Understanding Alpha-Synuclein Spread in Parkinson's Disease Through Blood Biomarkers and Neuroimaging

Parkinson's disease (PD) is characterized by pathological aggregation and propagation of alpha synuclein, leading to synaptic and mitochondrial dysfunction. Heterozygous mutations in the GBA1 gene represent the strongest genetic risk factor for PD and are associated with earlier onset, faster progression, and increased burden of misfolded alpha synuclein. Experimental evidence suggests that alpha synuclein toxicity may be amplified by its interaction with tau, promoting synergistic neurodegenerative mechanisms; however, in vivo human data on these processes remain limited.

This study aims to define, through a personalized and multimodal approach, how alpha synuclein accumulation and its interaction with tau influence synaptic and mitochondrial dysfunction and brain connectivity across the PD spectrum, from periclinal stages to established disease, with particular focus on the impact of GBA1 mutations. The study includes patients with PD, individuals with idiopathic REM sleep behavior disorder (iRBD), and subjects without PD, both carriers and non carriers of GBA1 mutations.

All participants will undergo comprehensive clinical and neuropsychological assessments to characterize motor, non motor, and cognitive manifestations across disease stages. Blood samples will be collected to define a fluid biomarker profile, including alpha synuclein, tau, markers of synaptic integrity, mitochondrial function, and neurodegeneration. In addition, all participants will undergo multimodal brain MRI, including structural, diffusion weighted, and resting state functional sequences, to evaluate brain structure, white matter integrity, and functional connectivity.

In a subset of participants, a skin biopsy will be performed to generate patient specific induced pluripotent stem cell (hiPSC) derived dopaminergic neurons. These cellular models will be used to investigate neuronal and synaptic function in relation to individual biomarker profiles and genetic background. In parallel, preclinical studies will be conducted in GBA-PD mouse models and wild type mice injected with saline, alpha synuclein or combined alpha synuclein/tau fibrils. Mice will undergo behavioral, in vivo MRI and PET imaging and post mortem assessment of synaptic, axonal and mitochondrial pathology. In addition, a novel alpha synuclein PET tracer, \[¹⁸F\]Syntacasyn, will undergo preclinical validation.

Multimodal human and animal data will be integrated using advanced statistical and computational approaches to identify vulnerable network hubs and generate subject specific "virtual brain" models of alpha synuclein pathology propagation.

The study is designed to provide a translational framework linking molecular pathology, brain network dysfunction and clinical heterogeneity in PD, supporting biomarker development and precision medicine strategies across the prodromal and clinical spectrum, with particular focus on genetically defined populations such as GBA1 mutation carriers.

• brain imaging (Diagnostic test): Participants will undergo a single brain MRI acquisition, including structural, diffusion-weighted, and resting-state functional MRI.
• blood draw (Procedure): Collection of a venous blood sample for biochemical analyses
• Skin biopsy (Procedure): A small punch skin biopsy (about 3-4 mm) will be performed under local anesthesia on a small sample of enrolled participants (n=10)

Primary outcomes

• Concentration of alpha-synuclein levels in plasma [Baseline visit]: Quantification of levels of Total alpha-synuclein and 129P-alpha-synuclein (pg/ml) in neuronal-derived extracellular vesicles using ultrasensitive immunoassays (NULISA).
• Concentration of tau levels in plasma [Baseline visit]: Quantification of total-tau and p-tau18 (pg/ml) in neuronal-derived extracellular vesicles using ultrasensitive immunoassays (NULISA).
• Investigation of glucocerebrosidase activity in Peripheral Blood Mononuclear Cells [Baseline visit]: Glucocereborsidase activity will be estimated in Peripheral Blood Mononuclear Cells usign a flourimetry assay

Secondary outcomes

• Assessment of mitochondrial damage in plasma [Baseline visit]: Quantification of circulating cell-free mitochondrial DNA (cf-mtDNA) in plasma, including total concentration and deletion fraction.
• Plasma synaptic protein concentration [Baseline visit]: Quantification of SNAP25, STX1A, and VAMP2 in neuronal-derived extracellular vesiclesusing ultrasensitive immunoassay (NULISA)
• Brain network connectivity in Parkinson's disease [Baseline visit]: We will exctract structural and functional connectivity information form MRI, resting-state functional MRI and diffusion weighted MRI. Connectivity metrics will include global and nodal efficiency, participation coefficient and indexes of withe matter tract integrity.
• In vitro neuronal responses in hiPSC-derived dopaminergic neurons [Baseline visit]: In vitro neuronal responses will be assessed in dopaminergic neurons derived from human induced pluripotent stem cells (hiPSC), following exposure to alpha-synuclein and alpha-synuclein+tau.
• Estimation of synaptic damage in Neuronal extracellular vesicle [Baseline visit]: Neurogranin concentration (pg/ml) will be quantified in neuronal-derived extracellular vesicles
• Concentration of neurofilament light chain [Baseline visit]: Neurofilament light chain concentration (pg/ml) will be quantified in plasma using Ella™.

Other outcomes

• Comprehensive clinical and neuropsychological assessement [Baseline visit]

Sex: All, ages: 18 Years to any, accepts healthy volunteers

Inclusion criteria for Parkinson's disease cohorts (GBA-PD and nonGBA-PD):

* Diagnosis of PD according to MDS-PD criteria and, for the GBA-PD group, presence of heterozygous GBA mutations (with a balanced distribution of severe, risk, mild, and complex variants);
* Disease duration between 3 and 7 years;
* Disease stage according to Hoehn \& Yahr ≤ 3;
* Absence of mutations in other known genes associated with PD susceptibility;
* Age > 18 Years;
* Ability to understand and voluntarily sign informed consent and to comply with study procedures.

Exclusion criteria for Parkinson's disease cohorts:

* Diagnosis of atypical and/or secondary parkinsonism;
* Diagnosis of dementia according to DSM-5 criteria;
* Presence of other neurological disorders and/or essential tremor;
* Presence of systemic inflammatory or infectious diseases, autoimmune diseases, or malignant tumors at the time of enrollment.

Inclusion criteria for unaffected subjects (GBA-nonPD and nonGBA-nonPD):

* Age > 18 Years;
* Ability to understand and voluntarily sign informed consent and to comply with study procedures;
* No diagnosis of PD or other neurological disorders;
* Presence of a heterozygous GBA mutation for the GBA-nonPD group and absence of such mutation for control subjects (nonGBA-nonPD);
* Absence of mutations in other known genes associated with PD susceptibility.

Exclusion criteria for unaffected subjects (GBA-nonPD and nonGBA-nonPD):

* Presence of systemic inflammatory or infectious diseases, autoimmune diseases, or malignant tumors at the time of enrollment;
* Diagnosis of atypical and/or secondary parkinsonism;
* Diagnosis of dementia according to DSM-5 criteria.

Inclusion criteria for subjects with idiopathic REM Sleep Behavior Disorder (GBA-iRBD and nonGBA-iRBD):

* Diagnosis of idiopathic REM Sleep Behavior Disorder according to ICSD-3;
* Age > 18 Years;
* Ability to understand and voluntarily sign informed consent and to comply with study procedures;
* No diagnosis of PD or other neurological disorders;
* Presence of a heterozygous GBA mutation for the GBA-iRBD group and absence of such mutation for the nonGBA-iRBD group;
* Absence of mutations in other known genes associated with PD susceptibility.

Exclusion criteria for subjects with idiopathic REM Sleep Behavior Disorder (GBA-iRBD and nonGBA-iRBD):

* Presence of systemic inflammatory or infectious diseases, autoimmune diseases, or malignant tumors at the time of enrollment;
* Diagnosis of atypical and/or secondary parkinsonism;
* Diagnosis of dementia according to DSM-5 criteria.

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