Sex-Biased Expression and Response of microRNAs in Neurological Diseases and Neurotrauma.

Neurological diseases and neurotrauma manifest significant sex differences in prevalence, progression, outcome, and therapeutic responses. Genetic predisposition, sex hormones, inflammation, and environmental exposures are among many physiological and pathological factors that impact the sex disparity in neurological diseases. MicroRNAs (miRNAs) are a powerful class of gene expression regulator that are extensively involved in mediating biological pathways. Emerging evidence demonstrates that miRNAs play a crucial role in the sex dimorphism observed in various human diseases, including neurological diseases. Understanding the sex differences in miRNA expression and response is believed to have important implications for assessing the risk of neurological disease, defining therapeutic intervention strategies, and advancing both basic research and clinical investigations. However, there is limited research exploring the extent to which miRNAs contribute to the sex disparities observed in various neurological diseases. Here, we review the current state of knowledge related to the sexual dimorphism in miRNAs in neurological diseases and neurotrauma research. We also discuss how sex chromosomes may contribute to the miRNA sexual dimorphism phenomenon. We attempt to emphasize the significance of sexual dimorphism in miRNA biology in human diseases and to advocate a gender/sex-balanced science.

Fluorinated Cell-Penetrating Peptide for Co-Delivering siHIF-1α and Sorafenib to Enhance In Vitro Anti-Tumor Efficacy.

Antiangiogenic therapy with sorafenib (SF) alone is ineffective in eradicating tumors, and its long-term application can exacerbate tumor hypoxia, which in turn restricts SF's therapeutic efficacy. Here, a redox-responsive fluorinated peptide (DEN-TAT-PFC) consisting of dendritic poly-lysine, cell-penetrating peptide TAT, and perfluorocarbon was designed and synthesized to co-load siRNA-targeting hypoxia-inducible factors (siHIF-1α) and SF. The unique architecture of the peptide and fluorinated modifications enhanced the siRNA delivery efficiency, including increased siRNA binding, GSH-responsive release, cellular uptake, endosomal escape, and serum resistance. Simultaneously, the DEN-TAT-PFC/SF/siHIF-1α co-delivery system achieved efficient knockdown of HIF-1α at mRNA and protein levels, thus alleviating hypoxia and further substantially reducing VEGF expression. Additionally, the excellent oxygen-carrying ability of DEN-TAT-PFC may facilitate relief of the hypoxic microenvironment. As a result of these synergistic effects, DEN-TAT-PFC/SF/siHIF-1α exhibited considerable anti-tumor cell proliferation and anti-angiogenesis effects. Therefore, DEN-TAT-PFC can be a versatile platform for fabricating fluorine-containing drugs/siRNA complex nano-systems.

Advances in cell-penetrating poly(disulfide)s for intracellular delivery of therapeutics.

Efficient intracellular delivery is essential for most therapeutic agents; however, existing delivery vectors face a dilemma between efficiency and toxicity, and always encounter the challenge of endolysosomal trapping. The cell-penetrating poly(disulfide) (CPD) is an effective tool for intracellular delivery, as it is taken up through thiol-mediated cellular uptake, thus avoiding endolysosomal entrapment and ensuring efficient cytosolic availability. Upon cellular uptake, CPD undergoes reductive depolymerization by glutathione inside cells and has minimal cytotoxicity. This review summarizes CPD's chemical synthesis approaches, cellular uptake mechanism, and recent advances in the intracellular delivery of proteins, antibodies, nucleic acids, and other nanoparticles. Overall, CPD is a promising candidate carrier for efficient intracellular delivery.

Impact of thyroid hormone perturbations in adult mice: brain weight and blood vessel changes, gene expression variation, and neurobehavioral outcomes.

Mouse models of hyper- and hypothyroidism were used to examine the effects of thyroid hormone (TH) dyshomeostasis on the aging mammalian brain. 13-14 month-old mice were treated for 4months with either levothyroxine (hyperthyroid) or a propylthiouracil and methimazole combination (PTU/Met; hypothyroid). Hyperthyroid mice performed better on Morris Water Maze than control mice, while hypothyroid mice performed worse. Brain weight was increased in thyroxine-treated, and decreased in PTU/Met-treated animals. The brain weight change was strongly correlated with circulating and tissue T4. Quantitative measurements of microvessels were compared using digital neuropathologic methods. There was an increase in microvessel area in hyperthyroid mice. Hypothyroid mice showed a trend for elevated glial fibrillary acidic protein-immunoreactive astrocytes, indicating an increase in neuroinflammation. Gene expression alterations were associated with TH perturbation and astrocyte-expressed transcripts were particularly affected. For example, expression of Gli2 and Gli3, mediators in the Sonic Hedgehog signaling pathway, were strongly impacted by both treatments. We conclude that TH perturbations produce robust neurobehavioral, pathological, and brain gene expression changes in aging mouse models.

Sex-Specific Alterations in Inflammatory MicroRNAs in Mouse Brain and Bone Marrow CD11b+ Cells Following Traumatic Brain Injury.

Sex is a key biological variable in traumatic brain injury (TBI) and plays a significant role in neuroinflammatory responses. However, the molecular mechanisms contributing to this sexually dimorphic neuroinflammatory response remain elusive. Here we describe a significant and previously unreported tissue enrichment and sex-specific alteration of a set of inflammatory microRNAs (miRNAs) in CD11b+ cells of brain and bone marrow isolated from naïve mice as well as mice subjected to TBI. Our data from naïve mice demonstrated that expression levels of miR-146a-5p and miR-150-5p were relatively higher in brain CD11b+ cells, and that miR-155-5p and miR-223-3p were highly enriched in bone marrow CD11b+ cells. Furthermore, while miR-150-5p and miR-155-5p levels were higher in male brain CD11b+ cells, no significant sexual difference was observed for miR-146a-5p and miR-223-3p. However, TBI resulted in sex-specific differential responses of these miRNAs in brain CD11b+ cells. Specifically, miR-223-3p levels in brain CD11b+ cells were markedly elevated in both sexes in response to TBI at 3 and 24 h, with levels in females being significantly higher than males at 24 h. We then focused on analyzing several miR-223-3p targets and inflammation-related marker genes following injury. Corresponding to the greater elevation of miR-223-3p in females, the miR-223-3p targets, TRAF6 and FBXW7 were significantly reduced in females compared to males. Interestingly, anti-inflammatory genes ARG1 and IL4 were higher in females after TBI than in males. These observations suggest miR-223-3p and other inflammatory responsive miRNAs may play a key role in sex-specific neuroinflammatory response following TBI.

RNA editing of microtubule-associated protein tau circular RNAs promotes their translation and tau tangle formation.

Aggregation of the microtubule-associated protein tau characterizes tauopathies, including Alzheimer's disease and frontotemporal lobar degeneration (FTLD-Tau). Gene expression regulation of tau is complex and incompletely understood. Here we report that the human tau gene (MAPT) generates two circular RNAs (circRNAs) through backsplicing of exon 12 to either exon 7 (12→7 circRNA) or exon 10 (12→10 circRNA). Both circRNAs lack stop codons. The 12→7 circRNA contains one start codon and is translated in a rolling circle, generating a protein consisting of multimers of the microtubule-binding repeats R1-R4. For the 12→10 circRNA, a start codon can be introduced by two FTLD-Tau mutations, generating a protein consisting of multimers of the microtubule-binding repeats R2-R4, suggesting that mutations causing FTLD may act in part through tau circRNAs. Adenosine to inosine RNA editing dramatically increases translation of circRNAs and, in the 12→10 circRNA, RNA editing generates a translational start codon by changing AUA to AUI. Circular tau proteins self-aggregate and promote aggregation of linear tau proteins. Our data indicate that adenosine to inosine RNA editing initiates translation of human circular tau RNAs, which may contribute to tauopathies.

Apolipoprotein E Proteinopathy Is a Major Dementia-Associated Pathologic Biomarker in Individuals with or without the APOE Epsilon 4 Allele.

The amygdala is vulnerable to multiple or "mixed" mis-aggregated proteins associated with neurodegenerative conditions that can manifest clinically with amnestic dementia; the amygdala region is often affected even at earliest disease stages. With the original intent of identifying novel dementia-associated proteins, the detergent-insoluble proteome was characterized from the amygdalae of 40 participants from the University of Kentucky Alzheimer's Disease Center autopsy cohort. These individuals encompassed a spectrum of clinical conditions (cognitively normal to severe amnestic dementia). Polypeptides from the detergent-insoluble fraction were interrogated using liquid chromatography-electrospray ionization-tandem mass spectrometry. As anticipated, portions of peptides previously associated with neurologic diseases were enriched from subjects with dementia. Among all detected peptides, Apolipoprotein E (ApoE) stood out: even more than the expected Tau, APP/Aß, and α-Synuclein peptides, ApoE peptides were strongly enriched in dementia cases, including from individuals lacking the APOE ε4 genotype. The amount of ApoE protein detected in detergent-insoluble fractions was robustly associated with levels of complement proteins C3 and C4. Immunohistochemical staining of APOE ε3/ε3 subjects' amygdalae confirmed ApoE co-localization with C4 in amyloid plaques. Thus, analyses of human amygdala proteomics indicate that rather than being only an "upstream" genetic risk factor, ApoE is an aberrantly aggregated protein in its own right, and show that the ApoE protein may play active disease-driving mechanistic roles in persons lacking the APOE ε4 allele.

MicroRNAs as Biomarkers for Predicting Complications following Aneurysmal Subarachnoid Hemorrhage.

Aneurysmal subarachnoid hemorrhage (aSAH) is a high mortality hemorrhagic stroke that affects nearly 30,000 patients annually in the United States. Approximately 30% of aSAH patients die during initial hospitalization and those who survive often carry poor prognosis with one in five having permanent physical and/or cognitive disabilities. The poor outcome of aSAH can be the result of the initial catastrophic event or due to the many acute or delayed neurological complications, such as cerebral ischemia, hydrocephalus, and re-bleeding. Unfortunately, no effective biomarker exists to predict or diagnose these complications at a clinically relevant time point when neurologic injury can be effectively treated and managed. Recently, a number of studies have demonstrated that microRNAs (miRNAs) in extracellular biofluids are highly associated with aSAH and complications. Here we provide an overview of the current research on relevant human studies examining the correlation between miRNAs and aSAH complications and discuss the potential application of using miRNAs as biomarkers in aSAH management.

Modulating Thyroid Hormone Levels in Adult Mice: Impact on Behavior and Compensatory Brain Changes.

Thyroid hormone (TH) perturbation is a common medical problem. Because of substantial public health impact, prior researchers have studied hyper- and hypothyroidism in animal models. Although most prior research focused on in utero and/or developmental effects, changes in circulating TH levels are commonly seen in elderly individuals: approximately 20% of persons older than 80 years have clinically impactful hypothyroidism and up to 5% have clinical hyperthyroidism, with women being more often affected than men. TH disease model methodology in mice have varied but usually focus on a single sex, and the impact(s) of TH perturbation on the adult brain are not well understood. We administered thyroxine to middle-aged (13 to 14 months) male and female mice to model hyperthyroidism and TH-lowering drugs propylthiouracil (PTU) and methimazole, to induce hypothyroidism. These pharmacological agents are used commonly in adult humans. Circulating TH-level changes were observed when thyroxine was dosed at 20 µg/mL in drinking water for two weeks. By contrast, PTU and methimazole did not elicit a consistent reproducible effect until two months of treatment. No substantial changes in TH levels were detected in brain tissues of treated animals; however, pronounced changes in gene expression, specifically for TH-processing transcripts, were observed following the treatment with thyroxine. Our study indicated a robust compensatory mechanism by which the brain tissue/cells minimize the TH fluctuation in CNS by altering gene expression. Neurobehavioral changes were related to the TH perturbation and suggested potential associations between cognitive status and hyper- and hypothyroidism.

A Highly Predictive MicroRNA Panel for Determining Delayed Cerebral Vasospasm Risk Following Aneurysmal Subarachnoid Hemorrhage.

Approximately one-third of aneurysmal subarachnoid hemorrhage (aSAH) patients develop delayed cerebral vasospasm (DCV) 3-10 days after aneurysm rupture resulting in additional, permanent neurologic disability. Currently, no validated biomarker is available to determine the risk of DCV in aSAH patients. MicroRNAs (miRNAs) have been implicated in virtually all human diseases, including aSAH, and are found in extracellular biofluids including plasma and cerebrospinal fluid (CSF). We used a custom designed TaqMan Low Density Array miRNA panel to examine the levels of 47 selected brain and vasculature injury related miRNAs in CSF and plasma specimens collected from 31 patients with or without DCV at 3 and 7 days after aSAH, as well as from eight healthy controls. The analysis of the first 18-patient cohort revealed a striking differential expression pattern of the selected miRNAs in CSF and plasma of aSAH patients with DCV from those without DCV. Importantly, this differential expression was observed at the early time point (3 days after aSAH), before DCV event occurs. Seven miRNAs were identified as reliable DCV risk predictors along with a prediction model constructed based on an array of additional 19 miRNAs on the panel. These chosen miRNAs were then used to predict the risk of DCV in a separate, testing cohort of 15 patients. The accuracy of DCV risk prediction in the testing cohort reached 87%. The study demonstrates that our novel designed miRNA panel is an effective predictor of DCV risk and has strong applications in clinical management of aSAH patients.

Multilayer surface construction for enhancing barrier properties of cellulose-based packaging.

It has been a consistent challenge to develop eco-friendly packaging in its entire life cycle with multiple barriers. Herein, a lignocellulose-derived strategy was developed for enhancing barrier properties of cellulose-based packaging. Porosity and hydrophilicity of paper packaging were remedied by the sequential deposition of oxalic acid modified microfibrillated cellulose (OMFC) and infiltration of nanosized alkaili lignin (NAL). OMFC deposition and NAL infiltration could fill the void among fibers and create hydrophobic micro/nano-roughness on paper surface, which showed synergetic effect on enhancing barrier and mechanical properties by self-bonding and crosslinking between cellulose and lignin. Water vapor transmission rate was reduced by 93 % with initial water contact angle at 113°. Besides, more than four-fold increase in tensile strength along with persisted water and grease resistance were achieved. The result suggests the barrier-enhanced packaging by multilayer surface construction has great potential in bio-based applications considering the biodegradability, biocompatibility, and recyclability.

Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease.

Iron homeostasis disturbance has been implicated in Alzheimer's disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of ferroptosis in the pathogenesis of AD remains elusive. Here, we report that ferroportin1 (Fpn), the only identified mammalian nonheme iron exporter, was downregulated in the brains of APPswe/PS1dE9 mice as an Alzheimer's mouse model and Alzheimer's patients. Genetic deletion of Fpn in principal neurons of the neocortex and hippocampus by breeding Fpnfl/fl mice with NEX-Cre mice led to AD-like hippocampal atrophy and memory deficits. Interestingly, the canonical morphological and molecular characteristics of ferroptosis were observed in both Fpnfl/fl/NEXcre and AD mice. Gene set enrichment analysis (GSEA) of ferroptosis-related RNA-seq data showed that the differentially expressed genes were highly enriched in gene sets associated with AD. Furthermore, administration of specific inhibitors of ferroptosis effectively reduced the neuronal death and memory impairments induced by Aß aggregation in vitro and in vivo. In addition, restoring Fpn ameliorated ferroptosis and memory impairment in APPswe/PS1dE9 mice. Our study demonstrates the critical role of Fpn and ferroptosis in the progression of AD, thus provides promising therapeutic approaches for this disease.

Temporal changes in inflammatory mitochondria-enriched microRNAs following traumatic brain injury and effects of miR-146a nanoparticle delivery.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function. We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation (i.e., miR-142-3p, miR-142-5p, and miR-146a) are altered in the hippocampus at 3-12 hours following a severe traumatic brain injury. In the present study, we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats. In addition, several inflammatory genes were analyzed in the cytosol fractions. The analysis showed that while elevated levels were observed in cytoplasm, the mitochondria-enriched miRNAs, miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury. Although not statistically significant, miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury, and recovered by 7 days. In contrast, miRNAs that are not enriched in mitochondria, including miR-124a, miR-150, miR-19b, miR-155, and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days. The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury. No major alterations were observed in sham operated animals. This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers. These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling. To test this, we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappaB inflammatory modulators in the injured brain. Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1), two important modulators of the nuclear factor-kappaB (NF-κB) pro-inflammatory pathway. Moreover, injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels. Taken together, our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain. All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee (IACUC protocol # 2014-1300) on August 17, 2017.

Dual-responsive carboxymethyl cellulose/dopamine/cystamine hydrogels driven by dynamic metal-ligand and redox linkages for controllable release of agrochemical.

The utilization of agrochemicals in crop production is often inefficient due to lack of appropriate carriers, raising in the significant concerns of ecological environment and public health. To enhance the efficiency of agrochemical delivery, a novel cellulose-based hydrogel was constructed in this work by cross-linking dopamine (DA)-modified carboxymethyl cellulose (CMC) with cystamine (CYS) in the presence of Fe3+ ions. The hydrogels displayed reversible sol-gel transitions upon exposure to stimulation of changes in pH and redox, leading to the controllable release of model agrochemical (6-benzyladenine). Compared with single-triggered condition, the hydrogel doubled the cumulative release when co-triggered by pH and redox. The dynamic metal/catechol complexation and disulfide bonding coexist in the hydrogel networks, enabling occurrence of dynamic reaction under a variety of environmental conditions. The finite element method (FEM) was employed to simulate the hydrogel to provide a theoretical insight into the tested drug delivery. Benefitting from the reversibly cross-linked networks and the excellent biodegradability of the hydrogels, we anticipate that this dual-responsive, polysaccharide-based hydrogel will offer diverse applications to reach the full potential in sustainable advancement of crop production.

The Mitochondria-Associated ER Membranes Are Novel Subcellular Locations Enriched for Inflammatory-Responsive MicroRNAs.

The mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are specific ER domains that contact the mitochondria and function to facilitate communication between ER and mitochondria. Disruption of contact between the mitochondria and ER is associated with a variety of pathophysiological conditions including neurodegenerative diseases. Considering the many cellular functions of MAMs, we hypothesized that MAMs play an important role in regulating microRNA (miRNA) activity linked to its unique location between mitochondria and ER. Here we present new findings from human and rat brains indicating that the MAMs are subcellular sites enriched for specific miRNAs. We employed subcellular fractionation and TaqMan® RT-qPCR miRNA analysis to quantify miRNA levels in subcellular fractions isolated from male rat brains and six human brain samples. We found that MAMs contain a substantial number of miRNAs and the profile differs significantly from that of cytosolic, mitochondria, or ER. Interestingly, MAMs are particularly enriched in inflammatory-responsive miRNAs, including miR-146a, miR-142-3p, and miR-142-5p in both human and rat brains; miR-223 MAM enrichment was observed only in human brain samples. Further, mitochondrial uncoupling or traumatic brain injury in male rats resulted in the alteration of inflammatory miRNA enrichment in the isolated subcellular fractions. These observations demonstrate that miRNAs are distributed differentially in organelles and may re-distribute between organelles and the cytosol in response to cellular stress and metabolic demands.

Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

Methodology for Subcellular Fractionation and MicroRNA Examination of Mitochondria, Mitochondria Associated ER Membrane (MAM), ER, and Cytosol from Human Brain.

Eukaryotic cell organelles exert unique functions individually but also interact with each other for essential cellular functions. This physical interface between the organelles serves as an important platform for biomolecule trafficking and signaling. Mitochondria are membrane-bound organelles and form a dynamic contact with other organelles. The interactions and communication between mitochondria and endoplasmic reticulum (ER) are facilitated by an ER specific domain, named mitochondria associated ER membrane (MAM). Due to its unique location, the MAM is a "hotspot" for important cell signaling and biochemical processes including calcium homeostasis, lipid synthesis/exchange, inflammasome and autophagosome formation, and mitochondria fission/fusion. Although techniques are available for isolation of organelle fractions including MAM, most utilize animal tissues and cell lines. Here we describe a protocol that is tailored to the isolation of highly purified MAM, mitochondria, ER, and cytosol from human brain. In addition, we include a protocol for the isolation of total RNA and subsequent analysis of microRNAs from these highly purified organelle fractions. Finally, we include a panel of protein markers that are useful for validating the enrichment and purity of each subcellular fraction.

Lignin Redistribution for Enhancing Barrier Properties of Cellulose-Based Materials.

Renewable cellulose-based materials have gained increasing interest in food packaging because of its favorable biodegradability and biocompatibility, whereas the barrier properties of hydrophilic and porous fibers are inadequate for most applications. Exploration of lignin redistribution for enhancing barrier properties of paper packaging material was carried out in this work. The redistribution of nanolized alkali lignin on paper surface showed excellent water, grease, and water vapor barrier. It provided persisted water (contact angle decrease rate at 0.05°/s) and grease (stained area undetectable at 72 h) resistance under long-term moisture or oil direct contact conditions, which also inhibited the bacterial growth to certain degree. Tough water vapor transmission rate can be lowered 82% from 528 to 97 g/m2/d by lignin redistribution. The result suggests that alkali lignin, with multiple barrier properties, has great potential in bio-based application considering the biodegradability, biocompatibility, and recyclability.

Enforced lysosomal biogenesis rescues erythromycin- and clindamycin-induced mitochondria-mediated cell death in human cells.

Antibiotics are the front-line treatment against many bacterial infectious diseases in human. The excessive and long-term use of antibiotics in human cause several side effects. It is important to understand the underlying molecular mechanisms of action of antibiotics in the host cell to avoid the side effects due to the prevalent uses. In the current study, we investigated the crosstalk between mitochondria and lysosomes in the presence of widely used antibiotics: erythromycin (ERM) and clindamycin (CLDM), which target the 50S subunit of bacterial ribosomes. We report here that both ERM and CLDM induced caspase activation and cell death in several different human cell lines. The activity of the mitochondrial respiratory chain was compromised in the presence of ERM and CLDM leading to bioenergetic crisis and generation of reactive oxygen species. Antibiotics treatment impaired autophagy flux and lysosome numbers, resulting in decreased removal of damaged mitochondria through mitophagy, hence accumulation of defective mitochondria. We further show that over-expression of transcription factor EB (TFEB) increased the lysosome number, restored mitochondrial function and rescued ERM- and CLDM-induced cell death. These studies indicate that antibiotics alter mitochondria and lysosome interactions leading to apoptotsis and may develop a novel approach for targeting inter-organelle crosstalk to limit deleterious antibiotic-induced side effects.