A highly sensitive blood test accurately identified people with synucleinopathies, disorders caused by the abnormal aggregation of the protein alpha-synuclein, such as Parkinson’s disease.
The test, called immunoprecipitation-based real-time quaking-induced conversion (IP/RT-QuIC), detected early-formed alpha-synuclein fibrils — or seeds — that eventually grew into disease-causing aggregates.
Microscopic analysis detected structural differences between aggregates derived from seeds isolated from patients with different synucleinopathies: Parkison’s disease, Lewy Body dementia (LBD), and multiple system atrophy (MSA). Notably, these differences were maintained after propagating patient-derived seeds in a mouse model.
“At present, a neurologist’s consultation is necessary to diagnose synucleinopathies,” study co-lead Nobutaka Hattori, MD, PhD, professor at Juntendo University, in Japan, said in a press release. “However, using IP/RTQuIC, a general internist can make the diagnosis,” he said.
“More patients with synucleinopathies may be diagnosed with precision and could receive appropriate treatment at an earlier stage,” Hattori noted.
The study, “Propagative α-synuclein seeds as serum biomarkers for synucleinopathies,” was published in the journal Nature Medicine.
Similar beginnings
Parkison’s disease, LBD, and MSA, the three most common synucleinopathies, all begin with the onset of Parkinson’s-like motor symptoms. LBD also is characterized by rapid cognitive decline, and MSA by widespread dysfunction of the autonomic nervous system, which controls automatic bodily functions.
In synucleinopathies, the aggregation process begins with the misfolding of alpha-synuclein and the formation of tiny fibril-like seeds. These seeds attract more alpha-synuclein proteins, which eventually grow into visible aggregates that interfere with normal biological processes and cause disease.
Because studies suggest alpha-synuclein seeds may be found in the bloodstream, researchers in Japan designed and validated IP/RT-QuIC to demonstrate their utility as a diagnostic biomarker.
“In this study, we validated the usefulness of our novel assay system, IP/RT-QuIC, as a diagnostic marker of synucleinopathies,” Hattori said. “We propose that the fibril morphology of serum [alpha]-synuclein seeds and aggregates derived by IP/RT-QuIC can discriminate between Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).”
With IP/RT-QuIC, seeds were first isolated from blood samples using selective antibodies (immunoprecipitation). Real-time rapid amplification of seeds into detectable aggregates was achieved with vigorous shaking in the presence of alpha-synuclein protein (quaking-induced conversion).
Using pre-formed seeds, the team first confirmed the test’s high sensitivity, with a detection limit as low as 1,000 picograms per mL.
Among the blood samples collected from 221 Parkinson’s patients, 210 (95%) tested positive for alpha-synuclein seeds. All samples collected from 34 Parkinson’s patients who participated in a previous study tested positive, as did 15 from 20 (75%) additional Parkinson’s patients who were tested blindly (without knowing the diagnosis).
IP/RT-QuIC’s ability to correctly identify people with Parkison’s was 94.6% (sensitivity), while its ability to rule out the condition was 92.1% (specificity). Also, a faster rate of aggregate formation correlated with longer disease duration and worse motor symptoms, as assessed by the Unified Parkinson’s Disease Rating Scale part 3.
Electron microscope analysis
A detailed examination of amplified seeds using an electron microscope revealed those from Parkinson’s and LBD patients formed paired filaments or bundled multiple filaments. By contrast, seeds from MSA patients had two distinct shapes: twisted filaments that could not be divided into two filaments, and straight filaments.
No differences were found between seeds from blood samples and those collected from Parkinson’s cerebrospinal fluid, the liquid surrounding the brain and spinal cord.
Amplified seeds from patient samples then were delivered into a cell line modified to produce a mutant form of alpha-synuclein that readily misfolds and forms aggregates.
Microscopic analysis of cells revealed seeds from different synucleinopathies formed aggregates with different structural features. Seeds from Parkinson’s patients generated significantly more fibrous aggregates, LBD seeds formed significantly more pale structures, while MSA had dense-core aggregates.
Injecting the seeds into mice
Injecting amplified seeds derived from Parkinson’s patients into the brains of mice led to the gradual accumulation of alpha-synuclein deposits. Brain samples all tested positive using IP/RT-QuIC, and seeds derived from these mice showed rapid rates of aggregate formation.
Microscopic analysis of new fibrils formed after injecting mice with Parkinson’s-derived seeds showed structures similar to cell experiments, but also MSA-type seed structures. By contrast, MSA-seeded mice formed MSA-type filaments and MSA-type aggregates.
“Our new IP/RT-QuIC assay may have many future applications as a biomarker for precise diagnosis and monitoring of treatment of neurodegenerative diseases in clinical trials,” Hattori said. “This simple diagnostic method will enable establishment of personalized therapy options for synucleinopathies.”
