Ermanna Turano

The olfactory neuroepithelium (ON) collected with nasal swabs (NS) is a valuable source for studying brain processes in physiological and pathological conditions, due to its anatomical proximity to the brain and heterogeneous neural cell population. In this proof-of-concept study, we developed a protocol for co-culturing distinct cellular populations from ON, establishing a human olfactory neuroepithelium cell culture (hON) model from healthy individuals (HIs) without differentiation factors. We characterized the cellular composition of ON samples at harvesting, identifying sustentacular cells (Pan-CK⁺), neuronal cells (PGP9.5⁺), and ciliated respiratory cells (βIV-tubulin⁺) by immunofluorescence and flow cytometry. We assessed the expression and distribution of TDP-43, Tau, and α-synuclein, together with their phosphorylated isoforms, to establish a physiological reference profile. Furthermore, Seeding Amplification Assay (SAA) analysis, including disease-positive and healthy control cohorts, confirmed the absence of aggregated protein species in HIs. While p-Tau and p-α-syn localization was preserved between collection and culture, p-TDP-43 displayed neuron-specific nuclear redistribution upon culture, suggesting distinct regulatory dynamics. Longitudinal monitoring revealed that hON cultures maintain a stable neuronal population for up to 21 days, after which cells transition toward a fibroblast-like phenotype. This study establishes NS-derived hON as a feasible model for investigating neurodegenerative mechanisms, providing a baseline for disease comparisons.

Magnetic Particle Imaging (MPI) detects superparamagnetic nanoparticles, enabling bimodal contrast with MRI. Resovist^®/Ferucarbotran, still used in research, has clinical safety compatibility but suboptimal MPI performance due to many small 5 nm SPIO cores. Magnetic fractionation can enrich larger cores, improving MPI signal and supporting cellular imaging applications. To enable bimodal MRI/MPI and assess in vivo extracellular vesicle (EV) labelling, we characterized the imaging sensitivity and cell-labelling performance of VivoTrax, a commercial formulation similar to Resovist^®, and VivoTrax Plus, obtained by magnetic fractionation. VivoTrax Plus showed higher MRI transverse relaxivity and MPI sensitivity than VivoTrax, and both formulations displayed low toxicity toward adipose-derived stem cells (ASCs). VivoTrax Plus allowed MRI detection of small cell numbers, around 100 cells, in agarose phantoms with greater sensitivity than VivoTrax. However, EVs of 30–150 nm isolated from ASCs labelled with VivoTrax Plus did not retain SPIONs, whereas EVs from VivoTrax-labelled ASCs did. Preliminary in vivo experiments in SOD-G93A mice showed MRI-detectable signal voids in the brain after intranasal EV administration, suggesting EV migration to lesioned areas. Overall, magnetic fractionation improves SPION imaging sensitivity but may alter relevant biological properties.

Adipose stem cells (ASCs) are a subset of mesenchymal stem cells with validated immunomodulatory and regenerative capabilities that make them attractive tools for treating neurodegenerative disorders, such as multiple sclerosis (MS). Several studies conducted on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, have clearly shown a therapeutic effect of ASCs. However, controversial data on their efficacy were obtained from I- and II-phase clinical trials in MS patients, highlighting standardization issues and limited data on long-term safety. In this context, ASC-derived extracellular vesicles from (ASC-EVs) represent a safer, more reproducible alternative for EAE and MS treatment. Moreover, their physical characteristics lend themselves to a non-invasive, efficient, and easy handling of intranasal delivery. Using an in vitro setting, we first verified ASC-EVs’ ability to cross the human nasal epithelium under an inflammatory milieu. Magnetic resonance corroborated these data in vivo in intranasally treated MOG35-55-induced EAE mice, showing a preferential accumulation of ASC-EVs in brain-inflamed lesions compared to a stochastic distribution in healthy control mice. Moreover, intranasal treatment of ASC-EVs at the EAE onset led to a long-term therapeutic effect using two different experimental protocols. A marked reduction in T cell infiltration, demyelination, axonal damage, and cytokine production were correlated to EAE amelioration in ASC-EV-treated mice compared to control mice, highlighting the immunomodulatory and neuroprotective roles exerted by ASC-EVs during EAE progression. Overall, our study paves the way for promising clinical applications of self-administered ASC-EV intranasal treatment in CNS disorders, including MS.

The objective of this study was to determine, at the time of diagnosis, a CSF and MRI profile of intrathecal compartmentalized inflammation predictive of progression independent of relapse activity (PIRA) in early relapsing-remitting multiple sclerosis (RRMS). This five-year prospective study included 80 treatment-naïve patients with RRMS enrolled at time of diagnosis. All patients underwent a lumbar puncture, regular neurologic evaluations including an Expanded Disability Status Scale (EDSS) assessment every 6 months, and an annual 3T brain MRI. PIRA was defined as having a confirmed disability progression independent of relapse activity. CSF levels of 68 inflammatory molecules were evaluated in combination with white matter and cortical lesion number (CLn) and volume, and regional gray matter thickness and volume. During the follow-up, 23 patients with RRMS (28.8%) experienced PIRA. At diagnosis, participants with PIRA were older (44.0 ± 10.7 vs 37.4 ± 12.4, p = 0.017) and with more disability (median EDSS score [interquartile range] of 3 [range 2-4] for PIRA vs 1.5 [range 1-2] for no PIRA group, p < 0.001). Random forest selected LIGHT, CXCL13, sTNFR1, sTNFR2, CCL7, MIF, sIL6Rbeta, IL35, CCL2, and IFNβ as the CSF markers best associated with PIRA. sTNFR1 (hazard ratio [HR] 10.11 [2.61-39.10], p = 0.001), sTNFR2 (HR 5.05 [1.63-15.64], p = 0.005), and LIGHT (HR 1.79 [1.11-2.88], p = 0.018) were predictors of PIRA at regression analysis. Baseline thalamus volume (HR 0.98 [0.97-0.99], p = 0.005), middle frontal gyrus thickness (HR 0.05 [0.01-0.72], p = 0.028), and CLn (HR 1.15 [1.05-1.25], p = 0.003) were MRI predictors of PIRA. A specific intrathecal inflammatory profile associated with TNF superfamily markers, CLn, and atrophy of several cortical and deep gray matter regions, assessed at time of diagnosis, is predictive of PIRA in early MS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons (MNs) in the brain and spinal cord, leading to progressive paralysis and death. Increasing evidence indicates that neuroinflammation plays an important role in ALS’s pathogenesis and disease progression. Neuroinflammatory responses, primarily driven by activated microglia and astrocytes, and followed by infiltrating peripheral immune cells, contribute to exacerbate/accelerate MN death. In particular, the role of the microglia in ALS remains unclear, partly due to the lack of experimental models that can fully recapitulate the complexity of ALS’s pathology. In this study, we developed and characterized a microglial cell line, SIM-A9-expressing human mutant protein Cu⁺/Zn⁺ superoxide dismutase_1 (SIM-A9hSOD1(G93A)), as a suitable model in vitro mimicking the microglia activity in ALS. The expression of hSOD1(G93A) in SIM-A9 cells induced a change in their metabolic activity, causing polarization into a pro-inflammatory phenotype and enhancing reactive oxygen species production, which is known to activate cell death processes and apoptosis. Afterward, we used our microglial model as an experimental set-up to investigate the therapeutic action of extracellular vesicles isolated from adipose mesenchymal stem cells (ASC-EVs). ASC-EVs represent a promising therapeutic treatment for ALS due to their neuroprotective and immunomodulatory properties. Here, we demonstrated that treatment with ASC-EVs is able to modulate activated ALS microglia, reducing their metabolic activity and polarizing their phenotype toward an anti-inflammatory one through a mechanism of reduction of reactive oxygen species.

Tumor necrosis factor (TNF) is a pleiotropic cytokine regulating many physiological and pathological immune-mediated processes. Although initially described as a tumor-cytotoxic agent, over time, it has also been recognized as an essential pro-inflammatory cytokine implicated in the pathogenesis of different autoimmune diseases, such as multiple sclerosis (MS). Several studies have shown an increase in TNF expression both in acute and chronic active MS brain lesions, as well as its involvement in maintaining a chronic inflammatory intrathecal process known as “compartmentalized inflammation”, which contributes to disease progression and disability accumulation in MS, even more than acute inflammatory activity. Indeed, high TNF levels were observed in the serum and cerebrospinal fluid of subjects with MS and correlated to disease severity, promoting axonal damage and, consequently, neurodegeneration. In this review we discuss the current knowledge of TNF and its receptors involvement in MS progression, focusing on disability progression independent of relapse activity (PIRA), meant as the disability accumulation evident in relapsing MS patients not related to acute inflammatory events.

Neurodegenerative diseases are fatal disorders of the central nervous system (CNS) which currently lack effective treatments. The application of mesenchymal stem cells (MSCs) represents a new promising approach for treating these incurable disorders. Growing evidence suggest that the therapeutic effects of MSCs are due to the secretion of neurotrophic molecules through extracellular vesicles. The extracellular vesicles produced by MSCs (MSC-EVs) have valuable innate properties deriving from parental cells and could be exploited as cell-free treatments for many neurological diseases. In particular, thanks to their small size, they are able to overcome biological barriers and reach lesion sites inside the CNS. They have a considerable pharmacokinetic and safety profile, avoiding the critical issues related to the fate of cells following transplantation. This review discusses the therapeutic potential of MSC-EVs in the treatment of neurodegenerative diseases, focusing on the strategies to further enhance their beneficial effects such as tracking methods, bioengineering applications, with particular attention to intranasal delivery as a feasible strategy to deliver MSC-EVs directly to the CNS in an effective and minimally invasive way. Current progresses and limiting issues to the extent of the use of MSC-EVs treatment for human neurodegenerative diseases will be also revised.

The impact of disease-modifying therapies (DMTs) on the immune response to coronavirus disease-2019 (COVID-19) vaccines in persons with multiple sclerosis (pwMS) needs further elucidation. We investigated BNT162b2 mRNA COVID-19 vaccine effects concerning antibody seroconversion, inflammatory mediators’ level and immunophenotype assessment in pwMS treated with cladribine (c-pwMS, n = 29), fingolimod (f-pwMS, n = 15) and ocrelizumab (o-pwMS, n = 54). Anti-spike immunoglobulin (Ig)-G detection was performed by an enzyme immunoassay; molecular mediators (GrB, IFN-γ and TNF-α) were quantified using the ELLA platform, and immunophenotype was assessed by flow cytometry. ANCOVA, Student’s t-test and Pearson correlation analyses were applied. Only one o-pwMS showed a mild COVID-19 infection despite most o-pwMS lacking seroconversion and showing lower anti-spike IgG titers than c-pwMS and f-pwMS. No significant difference in cytokine production and lymphocyte count was observed in c-pwMS and f-pwMS. In contrast, in o-pwMS, a significant increase in GrB levels was detected after vaccination. Considering non-seroconverted o-pwMS, a significant increase in GrB serum levels and CD4+ T lymphocyte count was found after vaccination, and a negative correlation was observed between anti-spike IgG production and CD4+ T cells count. Differences in inflammatory mediators’ production after BNT162b2 vaccination in o-pwMS, specifically in those lacking anti-spike IgG, suggest a protective cellular immune response.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder. There is no cure and current treatments fail to slow the progression of the disease. Epigenetic modulation in the acetylation state of NF-kB RelA and the histone 3 (H3) protein, involved in the development of neurodegeneration, is a drugable target for the class-I histone deacetylases (HDAC) inhibitors, entinostat or valproate, and the AMP-activated kinase (AMPK)-sirtuin 1 pathway activator, resveratrol. In this study, we demonstrated that the combination of valproate and resveratrol can restore the normal acetylation state of RelA in the SOD1(G93A) murine model of ALS, in order to obtain the neuroprotective form of NF-kB. We also investigated the sexually dimorphic development of the disease, as well as the sex-sensibility to the treatment administered. We showed that the combined drugs, which rescued AMPK activation, RelA and the histone 3 acetylation state, reduced the motor deficit and the disease pathology associated with motor neuron loss and microglial reactivity, Brain-Derived Neurotrophic Factor (BDNF) and B-cell lymphoma-extra large (Bcl-xL) level decline. Specifically, vehicle-administered males showed earlier onset and slower progression of the disease when compared to females. The treatment, administered at 50 days of life, postponed the time of onset in the male by 22 days, but not in a significant way in females. Nevertheless, in females, the drugs significantly reduced symptom severity of the later phase of the disease and prolonged the mice’s survival. Only minor beneficial effects were produced in the latter stage in males. Overall, this study shows a beneficial and sexually dimorphic response to valproate and resveratrol treatment in ALS mice.