Long non-coding RNAs and their role in muscle regeneration.

In mammals, most of the genome is transcribed to generate a large and heterogeneous variety of non-protein coding RNAs, that are broadly grouped according to their size. Long noncoding RNAs include a very large and versatile group of molecules. Despite only a minority of them has been functionally characterized, there is emerging evidence indicating long noncoding RNAs as important regulators of expression at multiple levels. Several of them have been shown to be modulated during myogenic differentiation, playing important roles in the regulation of skeletal muscle development, differentiation and homeostasis, and contributing to neuromuscular diseases. In this chapter, we have summarized the current knowledge about long noncoding RNAs in skeletal muscle and discussed specific examples of long noncoding RNAs (lncRNAs and circRNAs) regulating muscle stem cell biology. We have also discussed selected long noncoding RNAs involved in the most common neuromuscular diseases.

Report and Abstracts of the 20th Meeting of IIM, the Interuniversity Institute of Myology: Assisi, October 12-15, 2023.

The 2023 represented a milestone for the Interuniversity Institute of Myology (IIM) since it marked twenty years of IIM activity joined with the 20th annual meeting organized by the association. The 20th IIM meeting took place in the fascinating town of Assisi, in the heart of central Italy, from 12 to 15 October. The commemorative 20th edition of the meeting represented a success in terms of participation and contributions as it brought together 160 myologists, clinicians, pharmaceutical companies, and patient organization representatives from Italy, several European countries (especially France), the United Kingdom, Brazil, and the USA. Four main scientific sessions hosted 36 oral communications and 54 always-on-display posters reporting original and unpublished results. Four main lectures from internationally renowned invited speakers and talks from delegates of the Societé Française de Myologie gave particular interest and emphasis to the scientific discussion. In line with the traditional policy of the IIM to encourage the participation of young researchers, about 50% of the attendees were under 35 years old. Moreover, the 20th IIM meeting was part of the high-training course in "Advanced Myology Update 2023", reserved to young trainees and managed by the University of Perugia (Italy) in collaboration with the IIM. In addition to the meeting scientific sessions, the 29 attendees to the course had a dedicated round table and dedicated lessons with the IIM invited speakers as teachers. Awards for the best talk, best poster blitz, and best poster have been conferred to young attendees, who became part of the IIM Young Committee, involved in the scientific organization of the IIM meetings. To celebrate the 20th IIM anniversary, a special free-access educational convention on "Causes and mechanisms of muscle atrophy. From terrestrial disuse to Space flights" has been organized, in which IIM experts in the field have illustrated the current knowledge about the muscle atrophy process in several atrophying conditions, and the former Italian astronaut, Paolo Nespoli shared his incredible experience in Space fascinating the large audience attending both in presence and online live stream. The meeting was characterized by a vibrant, friendly, and inclusive atmosphere, and stimulated discussion on emerging areas of muscle research, fostering international collaborations, and confirming the IIM meeting as an ideal venue to discuss around muscle development, function, and diseases pointing to the development of efficacious therapeutic strategies. Here, the abstracts of the meeting illustrate the most recent results on basic, translational, and clinical research in the myology field. Some abstracts are missing as per authors' decision due to the patentability of the results.

WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

DUX4 Role in Normal Physiology and in FSHD Muscular Dystrophy.

In the last decade, the sequence-specific transcription factor double homeobox 4 (DUX4) has gone from being an obscure entity to being a key factor in important physiological and pathological processes. We now know that expression of DUX4 is highly regulated and restricted to the early steps of embryonic development, where DUX4 is involved in transcriptional activation of the zygotic genome. While DUX4 is epigenetically silenced in most somatic tissues of healthy humans, its aberrant reactivation is associated with several diseases, including cancer, viral infection and facioscapulohumeral muscular dystrophy (FSHD). DUX4 is also translocated, giving rise to chimeric oncogenic proteins at the basis of sarcoma and leukemia forms. Hence, understanding how DUX4 is regulated and performs its activity could provide relevant information, not only to further our knowledge of human embryonic development regulation, but also to develop therapeutic approaches for the diseases associated with DUX4. Here, we summarize current knowledge on the cellular and molecular processes regulated by DUX4 with a special emphasis on FSHD muscular dystrophy.

Curcumin Affects HSP60 Folding Activity and Levels in Neuroblastoma Cells.

The fundamental challenge in fighting cancer is the development of protective agents able to interfere with the classical pathways of malignant transformation, such as extracellular matrix remodeling, epithelial-mesenchymal transition and, alteration of protein homeostasis. In the tumors of the brain, proteotoxic stress represents one of the main triggering agents for cell transformation. Curcumin is a natural compound with anti-inflammatory and anti-cancer properties with promising potential for the development of therapeutic drugs for the treatment of cancer as well as neurodegenerative diseases. Among the mediators of cancer development, HSP60 is a key factor for the maintenance of protein homeostasis and cell survival. High HSP60 levels were correlated, in particular, with cancer development and progression, and for this reason, we investigated the ability of curcumin to affect HSP60 expression, localization, and post-translational modifications using a neuroblastoma cell line. We have also looked at the ability of curcumin to interfere with the HSP60/HSP10 folding machinery. The cells were treated with 6, 12.5, and 25 µM of curcumin for 24 h, and the flow cytometry analysis showed that the compound induced apoptosis in a dose-dependent manner with a higher percentage of apoptotic cells at 25 µM. This dose of curcumin-induced a decrease in HSP60 protein levels and an upregulation of HSP60 mRNA expression. Moreover, 25 µM of curcumin reduced HSP60 ubiquitination and nitration, and the chaperonin levels were higher in the culture media compared with the untreated cells. Furthermore, curcumin at the same dose was able to favor HSP60 folding activity. The reduction of HSP60 levels, together with the increase in its folding activity and the secretion in the media led to the supposition that curcumin might interfere with cancer progression with a protective mechanism involving the chaperonin.

Non-Invasive Transcranial Nano-Pulsed Laser Therapy Ameliorates Cognitive Function and Prevents Aberrant Migration of Neural Progenitor Cells in the Hippocampus of Rats Subjected to Traumatic Brain Injury.

Traumatic brain injury (TBI) can lead to chronic diseases, including neurodegenerative disorders and epilepsy. The hippocampus, one of the most affected brain region after TBI, plays a critical role in learning and memory and is one of the only two regions in the brain in which new neurons are generated throughout life from neural stem cells (NSC) in the dentate gyrus (DG). These cells migrate into the granular layer where they integrate into the hippocampus circuitry. While increased proliferation of NSC in the hippocampus is known to occur shortly after injury, reduced neuronal maturation and aberrant migration of progenitor cells in the hilus contribute to cognitive and neurological dysfunctions, including epilepsy. Here, we tested the ability of a novel, proprietary non-invasive nano-pulsed laser therapy (NPLT), that combines near-infrared laser light (808 nm) and laser-generated, low-energy optoacoustic waves, to mitigate TBI-driven impairments in neurogenesis and cognitive function in the rat fluid percussion injury model. We show that injured rats treated with NPLT performed significantly better in a hippocampus-dependent cognitive test than did sham rats. In the DG, NPLT significantly decreased TBI-dependent impaired maturation and aberrant migration of neural progenitors, while preventing TBI-induced upregulation of specific microRNAs (miRNAs) in NSC. NPLT did not significantly reduce TBI-induced microglia activation in the hippocampus. Our data strongly suggest that NPLT has the potential to be an effective therapeutic tool for the treatment of TBI-induced cognitive dysfunction and dysregulation of neurogenesis, and point to modulation of miRNAs as a possible mechanism mediating its neuroprotective effects.

Nano-Pulsed Laser Therapy Is Neuroprotective in a Rat Model of Blast-Induced Neurotrauma.

We have developed a novel, non-invasive nano-pulsed laser therapy (NPLT) system that combines the benefits of near-infrared laser light (808 nm) and ultrasound (optoacoustic) waves, which are generated with each short laser pulse within the tissue. We tested NPLT in a rat model of blast-induced neurotrauma (BINT) to determine whether transcranial application of NPLT provides neuroprotective effects. The laser pulses were applied on the intact rat head 1 h after injury using a specially developed fiber-optic system. Vestibulomotor function was assessed on post-injury days (PIDs) 1-3 on the beam balance and beam walking tasks. Cognitive function was assessed on PIDs 6-10 using a working memory Morris water maze (MWM) test. BDNF and caspase-3 messenger RNA (mRNA) expression was measured by quantitative real-time PCR (qRT-PCR) in laser-captured cortical neurons. Microglia activation and neuronal injury were assessed in brain sections by immunofluorescence using specific antibodies against CD68 and active caspase-3, respectively. In the vestibulomotor and cognitive (MWM) tests, NPLT-treated animals performed significantly better than the untreated blast group and similarly to sham animals. NPLT upregulated mRNA encoding BDNF and downregulated the pro-apoptotic protein caspase-3 in cortical neurons. Immunofluorescence demonstrated that NPLT inhibited microglia activation and reduced the number of cortical neurons expressing activated caspase-3. NPLT also increased expression of BDNF in the hippocampus and the number of proliferating progenitor cells in the dentate gyrus. Our data demonstrate a neuroprotective effect of NPLT and prompt further studies aimed to develop NPLT as a therapeutic intervention after traumatic brain injury (TBI).

The dissociation of the Hsp60/pro-Caspase-3 complex by bis(pyridyl)oxadiazole copper complex (CubipyOXA) leads to cell death in NCI-H292 cancer cells.

Cell survival and proliferation are central to carcinogenesis, involving various mechanisms among which those that impede apoptosis are important. In this, the role of the molecular chaperone Hsp60 is unclear since it has been reported that it can be both, pro- or anti-apoptotic. A solution to this riddle is crucial to the development of anti-cancer therapies targeting Hsp60. We addressed this question using a tumor cell line, NCI-H292, and [Cu(3,5-bis(2'-pyridyl)-1,2,4-oxadiazole)2(H2O)2](ClO4)2, CubipyOXA, a copper-containing compound with cytotoxic properties. We treated cells with various doses of the compound and measured cell viability; apoptosis indicators; and levels of Hsp60, pro-Caspase-3 (pC3), Caspase-3 (C3), and complex Hsp60/pC3, with complementary methods. The quantitative dose-response curves of the levels of Hsp60, activated C3, inactivated pC3, Hsp60/pC3 complex and indicators of cell apoptosis, and cell death, all coincided to show that CubipyOXA has pro-apoptotic activity and promotes cell death. The curves also indicate that the pro-apoptotic effects of CubipyOXA could likely be due to a lowering of Hsp60 levels and to its blocking the formation of the Hsp60/pC3 complex and/or its dissociating the complex when already formed, thus, interfering with the anti-apoptotic action of Hsp60. These findings shed some light on how a tumor cell may avert apoptosis using Hsp60 and point to the anti-cancer potential of drugs, such as CubipyOXA, which interfere with Hsp60/pC3 complex formation, and thus allow the apoptotic cascade to proceed. In view of these findings it becomes clear that the novel compound CubipyOXA should be considered a potential, high-efficiency antitumor agent deserving further testing.

Quantitative patterns of Hsps in tubular adenoma compared with normal and tumor tissues reveal the value of Hsp10 and Hsp60 in early diagnosis of large bowel cancer.

Large bowel carcinogenesis involves accumulation of genetic alterations leading to transformation of normal mucosa into dysplasia and, lastly, adenocarcinoma. It is pertinent to elucidate the molecular changes occurring in the pre-neoplastic lesions to facilitate early diagnosis and treatment. Heat shock proteins (Hsps), many of which are molecular chaperones, are implicated in carcinogenesis, and their variations with tumor progression encourage their study as biomarkers. There are many reports on Hsps and cancer but none to our knowledge on their systematic quantification in pre-neoplastic lesions of the large bowel. We performed immunohistochemical determinations of Hsp10, Hsp60, Hsp70, and Hsp90 in biopsies of large bowel tubular adenomas with moderate grade of dysplasia and compared to normal mucosa and adenocarcinoma with a moderate grade of differentiation (G2). A significant elevation of Hsp10 and Hsp60 only, i.e., in the absence of elevation of Hsp70 or Hsp90, in both epithelium and lamina propria was found in tubular adenoma by comparison with normal mucosa. In contrast, adenocarcinoma was characterized by the highest levels of Hsp10 and Hsp60 in epithelium and lamina propria, accompanied by the highest levels of Hsp70 only in epithelium and of Hsp90 only in lamina propria, by comparison with normal and tubular adenoma counterparts. Hsp10 and Hsp60 are promising biomarkers for early diagnosis of tubular adenoma and for its differentiation from more advanced malignant lesions. Hsp10 and Hsp60 may be implicated in carcinogenesis from its very early steps and, thus, are potentially convenient targets for therapy.

The histone deacetylase inhibitor SAHA induces HSP60 nitration and its extracellular release by exosomal vesicles in human lung-derived carcinoma cells.

HSP60 undergoes changes in quantity and distribution in some types of tumors suggesting a participation of the chaperonin in the mechanism of transformation and cancer progression. Suberoylanilide hydroxamic acid (SAHA), a member of a family of histone deacetylase inhibitors (HDACi), has anti-cancer potential but its interaction, if any, with HSP60 has not been elucidated. We investigated the effects of SAHA in a human lung-derived carcinoma cell line (H292). We analysed cell viability and cycle; oxidative stress markers; mitochondrial integrity; HSP60 protein and mRNA levels; and HSP60 post-translational modifications, and its secretion. We found that SAHA is cytotoxic for H292 cells, interrupting the cycle at the G2/M phase, which is followed by death; cytotoxicity is associated with oxidative stress, mitochondrial damage, and diminution of intracellular levels of HSP60; HSP60 undergoes a post-translational modification and becomes nitrated; and nitrated HSP60 is exported via exosomes. We propose that SAHA causes ROS overproduction and mitochondrial dysfunction, which leads to HSP60 nitration and release into the intercellular space and circulation to interact with the immune system. These successive steps might constitute the mechanism of the anti-tumor action of SAHA and provide a basis to design supplementary therapeutic strategies targeting HSP60, which would be more efficacious than the compound alone.