrils is more suitable than transgenic or rodent preformed fibril injected models for ex vivo and in vivo assessment.

Challenges in early detection: As alpha-synuclein pathology may begin years or even decades before clinical symptoms of PD appear. Whether current alpha-synuclein PET tracers can detect early preclinical stages of the disease, such as in patients with rapid eye movement remains to be demonstrated. Establishing the specificity of findings in well-defined cohorts of patients compared with healthy controls will be important for developing a clinically effective alpha-synuclein PET tracer for early detection.

Variable tracer uptake: PD presents significant heterogeneity in terms of age of onset, progression rates, and clinical manifestations and subtypes. There is variability in the uptake of alpha-synuclein PET tracers across different brain regions and among individuals based on the current available imaging data in PD patients. This variability can complicate the interpretation of results and affect the reliability of the diagnosis. The results from clinical evaluations of several tracers, including [¹¹C]MK-7337 (REC reference 23/WA/0306) and ACI-15916 (under investigational new drug-enabling studies), are anticipated. Similar to the centroid approach used in the amyloid and tau PET data analysis, further standardization and optimization of the analysis are warranted.

Resolution related challenge: The spatial resolution of the current PET scans may potentially lead to inaccuracies in the detection of alpha-synuclein deposits, especially in small brain structures such as the substantia nigra. In this regard, the development of a neuro-focused PET scanner with higher spatial resolution will enable better visualization of the small structures with alpha-synuclein deposition.

In summary, the development of alpha-synuclein imaging tracers as a potential PD imaging and biological diagnostic biomarker will facilitate the early detection, staging, and evaluation of alpha-synuclein-targeted treatments. Moreover, it will greatly improve the understanding of outstanding research questions such as the gut-first or brain-first hypothesis of PD and the temporal spatial association between the loss of nigrostriatal degeneration and the use of dopamine transporter imaging tools.

This work was supported by Swiss Center for Applied Human Toxicology (SCAHT AP22-01) (to RN).