There are currently no approved therapies to slow down the accumulation of neurological disability that occurs independently of relapses in MS. International agencies are engaging to expedite the development of novel strategies capable of modifying disease progression, abrogating persistent CNS inflammation, and support degenerating axons in people with progressive MS. Understanding why regeneration fails in the progressive MS brain and developing new regenerative approaches is a key priority for the Pluchino Lab.
We aim to elucidate how the immune system, in particular its cells called myeloid cells, affects brain structure and function under normal healthy conditions and in disease.
Our objective is to find how myeloid cells communicate with the central nervous system and affect tissue healing and functional recovery by stimulating mechanisms of brain plasticity mechanisms such as the generation of new nerve cells and the reduction of scar formation. To this aim, we use state-of-the-art transcriptomics, proteomics, and molecular approaches to study murine and human disease models of inflammation and neurodegeneration.
Our main goal is to develop experimental molecular medicines, including those with stem cells and gene therapy vectors, to slow down the accumulation of irreversible disabilities and improve functional recovery after multiple sclerosis, stroke and traumatic injuries.
By understanding the mechanisms of intercellular (neuro-immune) signalling, diseases of the brain and spinal cord may be treated more effectively, and significant neuroprotection may be achieved with new tailored molecular therapeutics.
Stem cells and Regenerative Medicine
Stem cells possess therapeutic potential as a cell-based therapy to promote tissue repair in neurodegenerative diseases.
We have made seminal discoveries that have established the potential of exogenous neural stem cell-based experimental therapies for progressive MS.
We have identified a critical role around the route of cell injection and the mechanisms of NSC accumulation in the chronically inflamed CNS, as well as revealed an unexpected ability of NSC therapies to provide neurotrophic support and inhibit detrimental host immune responses in vivo.
Our recent work has described a delayed accumulation of the pro-inflammatory tricarboxylic acid cycle intermediate succinate in the cerebrospinal fluid of mice with experimental autoimmune encephalomyelitis, a model of chronic MS. We have identified a new complementary mechanism by which directly induced NSCs (iNSCs) respond to endogenous inflammatory metabolic signals to inhibit the activation of type-1 mononuclear phagocytes in vivo after transplantation. Transplanted iNSCs respond to the succinate released by type-1 inflammatory macrophages and microglia in the CSF, which then signals to iNSCs via succinate receptor 1 (SUCNR1) and initiates the secretion of prostaglandin E2 and the scavenging of extracellular succinate.
Our research has therefore recalibrated the classical view that neural grafts only function through structural cell replacement and opened up a new therapeutic avenue by which to use exogenously delivered NSCs.
Alexandra Nicaise is supervising a range of projects in the lab that are evaluating the function, the fitness, the safety and the therapeutic potential of iNSCs from people with PMS in anticipation for use in first in men clinical trials.
Human induced neural stem cells transfected with fGFP (green) and stained with the neuronal marker Tuj1 (red). Cell nuclei (DAPI) are stained in blue. Credits: A Nicaise.
In progressive MS, a diffuse chronic activation of inflammatory myeloid cells, including microglia and macrophages, is one of the main processes associated to irreversible tissue damage in the central nervous system.
Cell metabolism influences the activity of myeloid cells by guiding their inflammatory function and several metabolites (or TCA cycle derivatives) have been identified to have key signalling roles in inflammation.
Luca Peruzzotti-Jametti is supervising a range of projects in the lab that aim at understanding how mitochondrial metabolism changes in myeloid cells in the context of chronic inflammation.
Our main goal is to modulate mitochondrial function and cell metabolism of myeloid cells to reduce secondary neurological damage and slow down the accumulation of disability in progressive MS.
Deconvolved projection of MHC-II (green) immunorectivity in Iba1+ microglia (red) in the mouse stroke brain. From Bacigaluppi, Pluchino et al. Brain 2009.
Inflammation plays a major role also in the evolution of secondary damage after traumatic injuries of the brain and spinal cord, with myeloid cells, both bone marrow derived macrophages and microglia as primary drivers. Upon CNS insult, BMDMs/MG initiate a complex and diverse activation profile. Both cell types are capable of contributing to the evolution of secondary damage by secreting inflammatory cytokines and complement components which further perpetuate recruitment of inflammatory cells while also being anti-inflammatory and neuroprotective. Clearly these two myeloid cells play dynamic roles as drivers of neuroinflammation post-SCI. Thus, a better understanding of their heterogeneity will allow us to identify and target the negative aspects of neuroinflammation, while preserving or fostering the beneficial ones.
A neural Stem Cell (green) interacting with a bone marrow derived macrophage (white). Mitochondria are labelled in red, nuclei are labelled in blue. Credits: L Peruzzotti-Jametti.
We are also applying advanced ex vivo single cell RNA sequencing (scRNAseq) techniques to uncover the complex heterogeneity of myeloid cell responses in animal models of chronic brain inflammation.
Analysis of microglia gene expression from healthy controls (blue), day 1 (oragnge), day 2 (green), and day 21 (red) post-spinal cord injury (SCI). Credits: R Hamel.
Ultimately, we aim to characterise the temporal evolution of their transcriptomic profile throughout the pathology with the goal of gaining a better insight into the complex secondary SCI mechanisms.
We anticipate our results will ultimately lead to the identification of novel therapeutic targets for future molecular approaches with significant translational value in complex SCIs and the potential to compare and contrast with other reactive myeloid cell profiles in the future.
Exosomes and Extracellular Vesicles
A key mechanism by which cells can communicate with each other is through the transfer of small fluid-filled membranous particles called extracellular vesicles (EVs). EVs are heterogenous in nature and contain a biomolecular cargo comprising nucleic acids, proteins, and lipids. The makeup of this cargo depends on the type of vesicle, cell source, and the conditions in which the EV was generated. The intercellular exchange of EVs and their contents is believed to play an essential role in the maintenance of homeostasis. Conversely, aberrations in the release or composition of EVs may otherwise reflect some cellular dysfunction, and thus EVs have potential significance as biomarkers of disease or injury.
Cellular therapies are thought to owe at least part of their therapeutic effect to the actions of secreted EVs on pathophysiological processes, and therefore there has been a growing interest in the use of EVs as a cell-free therapy. Indeed, recent work from our lab has shown that induced neural stem cell (iNSC)-derived EVs are metabolically active, trafficking an enzyme analogous to those used in anti-leukaemia applications, and that they can transfer functional mitochondria to metabolically-dysfunctional immune cells.
On this basis, our lab is further exploring the role of EVs – particularly those from neural stem cells – in health and disease, and elaborating their potential therapeutic use. Towards this end we are also collaborating with biotechnology companies on developing EVs a versatile, multifunctional drug delivery platform.
Left, Transmission electron microscopy (TEM)-based whole mount negative staining of NSC extracellular vesicles. Right, Scanning electron microscopy (SEM)-based visualisation of extracellular membrane vesicles associated with a long cellular process on a NSC [from Nature Chemical Biology volume 13, pages951–955(2017)]. Credits: JM Garcia-Verdugo (University of Valencia, Spain).