Cortical myelin loss and oligodendroglial dysfunction, hallmarks of numerous neurodegenerative and psychiatric disorders, compromise executive, sensory, and cognitive functions by altering circuit activity and integrity. Promoting cortical myelin repair therefore represents a critical therapeutic goal. In this frame, modulation of neuronal activity through non-invasive brain stimulation may represent a promising non-pharmacological approach to support remyelination. To address this issue, we investigated the effects of anodal transcranial direct current stimulation (A–tDCS) in a mouse model of unilateral myelin injury in the motor cortex. A–tDCS was delivered over the contralateral uninjured cortex during a bilateral motor task (locomotion), to enhance the physiological activity of interhemispheric M1–M1 projections and indirectly stimulate the lesioned cortex. When applied during the late phase of demyelination and the onset of repair, A–tDCS accelerated remyelination and increased oligodendroglial survival and maturation in the lesioned cortex, compared with unstimulated controls. Increased myelin was also detected in the directly stimulated uninjured cortex despite unchanged oligodendroglial cell numbers, suggesting de novo myelin formation or remodeling of existing internodes. These structural changes were accompanied by preservation of interhemispheric M1–M1 functional connectivity upon injury. Collectively, our findings demonstrate that A–tDCS promotes cortical myelin repair in vivo, supporting its therapeutic potential for disorders characterized by myelin damage. Notably, the observed effects of tDCS on myelin plasticity in the uninjured cortex – reported here for the first time – reveal a previously unrecognized mechanism underlying tDCS-mediated neuromodulation. ### Competing Interest Statement The authors have declared no competing interest. Ministero dell'università e della ricerca, https://ror.org/0341vw408, Dipartimenti di Eccellenza 2023–2027 to Dept of Neuroscience Rita Levi Montalcini (University of Turin) FISM - Fondazione Italiana Sclerosi Multipla, cod. 2020/PR-Single/028 to MC Ministero dell'università e della ricerca, “A multiscale integrated approach to the study of the nervous system in health and disease (MNESYS)”, CUP B33C22001060002, PE00000006 missione 4, componente 2, investimento 1.3

The application of anodal-transcranial direct current stimulation (A-tDCS) over the primary motor cortex (M1) increases its structural and functional plasticity, as also physical exercise. Combining both interventions has a boosting effect, thus revealing a crucial role of the brain state during stimulation. Although brain slice and anesthetized animal studies support this, further investigation in awake animals is necessary. In the present study, we analyzed the effects of coupling A-tDCS with low-intensity physical activity on the mouse M1 structural and functional plasticity. C57BL/6 mice were monolaterally treated with M1 A-tDCS while walking on a rotarod or at rest. To assess the impact of our interventions, we analyzed both motor cortices for changes in neuronal activation, dendritic spine density, and functional synchronisation as measured by local field potential coherence. The combination of physical activity and M1 stimulation revealed a synergistic interhemispheric effect on cortical activation in both layers II/III and V, not present when using a single type of intervention. These data were accompanied by increased M1-M1 synchrony in the low-theta frequency, a hallmark of motor network activity in mice. Dendritic spine density revealed an effect of the combo, which was significantly higher only in layer II/III, accompanied by increased post-synaptic density protein 95 expression in the same area. Based on our findings, we propose that the efficacy of tDCS hinges on brain state rather than being merely a direct causal factor. The observed outcomes contribute to a deeper comprehension of the mechanisms governing structural and functional reorganisation within the motor cortex under physiological conditions, with potential implications for research on learning, memory, and neurological disorders such as stroke.

Protein semisynthesis approaches are key for gaining insights into the effects of post-translational modifications (PTMs) on the structure and function of modified proteins. Among PTMs, ubiquitination involves the conjugation of a small protein modifier to a substrate amino acid residue and is unique in controlling a variety of cellular processes. Interest has grown in understanding the role of ubiquitination in neurodegenerative conditions, including tauopathies. The latter are characterized by the accumulation of the intrinsically disordered protein tau in the form of neurofibrillary tangles in the brains of patients. The presence of ubiquitinated tau in the pathological aggregates suggests that ubiquitination might play a role in the formation of abnormal protein deposits. In this study, we developed a new strategy, based on dehydroalanine chemistry, to install wild type ubiquitin on a tau repeat domain construct with site-specificity. We optimized a three-step reaction which yielded a good amount of highly pure tau repeat domain ubiquitinated in position 353. The structural features of the conjugate were examined by circular dichroism and NMR spectroscopy. The ubiquitinated tau was challenged in a number of assays: fibrils formation under aggregating conditions in vitro, chemical stability upon exposure to a variety of biological media including cell extracts, and internalization into astrocytes. The results demonstrated the wide applicability of the new semisynthetic strategy for the investigation of ubiquitinated substrates in vitro or in cell, and in particular for studying if ubiquitination has a role in the molecular mechanisms that underlie the aberrant transition of tau into pathological aggregates.

The association between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) has been extensively demonstrated, but despite this, the pathophysiological mechanisms underlying it are still unknown. In previous work, we discovered a central role for the autophagy pathway in the common alterations observed between AD and T2DM. In this study, we further investigate the role of genes belonging to this pathway, measuring their mRNA expression and protein levels in 3xTg-AD transgenic mice, an animal model of AD. Moreover, primary mouse cortical neurons derived from this model and the human H4Swe cell line were used as cellular models of insulin resistance in AD brains. Hippocampal mRNA expression showed significantly different levels for Atg16L1, Atg16L2, GabarapL1, GabarapL2, and Sqstm1 genes at different ages of 3xTg-AD mice. Significantly elevated expression of Atg16L1, Atg16L2, and GabarapL1 was also observed in H4Swe cell cultures, in the presence of insulin resistance. Gene expression analysis confirmed that Atg16L1 was significantly increased in cultures from transgenic mice when insulin resistance was induced. Taken together, these results emphasise the association of the autophagy pathway in AD-T2DM co-morbidity, providing new evidence about the pathophysiology of both diseases and their mutual interaction.

Transcranial direct current stimulation (tDCS) has been receiving increasing attention as an effective tool to modulate motor cortical excitability, although the underlying mechanisms still need to be clarified. Animal studies revealed that tDCS over the primary motor cortex (M1) affects functional plasticity, however, growing evidence suggests that tDCS might also affect the structural plasticity by modulating dendritic spine density and morphology. Similarly, physical activity is known to induce structural synaptic plasticity, also by increasing the dendritic spine density in M1 neurons. So far, the morphological effects of tDCS are poorly known, though there is a strong interest in coupling tDCS and physical activity as a strategy to further enhance cortical excitability in physiological and pathological conditions, ranging from neuroenhancement to aging and from brain ischemia to neurological disorders. The aim of our project is to study the effects of coupling physical activity and anodal tDCS over the M1 in mice. We investigated the effects of this combination on the morphological plasticity in layer II/III and layer V during aging in both young (2-3 months) and middle-age (14-16 months) mice. Our data indicates that, at both ages, the combination of stimulation and physical activity results in an increased number of activated cells and a higher spine density in basal and apical dendrites of both hemispheres, compared to tDCS or physical activity alone. Additionally, young mice displayed a higher number of mushroom spines, while middle-aged mice showed an increase of thin spines when the pairing occurs. Altogether, the combination of tDCS and the physiological activation of the motor cortex results in a boosting effect of neural plasticity. Research Category and Technology and Methods Basic Research: 9. Transcranial Direct Current Stimulation (tDCS) Keywords: neural plasticity, boosting , motor cortex, physical activity

EDITORIAL article Front. Syst. Neurosci., 06 February 2023 Volume 17 - 2023 | https://doi.org/10.3389/fnsys.2023.1128205

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites 1 . Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus , provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage 2 . Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and α-ketoglutarate-αKG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo . Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive neuromodulation technique, and it has been increasingly used as a nonpharmacological intervention for the treatment of various neurological and neuropsychiatric diseases, including depression. In humans, rTMS over the prefrontal cortex is used to induce modulation of the neural circuitry that regulates emotions, cognition, and depressive symptoms. However, the underlying mechanisms are still unknown. In this study, we investigated the effects of a short (5-day) treatment with high-frequency (HF) rTMS (15 Hz) on emotional behavior and prefrontal cortex morphological plasticity in mice. Mice that had undergone HF-rTMS showed an anti-depressant-like activity as evidenced by decreased immobility time in both the Tail Suspension Test and the Forced Swim Test along with increased spine density in both layer II/III and layer V apical and basal dendrites. Furthermore, dendritic complexity assessed by Sholl analysis revealed increased arborization in the apical portions of both layers, but no modifications in the basal dendrites branching. Overall, these results indicate that the antidepressant-like activity of HF-rTMS is paralleled by structural remodeling in the medial prefrontal cortex.

Early stroke therapeutic approaches rely on limited options, further characterized by a narrow therapeutic time window. In this context, the application of transcranial direct current stimulation (tDCS) in the acute phases after brain ischemia is emerging as a promising non-invasive tool. Despite the wide clinical application of tDCS, the cellular mechanisms underlying its positive effects are still poorly understood. Here, we explored the effects of cathodal tDCS (C-tDCS) 6 h after focal forelimb M1 ischemia in Cx3CR1GFP/+ mice. C-tDCS improved motor functionality of the affected forelimb, as assessed by the cylinder and foot-fault tests at 48 h, though not changing the ischemic volume. In parallel, histological analysis showed that motor recovery is associated with decreased microglial cell density in the area surrounding the ischemic core, while astrocytes were not affected. Deeper analysis of microglia morphology within the perilesional area revealed a shift toward a more ramified healthier state, with increased processes' complexity and a less phagocytic anti-inflammatory activity. Taken together, our findings suggest a positive role for early C-tDCS after ischemia, which is able to modulate microglia phenotype and morphology in parallel to motor recovery.

Tumor associated macrophages (TAMs) are the mostprevalent cells recruited in the tumor microenvironment (TME). Once recruited, TAMs acquire a pro-tumor phenotype characterized by a typical morphology: ameboid in the tumor core and with larger soma and thick branches in the tumor periphery. Targeting TAMs by reverting them to an anti-tumor phenotype is a promising strategy for cancer immunotherapy. Taking advantage of Cx3cr1GFP/WT heterozygous mice implanted with murine glioma GL261-RFP cells we investigated the role of Ca2+-activated K+ channel (KCa3.1) on the phenotypic shift of TAMs at the late stage of glioma growth through in vivo two-photon imaging. We demonstrated that TAMs respond promptly to KCa3.1 inhibition using a selective inhibitor of the channel (TRAM-34) in a time-dependent manner by boosting ramified projections attributable to a less hypertrophic phenotype in the tumor core. We also revealed a selective effect of drug treatment by reducing both glioma cells and TAMs in the tumor core with no interference with surrounding cells. Taken together, our data indicate a TRAM-34-dependent progressive morphological transformation of TAMs toward a ramified and anti-tumor phenotype, suggesting that the timing of KCa3.1 inhibition is a key point to allow beneficial effects on TAMs.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a −/− mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a −/− basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a −/− Lyn −/− showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a −/− hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.

Approaches utilizing multiple analysis techniques on a single sample are highly desirable in research, especially to reduce the number of animals and obtain the maximum information. Golgi-Cox staining is a widely used method for characterizing axon and dendritic morphology and several attempts to combine this technique with immunofluorescence and transmission electron microscopy have been proposed. With few exceptions, most of the protocols were characterized by a high degree of complexity and low reproducibility. Here we show a simplified procedure of perfusion, fixation and staining of brain tissues that allows Golgi-Cox staining, immunofluorescence and transmission electron microscopy in the same sample, to obtain high-quality images with a low-cost procedure. The main novelty in this protocol is the possibility of performing Golgi-Cox staining after the perfusion and post-fixation of brain tissue with a buffered solution containing, not only formaldehyde, but also glutaraldehyde. This renders the tissue suitable for electron microscopy, but it is also compatible with immunofluorescence staining. This combined protocol can be used in most neuroscience laboratories as it does not require special equipment and skills. This protocol will be useful in a broad range of neuroscience topics to study morphological changes during brain development and plasticity in physiological and pathological conditions.

High-frequency repeated transcranial magnetic stimulation (HF-rTMS) is a safe non-invasive neuromodulatory technique and there is a body of evidence shows that it can modulate plasticity in different brain areas. One of the most interesting application of HF-rTMS is the modulation of plasticity in primary motor cortex (M1) to promote recovery after brain injuries. However, the underlying mechanism by which HF-rTMS modulates motor cortex plasticity remain to be investigated. In this study, we investigated the effects of HF-rTMS treatment on morphological plasticity of pyramidal neurons in layer II/III (L2/3) of the primary motor cortex in mice. Our results show that the treatment did not increase anxiety in mice in the open field test and the elevated plus-maze test. Treated mice displayed increased total spine density in apical and basal dendrites, with a predominance of thin spines. The treatment also increased dendritic complexity, as assessed by Sholl analysis at both apical and basal dendrites. Collectively, the results show that HF-rTMS induced remarkable changes in dendritic complexity in primary motor cortex L2/3 connections which may strengthen corticocortical connections increasing integration of information across cortical areas. The data support the use of HF-rTMS as a circuit-targeting neuromodulation strategy.

Transcranial direct current stimulation (tDCS) is being increasingly considered as a non-pharmacological alternative for various psychiatric and neurological disorders. In major depressive disorder (MDD), anodal tDCS applied over the left dorsolateral prefrontal cortex (DL-PFC) improves symptoms in drug-resistant patients [1]. However, the molecular mechanisms underlying the efficacy of tDCS in MDD are still unknown. One of the main neurotransmitters involved in the pathophysiology and psychopharmacology of MDD is serotonin (5-HT), released by neurons of the dorsal raphe nucleus (DRN), which have widespread projections to cortical, subcortical and brainstem regions.

The critical role of neuroinflammation in favoring and accelerating the pathogenic process in Alzheimer's disease (AD) increased the need to target the cerebral innate immune cells as a potential therapeutic strategy to slow down the disease progression. In this scenario, mesenchymal stem cells (MSCs) have risen considerable interest thanks to their immunomodulatory properties, which have been largely ascribed to the release of extracellular vesicles (EVs), namely exosomes and microvesicles. Indeed, the beneficial effects of MSC-EVs in regulating the inflammatory response have been reported in different AD mouse models, upon chronic intravenous or intracerebroventricular administration. In this study, we use the triple-transgenic 3xTg mice showing for the first time that the intranasal route of administration of EVs, derived from cytokine-preconditioned MSCs, was able to induce immunomodulatory and neuroprotective effects in AD. MSC-EVs reached the brain, where they dampened the activation of microglia cells and increased dendritic spine density. MSC-EVs polarized in vitro murine primary microglia toward an anti-inflammatory phenotype suggesting that the neuroprotective effects observed in transgenic mice could result from a positive modulation of the inflammatory status. The possibility to administer MSC-EVs through a noninvasive route and the demonstration of their anti-inflammatory efficacy might accelerate the chance of a translational exploitation of MSC-EVs in AD.

Low-frequency repetitive transcranial magnetic stimulation (1-Hz rTMS) is a promising noninvasive tool for the treatment of depression. Hippocampal neuronal plasticity is thought to play a pivotal role in the pathophysiology of depressive disorders and the mechanism of action of antidepressant treatments. We investigated the effect of 1-Hz rTMS treatment on hippocampal dentate gyrus structural plasticity and related emotional behaviors modifications. Experimentally, adult male mice received either five days of 1-Hz rTMS or Sham stimulation. After stimulation, the mice underwent a battery of tests for anxiety-like and depression-like behaviors. We also tested the effect of treatment on mature and newly generated granule cell dendritic complexity. Our data showed that 1-Hz rTMS induced structural plasticity in mature granule cells, as evidenced by increased dendritic length and number of intersections. However, the stimulation did not increase the proliferation of the dentate gyrus progenitor cells. On the contrary, the stimulated mice showed increased dendritic complexity of newly generated neurons. Moreover, 1-Hz rTMS resulted in antidepressant-like effects in the tail suspension test, but it did not affect anxiety-like behaviors. Therefore, our results indicate that 1-Hz rTMS modulates dentate gyrus morphological plasticity in mature and newly generated neurons. Furthermore, our data provide some evidence of an association between the antidepressant-like activity of 1-Hz rTMS and structural plasticity in the hippocampus.

The olfactory neuroepithelium is located in the upper vault of the nasal cavity, lying on the olfactory cleft and projecting into the dorsal portion of the superior and middle turbinates beyond the mid-portion of the nasal septum. It is composed of a variety of cell types including olfactory sensory neurons, supporting glial-like cells, microvillar cells, and basal stem cells. The cells of the neuroepithelium are often intermingled with respiratory and metaplastic epithelial cells. Olfactory neurons undergo a constant self-renewal in the timespan of 2–3 months; they are directly exposed to the external environment, and thus they are vulnerable to physical and chemical injuries. The latter might induce metabolic perturbations and ultimately be the cause of cell death. However, the lifespan of olfactory neurons is biologically programmed, and for this reason, these cells have an accelerated metabolic cycle leading to an irreversible apoptosis. These characteristics make these cells suitable for research related to nerve cell degeneration and aging. Recent studies have shown that a non-invasive and painless olfactory brushing procedure allows an efficient sampling from the olfactory neuroepithelium. This approach allows to detect the pathologic prion protein in patients with sporadic Creutzfeldt–Jakob disease, using the real-time quaking-induced conversion assay. Investigating the expression of all the proteins associated to neurodegeneration in the cells of the olfactory mucosa is a novel approach toward understanding the pathogenesis of human neurodegenerative diseases. Our aim was to investigate the expression of α-synuclein, β-amyloid, tau, and TDP-43 in the olfactory neurons of normal subjects. We showed that these proteins that are involved in neurodegenerative diseases are expressed in olfactory neurons. These findings raise the question on whether a relationship exists between the mechanisms of protein aggregation that occur in the olfactory bulb during the early stage of the neurodegenerative process and the protein misfolding occurring in the olfactory neuroepithelium.

BACKGROUND Acute recurrent pancreatitis (ARP) is characterized by episodes of acute pancreatitis in an otherwise normal gland. When no cause of ARP is identifiable, the diagnosis of "idiopathic" ARP is given. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene increase the risk of ARP by 3- to 4-times compared to the general population, while cystic fibrosis (CF) patients present with a 40- to 80-times higher risk of developing pancreatitis. CASE SUMMARY In non-classical CF or CFTR-related disorders, CFTR functional tests can help to ensure a proper diagnosis. We applied an individualized combination of standardized and new CFTR functional bioassays for a patient referred to the Verona CF Center for evaluation after several episodes of acute pancreatitis. The CFTR genotype was G542X+/- with IVS8Tn:T7/9 polymorphism. The sweat (Cl-) values were borderline. Intestinal current measurements were performed according to the European Cystic Fibrosis Society Standardized Operating Procedure. Recent nasal surgery for deviated septum did not allow for nasal potential difference measurements. Lung function and sputum cultures were normal; azoospermia was excluded. Pancreas divisum was excluded by imaging but hypoplasia of the left hepatic lobe was detected. Innovative tests applied in this case include sweat rate measurement by image analysis, CFTR function in monocytes evaluated using a membrane potential-sensitive fluorescent probe, and the intestinal organoids forskolin-induced swelling assay. CONCLUSION Combination of innovative CFTR functional assays might support a controversial diagnosis when CFTR-related disorders and/or non-classical CF are suspected.