To Erase or Not to Erase: Non-Canonical Catalytic Functions and Non-Catalytic Functions of Members of Histone Lysine Demethylase Families.

Histone lysine demethylases (KDMs) play an essential role in biological processes such as transcription regulation, RNA maturation, transposable element control, and genome damage sensing and repair. In most cases, their action requires catalytic activities, but non-catalytic functions have also been shown in some KDMs. Indeed, some strictly KDM-related proteins and some KDM isoforms do not act as histone demethylase but show other enzymatic activities or relevant non-enzymatic functions in different cell types. Moreover, many studies have reported on functions potentially supported by catalytically dead mutant KDMs. This is probably due to the versatility of the catalytical core, which can adapt to assume different molecular functions, and to the complex multi-domain structure of these proteins which encompasses functional modules for targeting histone modifications, promoting protein-protein interactions, or recognizing nucleic acid structural motifs. This rich modularity and the availability of multiple isoforms in the various classes produced variants with enzymatic functions aside from histone demethylation or variants with non-catalytical functions during the evolution. In this review we will catalog the proteins with null or questionable demethylase activity and predicted or validated inactive isoforms, summarizing what is known about their alternative functions. We will then go through some experimental evidence for the non-catalytical functions of active KDMs.

A Plasma Circular RNA Profile Differentiates Subjects with Alzheimer's Disease and Mild Cognitive Impairment from Healthy Controls.

The most frequently used biomarkers to support the diagnosis of Alzheimer's Disease (AD) are Aß42, total-Tau, and phospho-tau protein levels in CSF. Moreover, magnetic resonance imaging is used to assess hippocampal atrophy, 18F-FDG PET to identify abnormal brain metabolism, and PET imaging for amyloid deposition. These tests are rather complex and invasive and not easily applicable to clinical practice. Circulating non-coding RNAs, which are inherently stable and easy to manage, have been reported as promising biomarkers for central nervous system conditions. Recently, circular RNAs (circRNAs) as a novel class of ncRNAs have gained attention. We carried out a pilot study on five participants with AD and five healthy controls (HC) investigating circRNAs by Arraystar Human Circular RNA Microarray V2.0. Among them, 26 circRNAs were differentially expressed (FC ≥ 1.5, p < 0.05) in participants with AD compared to HC. From a top 10 of differentially expressed circRNAs, a validation study was carried out on four up-regulated (hsa_circRNA_050263, hsa_circRNA_403959, hsa_circRNA_003022, hsa_circRNA_100837) and two down-regulated (hsa_circRNA_102049, hsa_circRNA_102619) circRNAs in a larger population. Moreover, five subjects with mild cognitive impairment (MCI) were investigated. The analysis confirmed the upregulation of hsa_circRNA_050263, hsa_circRNA_403959, and hsa_circRNA_003022 both in subjects with AD and in MCI compared to HCs. We also investigated all microRNAs potentially interacting with the studied circRNAs. The GO enrichment analysis shows they are involved in the development of the nervous system, and in the cellular response to nerve growth factor stimuli, protein phosphorylation, apoptotic processes, and inflammation pathways, all of which are processes related to the pathology of AD.

A Sex Perspective in Neurodegenerative Diseases: microRNAs as Possible Peripheral Biomarkers.

Sex is a significant variable in the prevalence and incidence of neurological disorders. Sex differences exist in neurodegenerative disorders (NDs), where sex dimorphisms play important roles in the development and progression of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the last few years, some sex specific biomarkers for the identification of NDs have been described and recent studies have suggested that microRNA (miRNA) could be included among these, as influenced by the hormonal and genetic background. Failing to consider the possible differences between males and females in miRNA evaluation could introduce a sex bias in studies by not considering some of these sex-related biomarkers. In this review, we recapitulate what is known about the sex-specific differences in peripheral miRNA levels in neurodegenerative diseases. Several studies have reported sex-linked disparities, and from the literature analysis miR-206 particularly has been shown to have a sex-specific involvement. Hopefully, in the near future, patient stratification will provide important additional clues in diagnosis, prognosis, and tailoring of the best therapeutic approaches for each patient. Sex-specific biomarkers, such as miRNAs, could represent a useful tool for characterizing subgroups of patients.

miR-218-5p and doxorubicin combination enhances anticancer activity in breast cancer cells through Parkin-dependent mitophagy inhibition.

Breast Cancer (BC) is one of the most common tumours, and is known for its ability to develop resistance to chemotherapeutic treatments. Autophagy has been linked to chemotherapeutic response in several types of cancer, highlighting its contribution to this process. However, the role of mitophagy, a selective form of autophagy responsible for damaged mitochondria degradation, in the response to therapies in BC is still unclear. In order to address this point, we analysed the role of mitophagy in the treatment of the most common anticancer drug, doxorubicin (DXR), in different models of BC, such as a luminal A subtype-BC cell line MCF7 cells, cultured in 2-Dimension (2D) or in 3-Dimension (3D), and the triple negative BC (TNBC) cell line MDA-MB-231. Through a microarray analysis, we identified a relationship between mitophagy gene expressions related to the canonical PINK1/Parkin-mediated pathway and DXR treatment in BC cells. Afterwards, we demonstrated that the PINK1/Parkin-dependent mitophagy is indeed induced following DXR treatment and that exogenous expression of a small non-coding RNA, the miRNA-218-5p, known to target mRNA of Parkin, was sufficient to inhibit the DXR-mediated mitophagy in MCF7 and in MDA-MB-231 cells, thereby increasing their sensitivity to DXR. Considering the current challenges involved in BC refractory to treatment, our work could provide a promising approach to prevent tumour resistance and recurrence, potentially leading to the development of an innovative approach to combine mitophagy inhibition and chemotherapy.

Can Repetitive Transcranial Magnetic Stimulation (rTMS) Promote Neurogenesis and Axonogenesis in Subacute Human Ischemic Stroke?

BACKGROUND: Ischemic stroke may trigger neuroplastic changes via proliferation, migration towards the lesion, and differentiation of neuroprogenitor cells into mature neurons. Repetitive Transcranial Magnetic Stimulation (rTMS) may promote brain plasticity. This study aimed to assess rTMS's effect on post-stroke endogenous neuroplasticity by dosing plasma miRs 17~92, Netrin-1, Sema3A, and BDNF. METHODS: In this case-controlled study, we randomized 19 ischemic stroke patients within five days from symptoms onset (T0) to neuronavigated-rTMS or sham stimulation. Stimulation was applied on the stroke hemisphere daily between the 7th and 14th day from stroke onset. Blood samples were collected at T0, before the first rTMS section (T7), and at the end of the last rTMS session (T14). Five healthy controls were also enrolled in this study. RESULTS: Of 19 patients, 10 received rTMS and 9 sham stimulation. Compared with the sham group, in the rTMS group, plasma levels of miRs17~92 and Ntn-1 significantly increased whereas Sema3A levels tended to decrease. In multivariate linear regression analyses, rTMS was independently related to Ntn-1 and miR-25 levels at T14. CONCLUSIONS: We found an association between rTMS and neurogenesis/axonogenesis biomarker enhancement. Our preliminary data suggest that rTMS may positively interfere with natural endogenous plasticity phenomena of the post-ischemic human brain.

miR-92a-3p and miR-320a are Upregulated in Plasma Neuron-Derived Extracellular Vesicles of Patients with Frontotemporal Dementia.

Despite the efforts to identify fluid biomarkers to improve diagnosis of Frontotemporal dementia (FTD), only a few candidates have been described in recent years. In a previous study, we identified three circulating miRNAs (miR-92a-3p, miR-320a and miR-320b) differentially expressed in FTD patients with respect to healthy controls and/or Alzheimer's disease (AD) patients. Now, we investigated whether those changes could be due to miRNAs contained in neuron-derived extracellular vesicles (NDEVs). We also evaluated miRNAs content in total plasma EVs and in CSF samples. The analysis of plasma NDEVs carried out on 40 subjects including controls (n = 13), FTD (n = 13) and AD (n = 14) patients, showed that both miR-92a-3p and miR-320a levels were triplicated in the FTD group if compared with CT and AD patients. Increased levels of the same miRNAs were found also in CSF derived from FTD group compared to CTs. No differences were observed in expression levels of miR-320b among the three groups. Worthy of note, all miRNAs analysed were increased in an FTD cell model, MAPT IVS10 + 16 neurons. Our results suggest that miR-92a and miR-320a in NDEVs could be proposed as FTD biomarkers.

Identification of miRNAs regulating MAPT expression and their analysis in plasma of patients with dementia.

Background: Dementia is one of the most common diseases in elderly people and hundreds of thousand new cases per year of Alzheimer's disease (AD) are estimated. While the recent decade has seen significant advances in the development of novel biomarkers to identify dementias at their early stage, a great effort has been recently made to identify biomarkers able to improve differential diagnosis. However, only few potential candidates, mainly detectable in cerebrospinal fluid (CSF), have been described so far. Methods: We searched for miRNAs regulating MAPT translation. We employed a capture technology able to find the miRNAs directly bound to the MAPT transcript in cell lines. Afterwards, we evaluated the levels of these miRNAs in plasma samples from FTD (n = 42) and AD patients (n = 33) and relative healthy controls (HCs) (n = 42) by using qRT-PCR. Results: Firstly, we found all miRNAs that interact with the MAPT transcript. Ten miRNAs have been selected to verify their effect on Tau levels increasing or reducing miRNA levels by using cell transfections with plasmids expressing the miRNAs genes or LNA antagomiRs. Following the results obtained, miR-92a-3p, miR-320a and miR-320b were selected to analyse their levels in plasma samples of patients with FTD and AD respect to HCs. The analysis showed that the miR-92a-1-3p was under-expressed in both AD and FTD compared to HCs. Moreover, miR-320a was upregulated in FTD vs. AD patients, particularly in men when we stratified by sex. Respect to HC, the only difference is showed in men with AD who have reduced levels of this miRNA. Instead, miR-320b is up-regulated in both dementias, but only patients with FTD maintain this trend in both genders. Conclusions: Our results seem to identify miR-92a-3p and miR-320a as possible good biomarkers to discriminate AD from HC, while miR-320b to discriminate FTD from HC, particularly in males. Combining three miRNAs improves the accuracy only in females, particularly for differential diagnosis (FTD vs. AD) and to distinguish FTD from HC.

Plasma Biomarker Profile and Clinical Correlations in Adult Patients With Tuberous Sclerosis Complex.

OBJECTIVES: Different pathophysiologic mechanisms, especially involving astrocytes, could contribute to tuberous sclerosis complex (TSC). We assessed neurodegeneration and astrocytopathy plasma biomarkers in adult patients with TSC to define TSC biomarker profile and investigate clinical-radiologic correlations. METHODS: Patients with TSC aged 15 years or older followed at Policlinico "Umberto I" of Rome were consecutively enrolled (July 2021-June 2022). The plasma levels of the following biomarkers were compared between patients and age/sex-matched healthy controls (HCs): tTau, pTau181, Abeta40, Abeta42, neurofilament light chain, and glial fibrillary acid protein (GFAP). RESULTS: Thirty-one patients (20 females/11 males; median age 30 years, interquartile range 24-47) and 38 HCs were enrolled. Only GFAP was significantly higher in the whole TSC population than in HCs (132.71 [86.14-231.06] vs 44.80 [32.87-66.76] pg/mL, p < 0.001), regardless of genotype. GFAP correlated with the disease clinical (ρ = 0.498, p = 0.005) and radiologic severity (ρ = 0.417, p = 0.001). It was significantly higher in patients with epileptic spasms (254.50 [137.54-432.96] vs 86.92 [47.09-112.76] pg/mL, p < 0.0001), moderate-severe intellectual disability (200.80 [78.40-427.6] vs 105.08 [46.80-152.58] pg/mL, p = 0.040), and autism spectrum disorder (306.26 [159.07-584.47] vs 109.34 [72.56-152.08] pg/mL, p = 0.021). DISCUSSION: Our exploratory study documented a significant increase of GFAP plasma concentration in adult patients with TSC, correlated with their neurologic severity, supporting the central role of astrocytopathy in TSC pathophysiology.

Cladribine and ocrelizumab induce differential miRNA profiles in peripheral blood mononucleated cells from relapsing-remitting multiple sclerosis patients.

Background and objectives: Multiple sclerosis (MS) is a chronic, progressive neurological disease characterized by early-stage neuroinflammation, neurodegeneration, and demyelination that involves a spectrum of heterogeneous clinical manifestations in terms of disease course and response to therapy. Even though several disease-modifying therapies (DMTs) are available to prevent MS-related brain damage-acting on the peripheral immune system with an indirect effect on MS lesions-individualizing therapy according to disease characteristics and prognostic factors is still an unmet need. Given that deregulated miRNAs have been proposed as diagnostic tools in neurodegenerative/neuroinflammatory diseases such as MS, we aimed to explore miRNA profiles as potential classifiers of the relapsing-remitting MS (RRMS) patients' prospects to gain a more effective DMT choice and achieve a preferential drug response. Methods: A total of 25 adult patients with RRMS were enrolled in a cohort study, according to the latest McDonald criteria before (pre-cladribine, pre-CLA; pre-ocrelizumab, pre-OCRE, time T0) and after high-efficacy DMTs, time T1, 6 months post-CLA (n = 10, 7 F and 3 M, age 39.0 ± 7.5) or post-OCRE (n = 15, 10 F and 5 M, age 40.5 ± 10.4) treatment. A total of 15 age- and sex-matched healthy control subjects (9 F and 6 M, age 36.3 ± 3.0) were also selected. By using Agilent microarrays, we analyzed miRNA profiles from peripheral blood mononuclear cells (PBMC). miRNA-target networks were obtained by miRTargetLink, and Pearson's correlation served to estimate the association between miRNAs and outcome clinical features. Results: First, the miRNA profiles of pre-CLA or pre-OCRE RRMS patients compared to healthy controls identified modulated miRNA patterns (40 and seven miRNAs, respectively). A direct comparison of the two pre-treatment groups at T0 and T1 revealed more pro-inflammatory patterns in the pre-CLA miRNA profiles. Moreover, both DMTs emerged as being capable of reverting some dysregulated miRNAs toward a protective phenotype. Both drug-dependent miRNA profiles and specific miRNAs, such as miR-199a-3p, miR-29b-3p, and miR-151a-3p, emerged as potentially involved in these drug-induced mechanisms. This enabled the selection of miRNAs correlated to clinical features and the related miRNA-mRNA network. Discussion: These data support the hypothesis of specific deregulated miRNAs as putative biomarkers in RRMS patients' stratification and DMT drug response.

The Encapsulation of Citicoline within Solid Lipid Nanoparticles Enhances Its Capability to Counteract the 6-Hydroxydopamine-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells.

(1) Backgrond: Considering the positive effects of citicoline (CIT) in the management of some neurodegenerative diseases, the aim of this work was to develop CIT-Loaded Solid Lipid Nanoparticles (CIT-SLNs) for enhancing the therapeutic use of CIT in parkinsonian syndrome; (2) Methods: CIT-SLNs were prepared by the melt homogenization method using the self-emulsifying lipid Gelucire® 50/13 as lipid matrix. Solid-state features on CIT-SLNs were obtained with FT-IR, thermal analysis (DSC) and X-ray powder diffraction (XRPD) studies. (3) Results: CIT-SLNs showed a mean diameter of 201 nm, -2.20 mV as zeta potential and a high percentage of entrapped CIT. DSC and XRPD analyses evidenced a greater amorphous state of CIT in CIT-SLNs. On confocal microscopy, fluorescent SLNs replacing unlabeled CIT-SLNs released the dye selectively in the cytoplasm. Biological evaluation showed that pre-treatment of SH-SY5Y dopaminergic cells with CIT-SLNs (50 µM) before the addition of 40 µM 6-hydroxydopamine (6-OHDA) to mimic Parkinson's disease's degenerative pathways counteracts the cytotoxic effects induced by the neurotoxin, increasing cell viability with the consistent maintenance of both nuclear and cell morphology. In contrast, pre-treatment with CIT 50 and 60 µM or plain SLNs for 2 h followed by 6-OHDA (40 µM) did not significantly influence cell viability. (4) Conclusions: These data suggest an enhanced protection exerted by CIT-SLNs with respect to free CIT and prompt further investigation of possible molecular mechanisms that underlie this difference.