The cell and stress-specific canonical and noncanonical tRNA cleavage.

Following stress, transfer RNA (tRNA) is cleaved to generate tRNA halves (tiRNAs). These tiRNAs have been shown to repress protein translation. Angiogenin was considered the main enzyme that cleaves tRNA at its anticodon to generate 35-45 nucleotide long tiRNA halves, however, the recent reports indicate the presence of angiogenin-independent cleavage. We previously observed tRNA cleavage pattern occurring away from the anticodon site. To explore this noncanonical cleavage, we analyze tRNA cleavage patterns in rat model of ischemia-reperfusion and in two rat cell lines. In vivo mitochondrial tRNAs were prone to this noncanonical cleavage pattern. In vitro, however, cytosolic and mitochondrial tRNAs could be cleaved noncanonically. Our results show an important regulatory role of mitochondrial stress in angiogenin-mediated tRNA cleavage. Neither angiogenin nor RNH1 appear to regulate the noncanonical tRNA cleavage. Finally, we verified our previous findings of the role of Alkbh1 in regulating tRNA cleavage and its impact on noncanonical tRNA cleavage.

Dysregulation of Rnf 213 gene contributes to T cell response via antigen uptake, processing, and presentation.

Growing evidence suggest the association between Moyamoya disease (MMD) and immune systems, such as antigen presenting cells in particular. Rnf213 gene, a susceptibility gene for MMD, is highly expressed in immune tissues, however, its function remains unclear. In addition, the physiological role of RNF213 gene polymorphism c.14576G > A (rs112735431), susceptibility variant for MMD, is also poorly understood. By studying Rnf213-knockout (Rnf213-KO) mice with deletion of largest exon32 and Rnf213-knockin (Rnf213-KI) mice with insertion of single-nucleotide polymorphism corresponding to c.14576G > A mutation in MMD patients, we aimed to investigate the role of RNF213 in dendritic cell development, and antigen processing and presentation. First, we found a high level of Rnf213 gene expression in conventional DCs and monocytes. Second, flow cytometric and confocal microscopic analysis revealed ovalbumin protein-pulsed Rnf213-KO and Rnf213-KI DCs showed impaired antigen uptake, proteolysis and reduced numbers of endosomes and lysosomes, and thereby failed to activate and proliferate antigen-specific T cells efficiently. In addition, Rnf213-KI DCs showed a similar phenotype to that of Rnf213-KO BMDCs. In conclusion, our findings suggest the critical role of RNF213 in antigen uptake, processing and presentation.

Epigenetic response of endothelial cells to different wall shear stress magnitudes: A report of new mechano-miRNAs.

Endothelial cells (ECs) respond to flow stress via a variety of mechanisms, leading to various intracellular responses that can modulate the vessel wall and lead to diseases if the flow is disturbed. Mechano-microRNAs (miRNAs) are a subset of miRNAs in the ECs that are flow responsive. Mechano-miRNAs were shown to be related to atherosclerosis pathophysiology, and a number of them were identified as pathologic. Here, we exposed human carotid ECs to different wall shear stresses (WSS), high and low, and evaluated the response of miRNAs by microarray and quantitative polymerase chain reaction analysis. We discovered five new mechano-miRNAs that were not reported in that context previously to the best of our knowledge. Moreover, functional pathway analysis revealed that under low WSS conditions, several pathways regulating apoptosis are affected. In addition, KLF2 and KLF4, known atheroprotective genes, were downregulated under low WSS and upregulated under high WSS. KLF2 and VCAM1, both angiogenic, were upregulated under high WSS. NOS3, which is vascular protective, was also upregulated with higher WSS. On the contrary, ICAM-1 and E-selectin, both atherogenic and proinflammatory, were upregulated with high WSS. Collectively, the epigenetic landscape with the gene expression analysis reveals that low WSS is associated with a proapoptotic state, while high WSS is associated with a proliferative and proinflammatory state.

The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation.

tiRNAs are small non-coding RNAs produced when tRNA is cleaved under stress. tRNA methylation modifications has emerged in recent years as important regulators for tRNA structural stability and sensitivity to cleavage and tiRNA generation during stress, however, the specificity and higher regulation of such a process is not fully understood. Alkbh1 is a m1A demethylase that leads to destabilization of tRNA and enhanced tRNA cleavage. We examined the impact of Alkbh1 targeting via gene knockdown or overexpression on B35 rat neuroblastoma cell line fate following stresses and on tRNA cleavage. We show that Alkbh1 impact on cell fate and tRNA cleavage is a stress specific process that is impacted by the demethylating capacity of the cellular stress in question. We also show that not all tRNAs are cleaved equally following Alkbh1 manipulation and stress, and that Alkbh1 KD fails to rescue tRNAs from cleavage following demethylating stresses. These findings shed a light on the specificity and higher regulation of tRNA cleavage and should act as a guide for future work exploring the utility of Alkbh1 as a therapeutic target for cancers or ischaemic insult.

Surgery for spinal intramedullary tumors: technique, outcome and factors affecting resectability.

Intramedullary spinal cord tumors (IMSCTs) are relatively infrequent lesions with ependymomas and astrocytomas representing the most common types. Microsurgical resection is established as the treatment of choice for these challenging lesions. We reviewed the surgical outcome of 29 cases operated for IMSCTs by the same surgeon between 2009 and 2015. The median follow-up period was 31 months, and all patients were followed up at least for 1 year. Among these 29 cases, 5 patients were previously operated for partial resection elsewhere. Age ranged from 9 to 62 years with a median of 39 years. All patients were symptomatic before surgery. The most common pathology was ependymoma (16 cases), and the most common tumor location was the cervical spine (18 cases). Gross total resection was achieved in 20 out of 29 cases (68.9%). Tumors were totally excised in all cases of ependymoma except in two patients; one was previously operated and irradiated and the second had an extensive anaplastic ependymoma. Sixteen cases experienced immediate post-operative worsening which was temporary in all but one case. At 1-year follow up, 23 patients (79.3%) maintained their pre-operative McCormick grade, 5 patients (17.2%) had a better grade, and 1 patient (3.5%) deteriorated. Surgery still represents the mainstay in the management of IMSCT. Gross total resection can be achieved safely in many cases especially in the presence of an identifiable plane of cleavage between the tumor and the normal spinal cord.

Stereotactic radiosurgery as a feasible treatment for intramedullary spinal arteriovenous malformations: a single-center observation.

Spinal cord intramedullary arteriovenous malformations are rare. For patients suffering from either hemorrhage or myelopathy, surgical or endovascular interventions are indicated. However, complete eradication of the nidus is often difficult because of its intramedullary location and complex angioarchitecture. In this report, we evaluate the feasibility and safety of stereotactic radiosurgery as a treatment modality for intramedullary spinal arteriovenous malformations (AVMs). Between 2010 and 2014, we performed stereotactic radiosurgery to treat four patients with intramedullary AVM and one with spinal arteriovenous metameric syndrome (one woman and four men; age range, 31-66 years). Three patients presented with myelopathy, and two suffered hemorrhages. Nidi were located in the cervical (three cases) and thoracic (two cases) spinal cord regions. Based on the angioarchitecture, surgery and endovascular embolization were indicated. When both modalities were deemed hazardous, radiosurgery using CyberKnife™ was offered. Radiation using marginal doses of 18 Gy was administered in three fractions. The mean follow-up period was 37.2 months (range, 16-62 months). After treatment, two of the three patients with myelopathy experienced either improvement or stabilization of their symptoms and one experienced worsening of dysesthesia. In two patients with hemorrhage, symptoms improved in one and remained stable in the other. No further hemorrhagic episodes were evident during follow-up. Follow-up angiograms showed marked shrinkage of the nidus located in the thoracic spinal cord in one case and angiographic stabilization in the others. As a treatment modality for intramedullary AVMs, CyberKnife™ is safe and can be considered when surgery or endovascular therapy is not indicated. To determine optimum radiation doses and protocols for treating spinal AVMs, further studies with more patients and long-term follow-up are required.

Uneven cerebral hemodynamic change as a cause of neurological deterioration in the acute stage after direct revascularization for moyamoya disease: cerebral hyperperfusion and remote ischemia caused by the 'watershed shift'.

Superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis is the standard surgical treatment for moyamoya disease (MMD). The main potential complications of this treatment are cerebral hyperperfusion (CHP) syndrome and ischemia, and their managements are contradictory to each other. We retrospectively investigated the incidence of the simultaneous manifestation of CHP and infarction after surgery for MMD. Of the 162 consecutive direct revascularization surgeries performed for MMD, we encountered two adult cases (1.2%) manifesting the simultaneous occurrence of symptomatic CHP and remote infarction in the acute stage. A 47-year-old man initially presenting with infarction developed CHP syndrome (aphasia) 2 days after left STA-MCA anastomosis, as assessed by quantitative single-photon emission computed tomography (SPECT). Although lowering blood pressure ameliorated his symptoms, he developed cerebral infarction at a remote area in the acute stage. Another 63-year-old man, who initially had progressing stroke, presented with aphasia due to focal CHP in the left temporal lobe associated with acute infarction at the tip of the left frontal lobe 1 day after left STA-MCA anastomosis, when SPECT showed a paradoxical decrease in cerebral blood flow (CBF) in the left frontal lobe despite a marked increase in CBF at the site of anastomosis. Symptoms were ameliorated in both patients with the normalization of CBF, and there were no further cerebrovascular events during the follow-up period. CHP and cerebral infarction may occur simultaneously not only due to blood pressure lowering against CHP, but also to the 'watershed shift' phenomenon, which needs to be elucidated in future studies.

Transient Global Cerebral Ischemia Induces RNF213, a Moyamoya Disease Susceptibility Gene, in Vulnerable Neurons of the Rat Hippocampus CA1 Subregion and Ischemic Cortex.

The RING finger protein 213 (RNF213) is an important susceptibility gene for moyamoya disease (MMD) and is also implicated in other types of intracranial major artery stenosis/occlusion (ICAS); however, the role of RNF213 in the development of ICAS including MMD is unclear. The constitutive expression of the RNF213 gene is relatively weak in brain tissue, while information regarding the expression patterns of the RNF213 gene under cerebral ischemia, which is one of characteristic pathologies associated with ICAS, is currently limited. Our objective was to address this critical issue, and we investigated Rnf213 mRNA expression in rat brains after 5 minutes of transient global cerebral ischemia (tGCI) by occluding the common carotid arteries coupled with severe hypotension. Rnf213 gene expression patterns were investigated with in situ RNA hybridization and a real-time polymerase chain reaction (PCR) from 1 to 72 hours after tGCI. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the hippocampus CA1 sub-region 48 hours after tGCI. The significant induction of the Rnf213 gene was also evident in the ischemic cortex. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression exclusively in neurons. The real-time PCR analysis confirmed the induction of the Rnf213 gene after tGCI. The up-regulation of the Rnf213 gene in vulnerable neurons in the hippocampus CA1 after tGCI suggests its involvement in forebrain ischemia, which is an underlying pathology of MMD. Further investigations are needed to elucidate its exact role in the pathophysiology of ICAS including MMD.

Blood Flow Into Basilar Tip Aneurysms: A Predictor for Recanalization After Coil Embolization.

BACKGROUND AND PURPOSE: Hemodynamic forces may play a role in the recanalization of coiled aneurysms. The purpose of this study was to investigate the influence of presurgical hemodynamics on the efficacy of coil embolization for basilar tip aneurysms. METHODS: We identified 82 patients who underwent endovascular coil embolization for basilar tip aneurysms with a follow-up of >1 year. Presurgical hemodynamics were investigated using computational fluid dynamics with 3-dimensional data derived from rotational angiography. During postprocessing, we quantified the rate of net flow entering the aneurysm through its neck and calculated the proportion of the aneurysmal inflow rate to the basilar artery flow rate. In addition, we investigated the correlation between the basilar bifurcation configuration and the hemodynamics. RESULTS: Twenty-five of the 82 patients were excluded because of difficult vascular geometry reconstruction. Among the 57 examined patients, angiographic recanalization was observed in 19 patients (33.3%). The proportion of the aneurysmal inflow rate to the basilar artery flow rate and a coil packing density <30% were independent and significant predictors for the recanalization of coiled aneurysms. Additional investigation revealed that a small branch angle formed by the basilar artery and the posterior cerebral artery increased blood flow into the aneurysm. CONCLUSIONS: The proportion of the aneurysmal inflow rate to the basilar artery flow rate, influenced by the basilar bifurcation configuration, was an independent and significant predictor for recanalization after coil embolization in basilar tip aneurysms.

Long-term follow-up of pediatric moyamoya disease treated by combined direct-indirect revascularization surgery: single institute experience with surgical and perioperative management.

Moyamoya disease (MMD) is a rare occlusive cerebrovascular disease that mainly presents in children as cerebral ischemia. Prompt treatment with either a direct or indirect revascularization procedure is necessary for children with MMD in order to prevent repeated ischemic events. We herein present our experience with combined direct and indirect bypass surgery for the treatment of pediatric MMD as well as our uniquely designed perioperative protocol. Twenty-three patients with MMD, aged between 2 and 16 years old (mean 9.36), underwent 38 combined bypass procedures between 2008 and 2015. All patients underwent single superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis combined with encephalo-duro-myo-synangiosis (EDMS). The perioperative management protocol was stratified into two unique eras: the first era with normotensive care and the second era with strict blood pressure control (systolic 100-130 mmHg) and routine aspirin administration. Patients were followed after surgery for a period ranging between 3 and 131 months (mean 77 months) in yearly clinical and radiological follow-ups. Three postoperative complications were observed: two cases of cerebral hyperperfusion (2/38, 5.3 %) and one case of perioperative minor stroke (1/38, 2.6 %), two of which were in the first era. No strokes, either ischemic or hemorrhagic, were observed in the follow-up period, and the activity of daily living as shown by the modified Rankin Scale improved in 20 patients, with no deterioration being observed in any of our patients. STA-MCA bypass with EDMS is safe and effective for the management of pediatric MMD and provides long-term favorable outcomes. Perioperative care with blood pressure control combined with the administration of aspirin may reduce the potential risk of surgical complications.

Endovascular Modalities for the Treatment of Cavernous Sinus Arteriovenous Fistulas: A Single-Center Experience.

BACKGROUND: Cavernous sinus (CS) fistulas are classified into traumatic and spontaneous. Traumatic carotid-cavernous fistulas (CCFs) are usually direct internal carotid artery (ICA) high-flow fistulas; whereas spontaneous CCFs are usually dural, low-flow fistulas and generally possess less severe symptoms than direct carotid-cavernous fistulas. METHODS: This study involved 34 patients who were classified into 2 groups: Group A included 26 patients with direct carotid-cavernous fistula; and Group B included 8 patients with indirect dural cavernous fistula. All patients had ocular manifestations. One patient had subarachnoid hemorrhage. Coils were used alone in 19 cases of direct fistula and in 1 case of dural fistulas. Coils and Onyx (Covidien, Mansfield, MA, USA) were used in 7 cases of direct fistula and in 2 cases of dural fistulas. Onyx alone was used to treat 5 cases with dural fistulas but none of the cases with direct fistulas. Covered stents and coils were used in 2 cases of direct fistulas. RESULTS: All patients in both groups showed full recovery of their clinical signs and symptoms. Only 1 procedure-related complication was observed (3%) in which a patient had an embolic event and trigeminal dysesthesia as a result of Onyx reflux through external carotid artery-ICA anastomosis. CONCLUSION: Coils are superior solid embolic agents used for the treatment of direct high-flow fistulas, while Onyx is more valuable in dural low-flow CCF. Onyx shortens the procedure time and decreases procedure cost. Onyx injection inside the CS proper through the transarterial or transvenous route may be safer than Onyx injected inside dural arteries supplying the CS. However, more cases are needed to determine this.

Therapeutic Clip Occlusion of the Anterior Choroidal Artery Involved with Partially Thrombosed Fusiform Aneurysm: A Case Report.

BACKGROUND: We describe a rare case with partially thrombosed fusiform anterior choroidal artery (AchA) aneurysm successfully treated with therapeutic occlusion of the AchA. CLINICAL PRESENTATION: A 58-year-old man presented with transient mild hemiparesis of the right side. Magnetic resonance imaging (MRI) showed an ischemic lesion in the posterior limb of the left internal capsule. Digital subtraction angiography (DSA) revealed a left internal carotid artery saccular aneurysm (14.5-mm diameter) arising from the supraclinoid segment. The left AchA was not detected in the initial DSA, and MRI showed the aneurysm to be partially thrombosed. The second DSA performed 2 weeks after the onset showed recanalization of the thrombosed portion of the aneurysm with the left AchA apparently arising from its tip. The aneurysm was diagnosed as a partially thrombosed fusiform AchA aneurysm. RESULTS: Open surgery was performed and a titanium clip was applied to the base of the fusiform aneurysm under motor evoked potential monitoring, which remained unchanged after clipping. Occlusion of the aneurysm was confirmed by Doppler ultrasound and intraoperative fluorescence angiography. Furthermore, Doppler ultrasound and fluorescence angiography showed that the blood flow supplying the pyramidal tract was reconstituted by the retrograde collateral flow from the choroidal segment. The aneurysm was completely obliterated in postoperative DSA, which demonstrated retrograde filling of the AchA through the posterior circulation. The patient manifested transient weakness of the right side postoperatively, which was completely recovered after short-term rehabilitation. CONCLUSIONS: This case illustrates the unique clinical course of a rare partially thrombosed fusiform AchA aneurysm, successfully treated with therapeutic clip occlusion of the AchA under the multimodal monitoring.

Carotid artery occlusion for the treatment of symptomatic giant carotid aneurysms: a proposal of classification and surgical protocol.

Giant intracranial aneurysms are rare disorders that represent only 5% of all intracranial aneurysms; they have a wide variety of presentations including rupture, embolic effects, and mass effect symptoms that can mislead the diagnosis to tumors rather than aneurysms. Their treatment is difficult and carries higher morbidity and mortality than usual aneurysms due to their complex nature. This study involved retrospective analysis of data of 28 patients, managed between 2006 and 2012, suffering from giant internal carotid artery (ICA) aneurysms with various presenting symptoms, none of which was hemorrhage. They were all evaluated by BOT prior to any intervention; they were subjected to various treatment strategies including selective coiling, parent artery occlusion with or without bypass, aneurysm trapping with or without bypass, and patients were followed for a period ranging from 6 months to 5 years. Out of 26 patients with giant aneurysms with mass effects, 16 patients showed full recovery (61.5 %), 5 showed partial improvement (19.2 %), and 5 showed no change in mass effect symptoms (19.2 %). One patient died (3.5 %). Symptoms such as TIA or epistaxis showed complete recovery. This study shows that a well-designed protocol aiming at parent artery sacrifice will yield good to excellent results in managing ICA giant aneurysms, and it also shows that parent artery sacrifice is superior to other forms of treatment of these lesions regarding recurrence rates, morbidity, and mortality.

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

ß-amyloid accumulation enhances microtubule associated protein tau pathology in an APPNL-G-F/MAPTP301S mouse model of Alzheimer's disease.

Introduction: The study of the pathophysiology study of Alzheimer's disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular -amyloid (A) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. Methods: The humanized APPNL-G-F knock-in mouse line was crossed to the PS19 MAPTP301S, over-expression mouse line to create the dual APPNL-G-F/PS19 MAPTP301S line. The resulting pathologies were characterized by immunochemical methods and PCR. Results: We now report on a double transgenic APPNL-G-F/PS19 MAPTP301S mouse that at 6 months of age exhibits robust A plaque accumulation, intense MAPT pathology, strong inflammation and extensive neurodegeneration. The presence of A pathology potentiated the other major pathologies, including MAPT pathology, inflammation and neurodegeneration. MAPT pathology neither changed levels of amyloid precursor protein nor potentiated A accumulation. Interestingly, study of immunofluorescence in cleared brains indicates that microglial inflammation was generally stronger in the hippocampus, dentate gyrus and entorhinal cortex, which are regions with predominant MAPT pathology. The APPNL-G-F/MAPTP301S mouse model also showed strong accumulation of N6-methyladenosine (m6A), which was recently shown to be elevated in the AD brain. m6A primarily accumulated in neuronal soma, but also co-localized with a subset of astrocytes and microglia. The accumulation of m6A corresponded with increases in METTL3 and decreases in ALKBH5, which are enzymes that add or remove m6A from mRNA, respectively. Discussion: Our understanding of the pathophysiology of Alzheimer's disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular -amyloid (A) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. The APPNL-G-F/MAPTP301S mouse recapitulates many features of AD pathology beginning at 6 months of aging, and thus represents a useful new mouse model for the field.

Accumulation of m6A exhibits stronger correlation with MAPT than ß-amyloid pathology in an APPNL-G-F /MAPTP301S mouse model of Alzheimer's disease.

The study for the pathophysiology study of Alzheimer's disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular ß-amyloid (Aß) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. We now report on a double transgenic APPNL-G-F MAPTP301S mouse that at 6 months of age exhibits robust Aß plaque accumulation, intense MAPT pathology, strong inflammation and extensive neurodegeneration. The presence of Aß pathology potentiated the other major pathologies, including MAPT pathology, inflammation and neurodegeneration. However, MAPT pathology neither changed levels of amyloid precursor protein nor potentiated Aß accumulation. The APPNL-G-F/MAPTP301S mouse model also showed strong accumulation of N6-methyladenosine (m6A), which was recently shown to be elevated in the AD brain. M6A primarily accumulated in neuronal soma, but also co-localized with a subset of astrocytes and microglia. The accumulation of m6A corresponded with increases in METTL3 and decreases in ALKBH5, which are enzymes that add or remove m6A from mRNA, respectively. Thus, the APPNL-G-F/MAPTP301S mouse recapitulates many features of AD pathology beginning at 6 months of aging.

Accumulation of m6A exhibits stronger correlation with MAPT than ß-amyloid pathology in an APPNL-G-F /MAPTP301S mouse model of Alzheimer's disease.

The study for the pathophysiology study of Alzheimer's disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular ß-amyloid (Aß) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. We now report on a double transgenic APPNL-G-F MAPTP301S mouse that at 6 months of age exhibits robust Aß plaque accumulation, intense MAPT pathology, strong inflammation and extensive neurodegeneration. The presence of Aß pathology potentiated the other major pathologies, including MAPT pathology, inflammation and neurodegeneration. However, MAPT pathology neither changed levels of amyloid precursor protein nor potentiated Aß accumulation. The APPNL-G-F/MAPTP301S mouse model also showed strong accumulation of N6-methyladenosine (m6A), which was recently shown to be elevated in the AD brain. M6A primarily accumulated in neuronal soma, but also co-localized with a subset of astrocytes and microglia. The accumulation of m6A corresponded with increases in METTL3 and decreases in ALKBH5, which are enzymes that add or remove m6A from mRNA, respectively. Thus, the APPNL-G-F/MAPTP301S mouse recapitulates many features of AD pathology beginning at 6 months of aging.

RNF213 loss of function reshapes vascular transcriptome and spliceosome leading to disrupted angiogenesis and aggravated vascular inflammatory responses.

RNF213 gene mutations are the cause behind Moyamoya disease, a rare cerebrovascular occlusive disease. However, the function of RNF213 in the vascular system and the impact of its loss of function are not yet comprehended. To understand RNF23 function, we performed gene knockdown (KD) in vascular cells and performed various phenotypical analysis as well as extensive transcriptome and epitranscriptome profiling. Our data revealed that RNF213 KD led to disrupted angiogenesis in HUVEC, in part due to downregulation of DNA replication and proliferation pathways. Furthermore, HUVEC cells became sensitive to LPS induced inflammation after RNF213 KD, leading to retarded cell migration and enhanced macrophage transmigration. This was evident at the level of transcriptome as well. Interestingly, RNF213 led to extensive changes in mRNA splicing that were not previously reported. In vascular smooth muscle cells (vSMCs), RNF213 KD led to alteration in cytoskeletal organization, contractility, and vSMCs function related pathways. Finally, RNF213 KD disrupted endothelial-to-vSMCs communication in co-culture models. Overall, our results indicate that RNF213 KD sensitizes endothelial cells to inflammation, leading to altered angiogenesis. Our results shed the light on the important links between RNF213 mutations and inflammatory/immune inducers of MMD and on the unexplored role of epitranscriptome in MMD.

Codon Usage and mRNA Stability are Translational Determinants of Cellular Response to Canonical Ferroptosis Inducers.

Ferroptosis is a non-apoptotic cell death mechanism characterized by the generation of lipid peroxides. While many effectors in the ferroptosis pathway have been mapped, its epitranscriptional regulation is not yet fully understood. Ferroptosis can be induced via system xCT inhibition (Class I) or GPX4 inhibition (Class II). Previous works have revealed important differences in cellular response to different ferroptosis inducers. Importantly, blocking mRNA transcription or translation appears to protect cells against Class I ferroptosis inducing agents but not Class II. In this work, we examined the impact of blocking transcription (via Actinomycin D) or translation (via Cycloheximide) on Erastin (Class I) or RSL3 (Class II) induced ferroptosis. Blocking transcription or translation protected cells against Erastin but was detrimental against RSL3. Cycloheximide led to increased levels of GSH alone or when co-treated with Erastin via the activation of the reverse transsulfuration pathway. RNA sequencing analysis revealed early activation of a strong alternative splice program before observed changes in transcription. mRNA stability analysis revealed divergent mRNA stability changes in cellular response to Erastin or RSL3. Importantly, codon optimality biases were drastically different in either condition. Our data also implicated translation repression and rate as an important determinant of the cellular response to ferroptosis inducers. Given that mRNA stability and codon usage can be influenced via the tRNA epitranscriptome, we evaluated the role of a tRNA modifying enzyme in ferroptosis stress response. Alkbh1, a tRNA demethylase, led to translation repression and increased the resistance to Erastin but made cells more sensitive to RSL3.

Stress Induced tRNA Halves (tiRNAs) as Biomarkers for Stroke and Stroke Therapy; Pre-clinical Study.

tiRNAs are small non-coding RNAs generated by angiogenin-mediated tRNA cleavage during cellular stress. Some tiRNAs were shown to be cytoprotective, while other reports indicate that the generation of tiRNAs is cytotoxic. We used rat model of focal cerebral ischemia-reperfusion (I/R) injury to study the generation and regulation of tiRNAs following in vivo I/R and the impact of neuroprotective therapy on their generation. tiRNAs were induced after I/R and Minocycline therapy reduced global tiRNA levels. Our results showed that tRNA cleavage is tRNA species specific, and neuroprotective treatment does not affect all tiRNA species. We also evaluated the temporal changes in several tRNA modifying enzymes and showed a correlation between their expression and tRNA cleavage. In conclusion, we show that tiRNAs can serve as biomarkers for stroke and stroke therapy, further adding them to the repertoire of tools that can be used to monitor and treat stroke.