Conference Agenda

Cdkl5 deficiency disorder (CDD) is a syndromic autism-spectrum disease (ASD) with a broad constellation of cognitive, motor, and autonomic deficits with complex clinical manifestations. These characteristics have hampered the identification of golden standard criteria for CDD diagnosis making it urgent to assess consistent, possibly non-invasive, and routinely manageable, biomarkers to monitor the disease progression and to objectively assess therapeutic intervention. Mounting evidence established that extracellular vesicles (EVs) represent a promising source of disease-specific biomarkers and therapeutic vectors for neurological disorders as neurodegenerative conditions. EVs are secreted by all brain cells, are involved in cell-to-cell communication, and their content reflects the physiological or pathological condition of the releasing cells. EVs cargoes are made of proteins, mRNAs and miRNAs with important implications for brain diseases and neuroprotection. Currently, very little is known on the potentials of EVs in the context of neurodevelopmental disorders such as ASD. During my talk I will illustrate the experimental pipeline established in our laboratory that allows for high-efficient isolation, purification and analysis of salivary EVs as a non-invasive source of brain/neuronal-derived multicomponent biomarkers for brain disorders. We isolate EVs from both saliva of CDD patients, acute murine neuronal CDKL5-KO cultures and corresponding controls. By identifying and characterizing EVs miRNomic profiles using bioinformatic tools we hope to disclose soon new pathogenetic markers and therapeutic targets for CDD. Moreover, Gene Ontology analyses will reveal novel molecular and cellular pathways that could be affected in CDD patients, the importance of which will be validated by testing functional, morphological and gene expression properties of acutely cultured neurons. Because therapeutic approaches are finally emerging for rare neurological diseases, often characterized by individuals with severe clinical conditions and unable to communicate, the need for powerful, objective and non-invasive biomarkers is immediate.

Alternative splicing (AS) is a post-transcriptional mechanism that increases the information content of the transcriptome through the expression of different mRNAs from single genes. AS directly competes with the backsplicing (BS) process, a mechanism leading to a non-canonical splicing of genes to generate non-coding, circular RNAs (circRNAs). AS and BS processes are intertwined, since their functions are based on the same spliceosomal machinery and trans-acting factors, and they may be tuned by genetic and epigenetic determinants, such as DNA methylation. A dysregulation of AS/BS has been implicated in several human disorders, including multiple sclerosis (MS), a chronic autoimmune neurodegenerative disease in which autoreactive T lymphocytes attack antigens of the central nervous system.
In our work we aimed at unraveling the mechanisms involved in the AS/BS dysregulation in MS by integrating different omics data and focusing on possible correlations with the genetic and epigenetic background. We first disclosed a significant association between MS-associated loci and the expression of selected circRNAs in peripheral blood mononuclear cells, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. We then collected data on gene expression, AS, BS, methylation status and genetic background of CD4+/CD8+ T lymphocytes of MS patients and controls, using sequencing and array approaches. A first analysis evidenced the presence of several differentially expressed genes (210, FDR<0.1), circRNAs (200, P<0.05), AS events (600 genes carrying dysregulated events, FDR<0.05), and differentially methylated positions (1200 genes, FDR<0.05) in MS cases. All the omics were integrated in the MOFA2 unified framework, and we identified a hidden factor that was strongly associated with the disease status (P<10^-5), mainly sustained by dysregulated AS events. An enrichment analysis run on the top features highlighted, among the top results, “T cell receptor signaling pathway”, “cellular response to stress”, and “TNF signaling pathway”.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression and cellular processes, with their dysregulation implicated in both neurodegenerative diseases and cancer. Cancer and neurodegenerative disorders represent major health burdens worldwide. While seemingly distinct in nature, accumulating evidence suggests that lncRNAs play important roles in the pathogenesis of both conditions. In neurodegenerative diseases, altered lncRNA expression and dysfunction have been associated with neuronal cell death, neuroinflammation, protein misfolding, and impaired cellular functions crucial for neuronal survival. Additionally, lncRNAs have been implicated in the regulation of neuronal development, synaptic plasticity, and mitochondrial function. In cancer, dysregulated lncRNA expression contributes to tumor initiation and progression, metastasis, and drug resistance. These lncRNAs can function as oncogenes or tumor suppressors by modulating gene expression, chromatin remodeling, and post-transcriptional processes. Interestingly, certain lncRNAs demonstrate dual roles, being involved in both cancer and neurodegenerative diseases. For instance, MALAT1 and HOTAIR, well-studied lncRNAs, have been found to be dysregulated in various cancer types, promoting tumor growth and metastasis, while also being implicated in neurodegenerative processes such as neuroinflammation and neurodegeneration. These dual-function lncRNAs highlight the interconnectedness between cancer and neurodegenerative diseases and underscore the need for further investigation into their intricate mechanisms of action. In this context, we provide initial insights into the function of the MYC-induced Long non-Coding RNA (MINCR), a lncRNA described as up-regulated in various cancers and downregulated in the blood and spinal cord of sporadic Amyotrophic Lateral Sclerosis patients. Understanding the complex interplay between lncRNAs and the molecular pathways underlying cancer and neurodegenerative diseases is crucial for developing effective diagnostic and therapeutic strategies. LncRNAs hold great promise as potential biomarkers for disease detection, prognosis, and therapeutic response prediction. Furthermore, targeted modulation of dysregulated lncRNAs may provide new avenues for therapeutic interventions in both cancer and neurodegenerative disorders.

While selective vulnerability at the network level has been largely explored in Alzheimer’s disease (AD) as a critical feature of neurodegenerative diseases, it is still unexplored at molecular and cellular level. Recent advancement in single cell genomics techniques offer the possibility to map the pathologic tissue at an unprecedentedly high resolution. Here, we aimed to investigate the cellular and molecular landscape of the human hippocampus in the early stage of the disease before the onset of cognitive impairment with the final goal to establish a gradient of susceptibility to degeneration of the different CNS-resident cells (i.e. neuronal, immune, glial, and endothelial).

To this end, we performed single nucleus RNA sequencing (snRNAseq) of the hippocampus from post-mortem human brain of subjects with mild cognitive impairment (MCI) with a neuropathological picture of low AD (n=6) vs subjects with low neuropathological AD without cognitive impairment (n=4), compared to age-matched non-neurological non-demented controls (n=3). Samples were provided by Golgi Cenci Foundation (Abbiategrasso, Milan).

snRNA-seq yielded a total of 120.158 single-nucleus transcriptomic profiles. Unsupervised clustering identified 13 initial clusters that were labelled on the basis of known lineage marker genes. We performed subclustering of neuronal, immune, OPC, and oligodendrocytes on the basis of differentially expressed genes and canonical cell-type-specific gene expression for each cell type. To define the molecular signatures of the hippocampus in MCI and low neuropathological AD brains, we built a detailed cellular blueprint and interactome of the human hypothalamus. Moreover, to unveil common and divergent features between AD and other neurodegenerative diseases, we leveraged existing and available snRNA-seq datasets and mapped our dataset onto their reference by identifying shared and/or disease-specific neurodegenerative phenotypes.

Results emerging from this study provide a vast amount of research data beyond this specific project and will inform future studies of the mechanistic basis of selective vulnerability.