Understanding Mechanisms of Synaptic Degeneration Underlying Clinical Symptoms in Patients With MDs and NDs.

SYNAPSING's POSTMORTEM AND IN VITRO STUDIES OF SYNAPSE DYSFUNCTION

Clinical Study A. Retrospective Neuropathological Study of Synapse dysfunction.

This is a cross-sectional study of patients retrospectively collected from existing postmortem collections and from existing collections of iPSC-derived neurons. Postmortem tissue and iPSC-derived neurons from age and sex-matched unaffected volunteers without a MD or ND diagnosis are used as controls.

This study will:

1. perform RNA sequencing of postmortem synapses from the prefrontal cortex of patients with schizophrenia or frontotemporal dementia and unaffected controls. The data will be combined with already available RNA sequencing data from established iPSC clones from schizophrenia or behavioural variant frontotemporal dementia patients and unaffected controls. The postmortem frontotemporal dementia samples have already been characterised in terms of frontal cortex pathology in a previous study and data from RNA sequencing of homogenates is available for comparison. The prefrontal cortex has been implicated as a core affected region across MDs and NDs making this the perfect region to determine common molecular drivers of synapse dysfunction. Pathway analysis of the RNA sequencing dataset will be performed to identify biological pathways or individual genes that are deregulated in the postmortem samples and iPSC lines from each condition. A total of 3 genes that converge between the postmortem and iPSC datasets will be selected for gene-editing. Each gene will be CRISPR/Cas9-repaired in 1 selected schizophrenia parental line and 1 selected frontotemporal dementia parental line. The impact of the induced transcriptional changes on functional and synaptic morphology phenotypes in neuronal cultures from each gene-edited line will be compared to the respective parental line and to lines from 3 healthy controls. A rescue of the disease phenotypes is expected such that the repaired edited line shows activity and synaptic morphology comparable to the healthy control lines and distinct from the unedited parental line. All experiments will include 3 biological replicates taking the total number of analyses to 420. The study will also determine the role of microglia on neuronal deficits in iPSC-derived neurons from the 20 available clones from patients with schizophrenia or behavioural variant frontotemporal dementia by comparing electrophysiological activity and synapse morphology in iPSC-derived neurons culture alone to those observed in iPSC-derived neurons co-cultured with iPSC-derived microglia differentiated from the same clones.
2. compare the molecular composition of postmortem synapses from the prefrontal cortex across 3 MDs (major depressive disorder, bipolar disorder and schizophrenia) and identify differentially expressed proteins and dysfunctional protein networks through pathway analyses. To determine the potential effects of psychotropic drugs on synaptic proteomes, the study will quantify the concentrations of >200 psychoactive drugs and metabolites in both blood and cerebellum postmortem samples from the same donors and determine the correlation with the differentially expressed proteins. While blood analyses provide information on drugs present at the time of death, toxicological data in brain tissue, which accumulates drugs for weeks or even months after the last intake, serves as a better indicator of subjects' adherence to the treatments. Groups larger than 15 subjects are recommended for proteomic studies such as this. Each subject was paired to a control subject, matching sex, age, and postmortem interval and with no evidence of psychiatric or neurological conditions, according to their medical records.
3. quantify and compare synapse loss in brain regions shown to be associated with reduced volumes in MD and ND patients postmortem. Specifically, the study will compare synapse density in the prefrontal cortex, hippocampus and striatum from patients with major depressive disorder, bipolar disorder, schizophrenia, Alzheimer's disease, frontotemporal dementia to age and sex-matched unaffected controls.

Inclusion criteria for psychiatric disorders:

• Age group: Midlife brain donors (age-at-death>40).
• Sex distribution: % Female in schizophrenia (23%), major depressive disorder and controls (40%), bipolar disorder and controls (55%). These percentages match those typically found in these disorders.
• Antemortem diagnosis of schizophrenia (paranoid-subtype diagnoses only to minimise the impact of potential confounding factors on the results and ensure homogeneity across schizophrenia subjects), major depression or bipolar disorder. Diagnoses are confirrmed antemortem by a psychiatrist using Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria, as recorded in the deceased individuals' medical records.

Exclusion criteria for psychiatric disorders:

For schizophrenia, cases displaying additional neurological or psychiatric conditions, such as histories of substance use disorder will be excluded. Postmortem delay >24h (to minimise perimortem confounding variables potentially affecting tissue quality.

Inclusion criteria for Neurodegenerative diseases:

• Age group: Latelife brain donors (age-at-death>50).
• Sex distribution: Alzheimer's disease (60%), frontotemporal dementia (65%). These percentages match those typically found in these diseases.
• Neuropathological confirmation of Alzheimer's disease or frontotemporal lobar degeneration (C9orf72 repeat expansion carrier)Alzheimer's disease

Exclusion criteria for Neurodegenerative diseases:

Postmortem delay >24h (to minimise perimortem confounding variables potentially affecting tissue quality.