Astrocyte Responses Influence Local Effects of Whole-Brain Magnetic Stimulation in Parkinsonian Rats.

BACKGROUND: Excessive glutamatergic transmission in the striatum is implicated in Parkinson's disease (PD) progression. Astrocytes maintain glutamate homeostasis, protecting from excitotoxicity through the glutamate-aspartate transporter (GLAST), whose alterations have been reported in PD. Noninvasive brain stimulation using intermittent theta-burst stimulation (iTBS) acts on striatal neurons and glia, inducing neuromodulatory effects and functional recovery in experimental parkinsonism. OBJECTIVE: Because PD is associated with altered astrocyte function, we hypothesized that acute iTBS, known to rescue striatal glutamatergic transmission, exerts regional- and cell-specific effects through modulation of glial functions. METHODS: 6-Hydroxydopamine-lesioned rats were exposed to acute iTBS, and the areas predicted to be more responsive by a biophysical, hyper-realistic computational model that faithfully reconstructs the experimental setting were analyzed. The effects of iTBS on glial cells and motor behavior were evaluated by molecular and morphological analyses, and CatWalk and Stepping test, respectively. RESULTS: As predicted by the model, the hippocampus, cerebellum, and striatum displayed a marked c-FOS activation after iTBS, with the striatum showing specific morphological and molecular changes in the astrocytes, decreased phospho-CREB levels, and recovery of GLAST. Striatal-dependent motor performances were also significantly improved. CONCLUSION: These data uncover an unknown iTBS effect on astrocytes, advancing the understanding of the complex mechanisms involved in TMS-mediated functional recovery. Data on numerical dosimetry, obtained with a degree of anatomical details never before considered and validated by the biological findings, provide a framework to predict the electric-field induced in different specific brain areas and associate it with functional and molecular changes. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Nano-Immunomodulation: A New Strategy for Skeletal Muscle Diseases and Aging?

The skeletal muscle has a very remarkable ability to regenerate upon injury under physiological conditions; however, this regenerative capacity is strongly diminished in physio-pathological conditions, such as those present in diseased or aged muscles. Many muscular dystrophies (MDs) are characterized by aberrant inflammation due to the deregulation of both the lymphoid and myeloid cell populations and the production of pro-inflammatory cytokines. Pathological inflammation is also observed in old muscles due to a systemic change in the immune system, known as "inflammaging". Immunomodulation represents, therefore, a promising therapeutic opportunity for different skeletal muscle conditions. However, the use of immunomodulatory drugs in the clinics presents several caveats, including their low stability in vivo, the need for high doses to obtain therapeutically relevant effects, and the presence of strong side effects. Within this context, the emerging field of nanomedicine provides the powerful tools needed to control the immune response. Nano-scale materials are currently being explored as biocarriers to release immunomodulatory agents in the damaged tissues, allowing therapeutic doses with limited off-target effects. In addition, the intrinsic immunomodulatory properties of some nanomaterials offer further opportunities for intervention that still need to be systematically explored. Here we exhaustively review the state-of-the-art regarding the use of nano-sized materials to modulate the aberrant immune response that characterizes some physio-pathological muscle conditions, such as MDs or sarcopenia (the age-dependent loss of muscle mass). Based on our learnings from cancer and immune tolerance induction, we also discuss further opportunities, challenges, and limitations of the emerging field of nano-immunomodulation.

Abdominal Wall Reconstruction with a Free Fibula Flap after Internal Extended Hemipelvectomy.

Major pelvic resections for malignant tumors are infrequent and have significant morbidity and mortality, for instance, incisional hernias are postoperative complications uncommonly reported probably because most cases are overshadowed by more serious complications. Reconstruction depends on the extent of the resection and overall prognosis of the patient. A case of a late complex hypogastric and femoral incisional hernia after extended hemipelvectomy for recurrent osteosarcoma treated with distal abdominal wall fixation into a free fibula flap is reported.

Levator Palpebrae Superioris Muscle Direct Neurotization.

Direct muscle neurotization has been proved to be a feasible technique for facial reanimation microsurgical procedures. Direct muscle neurotization is performed by implanting the interposition nerve graft directly into the substance of the muscle. The authors present the case of a 36-year-old male patient with upper eyelid dysfunction secondary to facial trauma. The levator palpebrae superioris muscle was macroscopically unaffected; however, neurophysiological test proved a selective denervation of the CN III motor branch to the levator palpebrae superioris muscle. Direct muscle neurotization was performed by means of 2 separate nerve procedures. The authors have made follow-up for 3 months after surgery. The authors have noted development of upper eyelid movement meaning adequate function of the neurotized muscle. The authors believe that this procedure could be integrated into the surgical options to treat selective nerve injuries should the right patient is encountered.

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.

BACKGROUND: Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts. METHODS: Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo. RESULTS: Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo. CONCLUSIONS: These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

Zika Virus: A Neurotropic Warrior against High-Grade Gliomas-Unveiling Its Potential for Oncolytic Virotherapy.

Gliomas account for approximately 75-80% of all malignant primary tumors in the central nervous system (CNS), with glioblastoma multiforme (GBM) considered the deadliest. Despite aggressive treatment involving a combination of chemotherapy, radiotherapy, and surgical intervention, patients with GBM have limited survival rates of 2 to 5 years, accompanied by a significant decline in their quality of life. In recent years, novel management strategies have emerged, such as immunotherapy, which includes the development of vaccines or T cells with chimeric antigen receptors, and oncolytic virotherapy (OVT), wherein wild type (WT) or genetically modified viruses are utilized to selectively lyse tumor cells. In vitro and in vivo studies have shown that the Zika virus (ZIKV) can infect glioma cells and induce a robust oncolytic activity. Consequently, interest in exploring this virus as a potential oncolytic virus (OV) for high-grade gliomas has surged. Given that ZIKV actively circulates in Colombia, evaluating its neurotropic and oncolytic capabilities holds considerable national and international importance, as it may emerge as an alternative for treating highly complex gliomas. Therefore, this literature review outlines the generalities of GBM, the factors determining ZIKV's specific tropism for nervous tissue, and its oncolytic capacity. Additionally, we briefly present the progress in preclinical studies supporting the use of ZIKV as an OVT for gliomas.

Fetal exposure to valproic acid dysregulates the expression of autism-linked genes in the developing cerebellum.

Autism spectrum disorder (ASD) includes a set of highly heritable neurodevelopmental syndromes characterized by social and communication impairment, repetitive behaviour, and intellectual disability. Although mutations in multiple genes have been associated to ASD, most patients lack detectable genetic alterations. For this reason, environmental factors are commonly thought to also contribute to ASD aetiology. Transcriptome analyses have revealed that autistic brains possess distinct gene expression signatures, whose elucidation can provide insights about the mechanisms underlying the effects of ASD-causing genetic and environmental factors. Herein, we have identified a coordinated and temporally regulated programme of gene expression in the post-natal development of cerebellum, a brain area whose defects are strongly associated with ASD. Notably, this cerebellar developmental programme is significantly enriched in ASD-linked genes. Clustering analyses highlighted six different patterns of gene expression modulated during cerebellar development, with most of them being enriched in functional processes that are frequently dysregulated in ASD. By using the valproic acid mouse model of ASD, we found that ASD-linked genes are dysregulated in the developing cerebellum of ASD-like mice, a defect that correlates with impaired social behaviour and altered cerebellar cortical morphology. Moreover, changes in transcript levels were reflected in aberrant protein expression, indicating the functional relevance of these alterations. Thus, our work uncovers a complex ASD-related transcriptional programme regulated during cerebellar development and highlight genes whose expression is dysregulated in this brain area of an ASD mouse model.

Nanomedicine, a valuable tool for skeletal muscle disorders: Challenges, promises, and limitations.

Muscular dystrophies are a group of rare genetic disorders characterized by progressive muscle weakness, which, in the most severe forms, leads to the patient's death due to cardiorespiratory problems. There is still no cure available for these diseases and significant effort is being placed into developing new strategies to either correct the genetic defect or to compensate muscle loss by stimulating skeletal muscle regeneration. However, the vast anatomical extension of the target tissue poses great challenges to these goals, highlighting the need for complementary strategies. Nanomedicine is an actively evolving field that merges nanotechnologies with biomedical and pharmaceutical sciences. It holds great potential in regenerative medicine, both in supporting tissue engineering and regeneration, and in optimizing drug and oligonucleotide delivery and gene therapy strategies. In this review, we will summarize the state-of-the-art in the field of nanomedicine applied to skeletal muscle regeneration. We will discuss the recent work toward the development of nanopatterned scaffolds for tissue engineering, the efforts in the synthesis of organic and inorganic nanoparticles for gene therapy and drug delivery applications, as well as their use as immune modulators. Although nanomedicine holds great promise for muscle and other degenerative diseases, many challenges still need to be systematically addressed to assure a smooth transition from the bench to the bedside. This article is categorized under: Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement.

Early Life Social Stress Causes Sex- and Region-Dependent Dopaminergic Changes that Are Prevented by Minocycline.

Early life stress (ELS) is known to modify trajectories of brain dopaminergic development, but the mechanisms underlying have not been determined. ELS perturbs immune system and microglia reactivity, and inflammation and microglia influence dopaminergic transmission and development. Whether microglia mediate the effects of ELS on dopamine (DA) system development is still unknown. We explored the effects of repeated early social stress on development of the dopaminergic system in male and female mice through histological, electrophysiological, and transcriptomic analyses. Furthermore, we tested whether these effects could be mediated by ELS-induced altered microglia/immune activity through a pharmacological approach. We found that social stress in early life altered DA neurons morphology, reduced dopamine transporter (DAT) and tyrosine hydroxylase expression, and lowered DAT-mediated currents in the ventral tegmental area but not substantia nigra of male mice only. Notably, stress-induced DA alterations were prevented by minocycline, an inhibitor of microglia activation. Transcriptome analysis in the developing male ventral tegmental area revealed that ELS caused downregulation of dopaminergic transmission and alteration in hormonal and peptide signaling pathways. Results from this study offer new insight into the mechanisms of stress response and altered brain dopaminergic maturation after ELS, providing evidence of neuroimmune interaction, sex differences, and regional specificity.

Restoration of ER proteostasis attenuates remote apoptotic cell death after spinal cord injury by reducing autophagosome overload.

The pathogenic mechanisms that underlie the progression of remote degeneration after spinal cord injury (SCI) are not fully understood. In this study, we examined the relationship between endoplasmic reticulum (ER) stress and macroautophagy, hereafter autophagy, and its contribution to the secondary damage and outcomes that are associated with remote degeneration after SCI. Using a rat model of spinal cord hemisection at the cervical level, we measured ER stress and autophagy markers in the axotomized neurons of the red nucleus (RN). In SCI animals, mRNA and protein levels of markers of ER stress, such as GRP78, CHOP, and GADD34, increased 1 day after the injury, peaking on Day 5. Notably, in SCI animals, the increase of ER stress markers correlated with a blockade in autophagic flux, as evidenced by the increase in microtubule-associated protein 2 light chain 3 (LC3-II) and p62/SQSTM1 (p62) and the decline in LAMP1 and LAMP2 levels. After injury, treatment with guanabenz protected neurons from UPR failure and increased lysosomes biogenesis, unblocking autophagic flux. These effects correlated with greater activation of TFEB and improved neuronal survival and functional recovery-effects that persisted after suspension of the treatment. Collectively, our results demonstrate that in remote secondary damage, impairments in autophagic flux are intertwined with ER stress, an association that contributes to the apoptotic cell death and functional damage that are observed after SCI.

Shaping modern human skull through epigenetic, transcriptional and post-transcriptional regulation of the RUNX2 master bone gene.

RUNX2 encodes the master bone transcription factor driving skeletal development in vertebrates, and playing a specific role in craniofacial and skull morphogenesis. The anatomically modern human (AMH) features sequence changes in the RUNX2 locus compared with archaic hominins' species. We aimed to understand how these changes may have contributed to human skull globularization occurred in recent evolution. We compared in silico AMH and archaic hominins' genomes, and used mesenchymal stromal cells isolated from skull sutures of craniosynostosis patients for in vitro functional assays. We detected 459 and 470 nucleotide changes in noncoding regions of the AMH RUNX2 locus, compared with the Neandertal and Denisovan genomes, respectively. Three nucleotide changes in the proximal promoter were predicted to alter the binding of the zinc finger protein Znf263 and long-distance interactions with other cis-regulatory regions. By surface plasmon resonance, we selected nucleotide substitutions in the 3'UTRs able to affect miRNA binding affinity. Specifically, miR-3150a-3p and miR-6785-5p expression inversely correlated with RUNX2 expression during in vitro osteogenic differentiation. The expression of two long non-coding RNAs, AL096865.1 and RUNX2-AS1, within the same locus, was modulated during in vitro osteogenic differentiation and correlated with the expression of specific RUNX2 isoforms. Our data suggest that RUNX2 may have undergone adaptive phenotypic evolution caused by epigenetic and post-transcriptional regulatory mechanisms, which may explain the delayed suture fusion leading to the present-day globular skull shape.

Treatment of Neuroma of the Dorsal Branch of the Ulnar Nerve With Transfer to the Distal Anterior Interosseous Nerve.

Painful neuromas of the dorsal branch of the ulnar nerve may be difficult to treat. Proximal transposition is the standard treatment, but pain may recur. Sensory-to-motor nerve transfer as an evolution of targeted muscle reinnervation is a recently described technique to reduce neuroma formation in the treatment of painful neuromas. This report describes sensory-to-motor transfer of the dorsal branch of the ulnar nerve to the distal anterior interosseous nerve to treat a painful neuroma.

Exposure to different early-life stress experiences results in differentially altered DNA methylation in the brain and immune system.

The existence of a proportional relationship between the number of early-life stress (ELS) events experienced and the impoverishment of child mental health has been hypothesized. However, different types of ELS experiences may be associated with different neuro-psycho-biological impacts, due to differences in the intrinsic nature of the stress. DNA methylation is one of the molecular mechanisms that have been implicated in the "translation" of ELS exposure into neurobiological and behavioral abnormalities during adulthood. Here, we investigated whether different ELS experiences resulted in differential impacts on global DNA methylation levels in the brain and blood samples from mice and humans. ELS exposure in mice resulted in observable changes in adulthood, with exposure to social isolation inducing more dramatic alterations in global DNA methylation levels in several brain structures compared with exposure to a social threatening environment. Moreover, these two types of stress resulted in differential impacts on the epigenetic programming of different brain regions and cellular populations, namely microglia. In a pilot clinical study, blood global DNA methylation levels and exposure to childhood neglect or abuse were investigated in patients presenting with major depressive disorder or substance use disorder. A significant effect of the mental health diagnosis on global methylation levels was observed, but no effect of either childhood abuse or neglect was detected. These findings demonstrate that different types of ELS have differential impacts on epigenetic programming, through DNA methylation in specific brain regions, and that these differential impacts are associated with the different behavioral outcomes observed after ELS experiences.

Histone deacetylase 4 protects from denervation and skeletal muscle atrophy in a murine model of amyotrophic lateral sclerosis.

BACKGROUND: Histone deacetylase 4 (HDAC4) has been proposed as a target for Amyotrophic Lateral Sclerosis (ALS) because it mediates nerve-skeletal muscle interaction and since its expression in skeletal muscle correlates with the severity of the disease. However, our recent studies on the skeletal muscle response upon long-term denervation highlighted the importance of HDAC4 in maintaining muscle integrity. METHODS: To fully identify the yet uncharacterized HDAC4 functions in ALS, we genetically deleted HDAC4 in skeletal muscles of a mouse model of ALS. Body weight, skeletal muscle, innervation and spinal cord were analyzed over time by morphological and molecular analyses. Transcriptome analysis was also performed to delineate the signaling modulated by HDAC4 in skeletal muscle of a mouse model of ALS. FINDINGS: HDAC4 deletion in skeletal muscle caused earlier ALS onset, characterized by body weight loss, muscle denervation and atrophy, and compromised muscle performance, although the main catabolic pathways were not activated. Transcriptome analysis identified the gene networks modulated by HDAC4 in ALS, revealing UCP1 as a top regulator that may be implicated in worsening ALS features. INTERPRETATION: HDAC4 plays an important role in preserving innervations and skeletal muscle in ALS, likely by modulating the UCP1 gene network. Our study highlights a possible risk in considering HDAC inhibitors for the treatment of ALS. FUND: This work was supported by FIRB grant (RBFR12BUMH) from Ministry of Education, Universities and Research, by Fondazione Veronesi, by Sapienza research project 2017 (RM11715C78539BD8) and Polish National Science Center grant (UMO-2016/21/B/NZ3/03638).

HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes.

Skeletal muscle exhibits a high regenerative capacity, mainly due to the ability of satellite cells to replicate and differentiate in response to appropriate stimuli. Epigenetic control is effective at different stages of this process. It has been shown that the chromatin-remodeling factor HDAC4 is able to regulate satellite cell proliferation and commitment. However, its molecular targets are still uncovered. To explain the signaling pathways regulated by HDAC4 in satellite cells, we generated tamoxifen-inducible mice with conditional inactivation of HDAC4 in Pax7+ cells (HDAC4 KO mice). We found that the proliferation and differentiation of HDAC4 KO satellite cells were compromised, although similar amounts of satellite cells were found in mice. Moreover, we found that the inhibition of HDAC4 in satellite cells was sufficient to block the differentiation process. By RNA-sequencing analysis we identified P21 and Sharp1 as HDAC4 target genes. Reducing the expression of these target genes in HDAC4 KO satellite cells, we also defined the molecular pathways regulated by HDAC4 in the epigenetic control of satellite cell expansion and fusion.

A permissive retinoid X receptor/thyroid hormone receptor heterodimer allows stimulation of prolactin gene transcription by thyroid hormone and 9-cis-retinoic acid.

Heterodimers of the retinoid X receptor (RXR) with the thyroid hormone receptor (TR) are considered to be nonpermissive. It is believed that within these complexes RXR acts as a "silent partner." We demonstrate here that a permissive heterodimer mediates stimulation of prolactin expression by the thyroid hormone T3 and by 9-cis retinoic acid (9-cis-RA). A response element located in the prolactin distal enhancer mediates transactivation by both ligands in pituitary cells, and RXR recruits coactivators when bound to this element as a heterodimer with TR. Furthermore, transcription by the RXR agonist can be obtained in CV-1 cells only after overexpression of coactivators, and overexpression of corepressors inhibits the response in pituitary cells. Thus, cell type-specific differences in coregulator recruitment can determine the cellular response to both ligands. Coactivator recruitment by 9-cis-RA requires the ligand-dependent transactivation domains (AF-2) of both heterodimeric partners. Interestingly, the presence of the RXR ligand can overcome the deleterious effect of the AF-2 mutation E401Q on association with coactivators and transactivation. These results demonstrate an unexpected role for RXR in TR signaling and show that in particular cellular environments this receptor can act as a "nonsilent" partner of TR, allowing stimulation by RXR agonists.

The repressor DREAM acts as a transcriptional activator on Vitamin D and retinoic acid response elements.

DREAM (downstream regulatory element antagonist modulator) is a transcriptional repressor, which binds DREs (downstream response elements) in a Ca2+-regulated manner. The DREs consist of core GTCA motifs, very similar to binding motifs for non-steroid nuclear receptors. In this work, we find that DREAM stimulates basal and ligand-dependent activation of promoters containing vitamin D and retinoic acid response elements (VDREs and RAREs), consisting of direct repeats of the sequence AGT/GTCA spaced by 3 or 5 nt, respectively. Stimulation occurs when the element is located upstream, but not downstream, the transcription initiation site. Activation requires both Ca2+ binding to the EF-hands and the leucine-charged domains (LCDs), analogous to those responsible for the interaction of the nuclear receptors with coregulators. Further more, DREAM can bind both 'in vitro' and in chromatin immunoprecipitation assays to these elements. Importantly, 'in vivo' binding is only observed in vitamin D- or RA-treated cells. These results show that DREAM can function as an activator of transcription on certain promoters and demonstrate a novel role for DREAM acting as a potential modulator of genes containing binding sites for nuclear receptors.

Praja1 E3 ubiquitin ligase promotes skeletal myogenesis through degradation of EZH2 upon p38α activation.

Polycomb proteins are critical chromatin modifiers that regulate stem cell differentiation via transcriptional repression. In skeletal muscle progenitors Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), contributes to maintain the chromatin of muscle genes in a repressive conformation, whereas its down-regulation allows the progression through the myogenic programme. Here, we show that p38α kinase promotes EZH2 degradation in differentiating muscle cells through phosphorylation of threonine 372. Biochemical and genetic evidence demonstrates that the MYOD-induced E3 ubiquitin ligase Praja1 (PJA1) is involved in regulating EZH2 levels upon p38α activation. EZH2 premature degradation in proliferating myoblasts is prevented by low levels of PJA1, its cytoplasmic localization and the lower activity towards unphosphorylated EZH2. Our results indicate that signal-dependent degradation of EZH2 is a prerequisite for satellite cells differentiation and identify PJA1 as a new player in the epigenetic control of muscle gene expression.

The Dynamics of Polycomb Complexes.

Polycomb complexes are essential regulators of embryonic and adult stem cells, highly conserved from flies to mammals. Traditionally, their study was based on biochemical and genetic approaches. More recently, the development of novel technologies and the improvement and standardization of existing ones has allowed to address previously unexplored aspects of Polycomb biology, such as dynamics and regulation. In this chapter, relevant researchers in the field discuss novel technologies aimed at dissecting the dynamics of Polycomb complexes in normal and pathological conditions.