Discovery and Validation of Protein Structural Complexes in Circulating Biofluids As Novel Biomarkers for Early Diagnosis, Prognosis and Therapeutic Management of Patients Affected by Neurodegenerative Disorders

Overview

Neurodegenerative disorders (NDDs), such as Parkinson¿s disease (PD), Alzheimer¿s disease (AD), Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are characterized by aggregation and intracellular accumulation of misfolded proteins, which are believed to play a key role in synaptic dysfunction and neuronal death. Protein structural complexes in biofluids have been proposed to mirror pathological conditions suggesting their use as biomarkers for NDDs characterized by protein aggregation. In this framework, we plan to: i) collect a large cohort of NDD and prodromal patients and healthy subjects using standardized clinical and genetics procedures; ii) apply a novel method based on genomics, proteomics and bioinformatic analysis to map protein complexes in biofluids; iii) identify novel circulating biomarkers and correlate them to genetic profiling and disease endophenotypes, and; iv) validate the biological properties in human brain tissue and dopaminergic cultures.

Detailed description

The project is a multicrentric observational study. Institutions involved are:

OU1 - IRCCS INM Neuromed OU2 -University of Piemonte Orientale The project takes advantage from the availability of a large collection of PD samples from familiar and sporadic cases, recruited at IRCCS Neuromed, for which we already collected and stored clinical information, genetic data as well as DNA, serum, plasma and peripheral blood mononuclear cells (PBMCs) for the entire study cohort. induced pluripotent stem cells (iPSC) are available for 6 PD patients and 5 healthy subjects.

The activities of the Operating Unit (OU1) are:

1. Stratification of NDD patients based on genetics and clinical records; 2. Recruitment of CSF sample of NDD patients; 3. Recruitment, WES and bioinformatic analysis of a selected cohort of NDD relatives (200 samples); 4. Bioinformatic analysis to identify protein structural alterations, candidate genes, genetic profiles and deregulatedpathways (OU1/ OU2); 5. Correlation of circulating complex with PD endophenotypes to identify disease biomarkers (OU1/ OU2); 6. Validation in RBD patients and PD relatives to identify early biomarkers for PD (OU1/ OU2); 7. Validation of the protein complexes in human brain slices and analysis of the biological activity of the most relevant CSF and plasmatic complexes in iPSC-derived mdDA neurons (OU1/ OU2).

The activities of the Operating Unit (OU2) are:

1. CSF and plasma sample collection and storage for patients with NDDs; 2. Stratification of patients based on genetics and clinical records (in collaboration with OU1); 3. Detect alterations in protein complexes in CSF samples (100 samples); 4. Identification and validation of CSF biomarkers for PD (100 samples) (in collaboration with OU1); 5. Validate complex biomarkers using immunochemical or targeted analysis on plasma samples (450 samples); 6. Identify potential drug targetable complexes (in collaboration with OU1); 7. Assess whether proteins with structural changes in the CSF/plasma have those changes in the brain as well; 8. Correlate protein structural information with genetics and clinical data (in collaboration with OU1).

Interventions

• Genetic Genetic: whole genome sequencing
  Genetic: whole genome sequencing PD Partecipants will be assessed for disease progression: Stadio di Hoehn and Yahr, MDS-UPDRS part III, MOCA test, no motor symptoms, therapy and LID occurrence, Sleep disorders. Partecipants will be subjected to peripheral blood sampling for the purification of DNA, plasma, serum, PBMC and generation of hiPSC. DNA of each partecipants we will analysed by whole genome sequencing by next generation sequencing to identify any variant in candidate PD genes
• Genetic whole exome sequencing
  Genetic: whole genome sequencing AD Partecipants will be assessed for disease progression: * assessment of cognitive disorders (MMSE, MOCA test, clock test); * assessment of language disorders; * current drug therapy (and possible start date of treatment); * date of onset of cognitive disorders; * acquisition and assessment of imaging data where present (MRI, CT, PET). DNA of each partecipants we will analysed by whole genome sequencing by next generation sequencing to identify any variant in
• Genetic whole exome sequencing
  Genetic: whole genome sequencing ALS/FTD Partecipants will be assessed for disease progression: * clinical classification according to El Escorial - revised; * assessment of cognitive disorders (and classification according to Strong criteria, 2017); * assessment of language disorders; * ongoing pharmacological therapy (and possible start date of treatment); * date of onset of motor and cognitive disorders; * acquisition and assessment of imaging data where present (MRI, CT, PET). DNA of each

Primary outcome measures

• motor symptoms of PD patients will be evaluated with Hoehn and Yahr (HY) score [Time frame: 2 years]
• motor and non motor symptoms of PD patients will be evaluated with MDS-UPDRS [Time frame: 2 years]
• clinical evaluation of cognitive impairment of PD, AD, ALS/FTD patients [Time frame: 2 years]
• clinical evaluation of ALS/FTD patients [Time frame: 2 years]
• identification of variants/mutations [Time frame: 2 years]
• Identification of protein complexes in CSF [Time frame: 2 years]
• Bioinformatic analysis for the identification of proteins and molecular pathways involved [Time frame: 2 years]
• Validation of protein complexes in plasma [Time frame: 2 years]
• Association analysis with endophenotypes [Time frame: 2 years]
• Analysis of the biological impact of protein complexes identified in CSF on the viability of mdDA dopaminergic neurons obtained from iPSCs of patients and controls [Time frame: 2 years]

Eligibility criteria

• Inclusion Criteria:
• Inclusion criteria for PD patients.

For the IRCCS INM Neuromed, patients will be recruited from those affiliated with the Center for the Study and Treatment of Parkinson's Disease of the Neuromed Institute of Pozzilli. Affected subjects will be selected according to the criteria proposed by Gelb et al in 1999. This is a very pragmatic scheme based on the presence of four cardinal signs, the response to a test administration of Levodopa and the absence of atypical signs:

A) Presence of at least 2 of the 4 cardinal signs (tremor, rigidity, bradykinesia, asymmetric onset) one of which must be tremor or bradykinesia; B) Absence of atypical symptoms such as: i) early postural instability, freezing phenomena, cognitive deterioration, hallucinations, pathological involuntary movements, vertical gaze paralysis; ii) proven causes of secondary parkinsonism (focal lesions, drugs, toxic substances); C) Documented response to the use of L-dopa or dapamine agonists (or lack of an adequate therapeutic attempt with L-dopa or dopamine agonists).

• Inclusion criteria forAD patients. Patients will be selected at the Center for Cognitive Disorders and Dementias (CDCD) SCDU Neurology, AOU Maggiore della Carità, Novara. Patients with AD will be included after diagnosis of probable Alzheimer's disease according to the McKhann criteria (2011) and supported by positive biomarkers for amyloidopathy (PET with amyloid tracer or amyloid cerebrospinal fluid dosage).
• Inclusion criteria for FTD/ALS patients. For UPO, patients will be selected at the tertiary center Amyotrophic Lateral Sclerosis - SCDU Neurology, AOU Maggiore della Carità, Novara. Patients with amyotrophic lateral sclerosis will be included following a diagnosis according to the El Escorial criteria - revised (2015): in this regard, only patients with a definite or probable diagnosis supported by laboratory will be included. For patients with FTD, they will be selected at both centers mentioned above with a diagnosis according to the Rascovsky criteria (2011). In this group, the diagnosis will be supported by negative biomarkers for amyloidopathy (PET with amyloid tracer or amyloid cerebrospinal fluid dosage). For patients with concomitant ALS and FTD, the El Escorial criteria (2015) associated with the Strong criteria (2017) will be used.

Exclusion criteria

• PD PATIENTS

• pre-existing psychiatric pathologies;
• neurodegenerative neurological diseases such as multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's, neuromuscular diseases, epilepsy;
• diagnosis of dementia;
• AD/FTD/ALS PATIENTS

• pre-existing psychiatric pathologies;
• previous diagnosis of other neurodegenerative neurological diseases;
• patients unable to sign informed consent.
• CONTROLS

• pre-existing psychiatric pathologies;
• neurodegenerative neurological diseases such as Parkinson's, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's, neuromuscular diseases, epilepsy;
• diagnosis of dementia;
• depression;
• prolonged intake of anxiolytic, antidepressant, antipsychotic, sleep-inducing, cognitive stimulant drugs.

Criteria are shown verbatim from the registry (in English). Final eligibility is always assessed by the study center.

Healthy volunteers: Yes

Study design

Observational model

Case-control

Study locations

Italy · 1 center

• IRCCS INM Neuromed — Pozzilli

Identifiers

NCT: NCT06803784 · PNRR-MCNT2-2023-12377375

Primary sources (government registries)

• ClinicalTrials.gov (US NIH)

View this study on ClinicalTrials.gov ↗