Pharmacological inhibition of the integrated stress response accelerates disease progression in an amyotrophic lateral sclerosis mouse model.

BACKGROUND AND PURPOSE: The integrated stress response (ISR) regulates translation in response to diverse stresses. ISR activation has been documented in amyotrophic lateral sclerosis (ALS) patients and ALS experimental models. In experimental models, both ISR stimulation and inhibition prevented ALS neurodegeneration; however, which mode of ISR regulation would work in patients is still debated. We previously demonstrated that the ISR modulator ISRIB (Integrated Stress Response InhiBitor, an eIF2B activator) enhances survival of neurons expressing the ALS neurotoxic allele SOD1 G93A. Here, we tested the effect of two ISRIB-like eIF2B activators (2BAct and PRXS571) in the disease progression of transgenic SOD1G93A mice. EXPERIMENTAL APPROACH: After biochemical characterization in primary neurons, SOD1G93A mice were treated with 2BAct and PRXS571. Muscle denervation of vulnerable motor units was monitored with a longitudinal electromyographic test. We used a clinical score to document disease onset and progression; force loss was determined with the hanging wire motor test. Motor neuronal survival was assessed by immunohistochemistry. KEY RESULTS: In primary neurons, 2BAct and PRXS571 relieve the ISR-imposed translational inhibition while maintaining high ATF4 levels. Electromyographic recordings evidenced an earlier and more dramatic muscle denervation in treated SOD1G93A mice that correlated with a decrease in motor neuron survival. Both compounds anticipated disease onset and shortened survival time. CONCLUSION AND IMPLICATIONS: 2BAct and PRXS571 anticipate disease onset, aggravating muscle denervation and motor neuronal death of SOD1G93A mice. This study reveals that the ISR works as a neuroprotective pathway in ALS motor neurons and reveals the toxicity that eIF2B activators may display in ALS patients.

Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1α.

Endoplasmic reticulum (ER) to nucleus homeostatic signaling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1α (inositol-requiring enzyme 1 alpha). While IRE1α can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1α complexes as well as the targeting mechanism that recruits XBP1 to IRE1α oligomers remains unknown. Here, we have developed a single-molecule imaging approach to monitor the recruitment of individual XBP1 transcripts to the ER surface. Using this methodology, we confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2 (hydrophobic region 2)-dependent targeting and relies on active translation. In addition, we show that IRE1α-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1α clusters, which are only observed upon overexpression of fluorescently tagged IRE1α during ER stress. Our findings support a model where ribosome-engaged, immobilized XBP1 mRNA is processed by small IRE1α assemblies that could be dynamically recruited for processing of mRNA transcripts on the ER.

Linking the Expression of Therapeutic Genes to Unfolded Protein Response: A New Option for Anti-Hepatitis B Virus Gene Therapy.

Tight control of transgene expression is key to ensure the efficacy of a wide range of gene therapy interventions, in which the magnitude and duration of gene expression have to be adjusted to therapeutic needs, thereby limiting secondary effects. The development of upgraded strategies to link transgene expression to pathological stress episodes is an unmet need in gene therapy. Here, we propose an expression strategy that associates transgene expression to an intracellular stress coping mechanism, the unfolded protein response. Specifically, we harnessed the cis elements required to sustain the noncanonical splicing of X-box binding protein 1 (XBP1) messenger RNA (mRNA) in response to the dysfunction of the endoplasmic reticulum (ER), a situation commonly known as ER stress, to drive the expression of heterologous genes. Since ER stress features a wide variety of pathological conditions, including viral infections, cancer, or metabolic disorders, this new expression module stimulates the synthesis of therapeutic genes as a response to cellular damage, and ensures their expression only when necessary. Validation of this inducible expression system was performed in vitro and in vivo, and its potential to limit/inhibit viral infections has been shown in proof-of principle experiments.

Fine tuning of the unfolded protein response by ISRIB improves neuronal survival in a model of amyotrophic lateral sclerosis.

Loss of protein folding homeostasis features many of the most prevalent neurodegenerative disorders. As coping mechanism to folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate a gene expression program to restore proteostasis, or when stress is chronic or overwhelming promote neuronal death. This fate-defining capacity of the UPR has been proposed to play a key role in amyotrophic lateral sclerosis (ALS). However, the several genetic or pharmacological attempts to explore the therapeutic potential of UPR modulation have produced conflicting observations. In order to establish the precise relationship between UPR signaling and neuronal death in ALS, we have developed a neuronal model where the toxicity of a familial ALS-causing allele (mutant G93A SOD1) and UPR activation can be longitudinally monitored in single neurons over the process of neurodegeneration by automated microscopy. Using fluorescent UPR reporters we established the temporal and causal relationship between UPR and neuronal death by Cox regression models. Pharmacological inhibition of discrete UPR processes allowed us to establish the contribution of PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme-1) mechanisms to neuronal fate. Importantly, inhibition of PERK signaling with its downstream inhibitor ISRIB, but not with the direct PERK kinase inhibitor GSK2606414, significantly enhanced the survival of G93A SOD1-expressing neurons. Characterization of the inhibitory properties of both drugs under ER stress revealed that in neurons (but not in glial cells) ISRIB overruled only part of the translational program imposed by PERK, relieving the general inhibition of translation, but maintaining the privileged translation of ATF4 (activating transcription factor 4) messenger RNA. Surprisingly, the fine-tuning of the PERK output in G93A SOD1-expressing neurons led to a reduction of IRE1-dependent signaling. Together, our findings identify ISRIB-mediated translational reprogramming as a new potential ALS therapy.

The exposure to uteroplacental insufficiency is associated with activation of unfolded protein response in postnatal life.

Early life events are associated with the susceptibility to chronic diseases in adult life. Perturbations of endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which contributes to the development of metabolic alterations. Our aim was to evaluate liver UPR in an animal model of intrauterine growth restriction (IUGR). A significantly increased expression of X-box binding protein-1 spliced (XBP1s) mRNA (p<0.01), Endoplasmic Reticulum-localized DnaJ homologue (Erdj4) mRNA (p<0.05) and Bip/GRP78-glucose-regulated protein 78 (Bip) mRNA (p<0.05) was observed in the liver of IUGR rats at birth. Furthermore, the expression of gluconeogenesis genes and lipogenesis genes were significantly upregulated (p<0.05) in IUGR pups. At 105 d, IUGR male rats showed significantly reduced glucose tolerance (p<0.01). A significant decreased expression of XBP1s mRNA (p<0.01) and increased expression of double-stranded RNA-dependent protein kinase-like ER kinase (PERK) and Asparagine synthetase (ASNS) (p<0.05) was observed in the liver of IUGR male adult rats. Liver focal steatosis and periportal fibrosis were observed in IUGR rats. These findings show for the first time that fetal exposure to uteroplacental insufficiency is associated with the activation of hepatic UPR and suggest that UPR signaling may play a role in the metabolic risk.

TMEM173 Alternative Spliced Isoforms Modulate Viral Replication through the STING Pathway.

The innate immune system provides a primary line of defense against pathogens. Stimulator of IFN genes (STING), encoded by the TMEM173 gene, is a critical protein involved in IFN-ß induction in response to infection by different pathogens. In this study, we describe the expression of three different alternative-spliced human (h) TMEM173 mRNAs producing STING truncated isoforms 1, 2, and 3 in addition to the full-length wild-type (wt) hSTING. All of the truncated isoforms lack exon 7 and share the N-terminal transmembrane region with wt hSTING. Overexpression of the three STING truncated isoforms failed to induce IFN-ß, and they acted as selective pathway inhibitors of wt hSTING even in combination with upstream inducer cyclic-di-GMP-AMP synthase. Truncated isoforms alter the stability of wt hSTING, reducing protein t 1/2 to some extent by the induction of proteasome-dependent degradation. Knocking down expression of truncated isoforms increased production of IFN-ß by THP1 monocytes in response to intracellular cytosolic DNA or HSV-1 infection. At early stages of infection, viruses like HSV-1 or vesicular stomatitis virus reduced the ratio of full-length wt hSTING/truncated STING isoforms, suggesting the skewing of alternative splicing of STING toward truncated forms as a tactic to evade antiviral responses. Finally, in silico analysis revealed that the human intron-exon gene architecture of TMEM173 (splice sites included) is preserved in other mammal species, predominantly primates, stressing the relevance of alternative splicing in regulating STING antiviral biology.

X-box Binding Protein 1 Regulates Unfolded Protein, Acute-Phase, and DNA Damage Responses During Regeneration of Mouse Liver.

BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.

Safety and liver transduction efficacy of rAAV5-cohPBGD in nonhuman primates: a potential therapy for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.

[Suprapubic cystostomy catheter knotting. Presentation of the first national case and review of the literature].

OBJECTIVE: This study presents the first Spanish case of a spontaneous knot in the catheter of a suprapubic cystostomy and review the national and international literature. METHODS: The case of an 87-year-old patient who was diagnosed with adenocarcinoma of the prostate is presented. A suprapubic vesical puncture for urinary retention was urgently performed in this patient because of the impossibility of urethral catheterisation. A spontaneous knot in the catheter was detected upon removal; the tightened knot could be removed by gentle and sustained traction without surgery. RESULTS: Knotting or calcification of the catheter was suspected when the catheter remained anchored in the bladder during a removal attempt 5 days after initial catheterisation. Plain pelvis x-ray was taken, but no calcification or knots were observed because the catheter was radiolucent. An ultrasound would have offered more information, but it was not requested. Gentle and sustained traction of the catheter reduced the knot size and allowed catheter removal without complications. Worldwide cases and national publications were reviewed. CONCLUSION: The formation of spontaneous or manipulation-induced knots in urinary cystostomy catheters is an extremely rare complication. The presented case is the first Spanish case of catheter knotting; it is only the 17th reported case worldwide.