Therapeutic Induction of Energy Metabolism Reduces Neural Tissue Damage and Increases Microglia Activation in Severe Spinal Cord Injury

: Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged, tissue suffers from a severe metabolic impairment, which aggravates axonal degeneration and, neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative, phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring the spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and ROS defense which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase (ChAT)-expressing motor neurons and increased neurofilament positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated microglia expressing the CD206 marker suggestive of a, pro-resolutive, M2-like phenotype. These molecular and histological modifications observed in treated animals ultimately led to a significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism can represent an effective strategy to treat SCI

The role of meninges and meningeal neural progenitor cells in a paradigm of enriched environment and in multiple sclerosis

Leptomeninges have been indicated as a novel neurogenic niche hosting neural stem cells (NSCs) able to generate neurons which can migrate and integrate into the brain cortex (Bifari et al. 2015; Bifari et al. 2009; Bifari et al. 2017; Decimo et al. 2011; Nakagomi et al. 2011; Pino et al. 2017). Nowadays, NSC populations are characterized by dynamic behaviour in response to environmental stimuli suggesting a possible role in physiologic and pathologic conditions (Decimo et al. 2012a; Decimo et al. 2020). In literature, is known that pro neurogenic paradigms such as EE and anti-depressant treatments affect hippocampal neurogenesis. However, the effect on meninges is still unexplored (David et al. 2009; Eisinger and Zhao 2018; Kempermann 2019). Furthermore, meninges are able to promptly respond also to neural pathological states (Lin et al. 2015; Nakagomi et al. 2011; Nakagomi et al. 2012; Ninomiya et al. 2013). In fact, it has been reported that the meningeal NSCs actively reacts to CNS insults (Dang et al. 2019; Decimo et al. 2011; Ninomiya et al. 2013). However, the specific role of meningeal NSCs in autoimmune disease is still unknown. The overall aim of my thesis was to investigate how meningeal niche can respond to neurogenic stimuli contributing to brain plasticity in physiological conditions and whether and how the meningeal NSC niche can be actively involved in response to autoinflammatory pathological stimuli. Enriched environment (EE), a particular housing condition which can offer enhanced sensory, cognitive or motor stimulation to the animal, and antidepressant treatment, are pro-neurogenic stimuli capable of increasing the number of newborn neurons in the DG of treated animals (Eisinger and Zhao 2018; Khodanovich et al. 2018). In the first part of the thesis, I characterized meningeal stem cell niche of young mice after exposure to EE and following administration of the selective serotonin reuptake inhibitor antidepressant Fluoxetine. By using immunofluorescence confocal analysis, western blot and RT-PCR we found that neural progenitors (GLAST+ cells) and immature neurons (β3-tubulin+ cells) were increased after the treatments and that BDNF played a pivotal role in this context, suggesting that the EE exposure was able to activate the meningeal niche. Interestingly, meningeal niche was found to be responsive also to a paradigm of Fluoxetine administration by increasing, similarly to EE exposure observations, GLAST+ neural precursors and β3-Tubulin+ immature neurons. These results confirm a reaction of meningeal niche to a different pro-neurogenic stimulus. 12 Then, we questioned about the role of meningeal niche in pathological stimuli. Exploiting the Experimental Autoimmune Encephalomyelitis (EAE) animal model, we studied the impact of the neurodegenerative Multiple Sclerosis (MS) disease on NSC meningeal population. Specifically, due to the inflammatory condition established following the development of MS disease, we focused the attention on the relationship between immune cells and NSCs and on the role of brain and spinal cord meninges as possible entry way for immune cells to the CNS parenchyma following inflammatory stimuli. By means of immunofluorescence and histological staining we found an increased number of NSCs and immune cells in meninges of brain and spinal cord animals and, in these sites, a strong presence of infiltrates, according to pathological cell recruitment of MS (Jordão et al. 2019; Wu et al. 2010). Moreover, NSCs expressing immunological marker have been found in meninges, suggesting an involvement of meninges in trophic and immune modulatory activity (Decimo et al. 2012b). Investigating in more detail the relationship between immune cells and NSCs by a genomic approach (scRNAseq) and in vitro techniques (co-culture and FACS analysis), we found a strong link between neutrophils and NSCs suggesting that NSCs could have immunomodulatory properties (Decimo et al. 2012a; Kokaia et al. 2012). All together these results indicate that meninges not only harbour a neural stem cell niche but also highly and actively respond to different stimuli. Meninges may be modulated by neurogenic stimuli such as EE and Fluoxetine and may be one of crucial checkpoint at which auto-reactive T cells are licensed to enter CNS parenchyma during MS, thanks to a dynamic interplay between different cell populations that include NSCs. Collectively, a better understanding of NSCs role in different paradigms may help to discover the mechanism that underpins the physiological and pathological processes and can help to consider meninges as a potential pharmacological target for regenerative medicine of the CNS

Environmental Enrichment Induces Meningeal Niche Remodeling through TrkB-Mediated Signaling

Neural precursors (NPs) present in the hippocampus can be modulated by several neurogenic stimuli, including environmental enrichment (EE) acting through BDNF-TrkB signaling. We have recently identified NPs in meninges; however, the meningeal niche response to pro-neurogenic stimuli has never been investigated. To this aim, we analyzed the effects of EE exposure on NP distribution in mouse brain meninges. Following neurogenic stimuli, although we did not detect modification of the meningeal cell number and proliferation, we observed an increased number of neural precursors in the meninges. A lineage tracing experiment suggested that EE-induced \u3b23-Tubulin+ immature neuronal cells present in the meninges originated, at least in part, from GLAST+ radial glia cells. To investigate the molecular mechanism responsible for meningeal reaction to EE exposure, we studied the BDNF-TrkB interaction. Treatment with ANA-12, a TrkB non-competitive inhibitor, abolished the EE-induced meningeal niche changes. Overall, these data showed, for the first time, that EE exposure induced meningeal niche remodeling through TrkB-mediated signaling. Fluoxetine treatment further confirmed the meningeal niche response, suggesting it may also respond to other pharmacological neurogenic stimuli. A better understanding of the neurogenic stimuli modulation for meninges may be useful to improve the effectiveness of neurodegenerative and neuropsychiatric treatments