Converging studies demonstrate the dysfunction of the dopaminergic neurons following chronic opioid administration. However, the therapeutic strategies targeting opioid-responsive dopaminergic ensembles that contribute to the development of opioid withdrawal remain to be elucidated. Here, we used the neuronal activity-dependent Tet-Off system to label dopaminergic ensembles in response to initial morphine exposure (Mor-Ens) in the ventral tegmental area (VTA). Fiber optic photometry recording and transcriptome analysis revealed downregulated spontaneous activity, dysregulated mitochondrial respiratory, ultrastructure, and oxidoreductase signal pathways after chronic morphine administration in these dopaminergic ensembles. Mitochondrial fragmentation and the decreased mitochondrial fusion gene mitofusin 1 (Mfn1) were found in these ensembles after prolonged opioid withdrawal. Restoration of Mfn1 in the dopaminergic Mor-Ens attenuated excessive oxidative stress and the development of opioid withdrawal. Administration of Mdivi-1, a mitochondrial fission inhibitor, ameliorated the mitochondrial fragmentation and maladaptation of the neuronal plasticity in these Mor-Ens, accompanied by attenuated development of opioid withdrawal after chronic morphine administration, without affecting the analgesic effect of morphine. These findings highlighted the plastic architecture of mitochondria as a potential therapeutic target for opioid analgesic-induced substance use disorders.
Changyou Jiang, Han Huang, Xiao Yang, Qiumin Le, Xing Liu, Lan Ma, Feifei Wang
Spinocerebellar ataxia type 3 (SCA3) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the ataxin-3 (ATXN3) gene. No effective treatment is available for this disorder, other than symptom-directed approaches. Bile acids have shown therapeutic efficacy in neurodegenerative disease models. Here, we pinpointed tauroursodeoxycholic acid (TUDCA) as an efficient therapeutic, improving the motor and neuropathological phenotype of SCA3 nematode and mouse models. Surprisingly, transcriptomic and functional in vivo data showed that TUDCA acts in neuronal tissue through the glucocorticoid receptor (GR), but independently of its canonical receptor, the FXR. TUDCA was predicted to bind to the GR, similarly to corticosteroid molecules. GR levels were decreased in disease-affected brain regions, likely due to increased protein degradation as a consequence of ATXN3 dysfunction, being restored by TUDCA treatment. Analysis of a SCA3 clinical cohort showed intriguing correlations between the peripheral expression of GR and the predicted age at disease onset, in pre-symptomatic subjects, and of FKBP5 expression with disease progression, suggesting this pathway as a potential source of biomarkers for future study. We have established a novel in vivo mechanism for the neuroprotective effects of TUDCA in SCA3, and propose this readily available drug for clinical trials in SCA3 patients.
Sara Duarte-Silva, Jorge Diogo Da Silva, Daniela Monteiro-Fernandes, Marta Daniela Costa, Andreia Neves-Carvalho, Mafalda Raposo, Carina Soares-Cunha, Joana S. Correia, Gonçalo Nogueira-Gonçalves, Henrique S. Fernandes, Stéphanie Oliveira, Ana Rita Ferreira-Fernandes, Fernando Rodrigues, Joana Pereira-Sousa, Daniela Vilasboas-Campos, Sara Guerreiro, Jonas Campos, Liliana Meireles-Costa, Cecilia M.P. Rodrigues, Stephanie Cabantous, Sérgio F. Sousa, Manuela Lima, Andreia Teixeira-Castro, Patricia Maciel
Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling in mice. Mice with forced skipping of exon 29 in the CaV1.1 calcium channel combined with loss of ClC-1 chloride channel function displayed markedly reduced lifespan, whereas other combinations of splicing mimics did not affect survival. The Ca2+/Cl– bi-channelopathy mice exhibited myotonia, weakness, and impairment of mobility and respiration. Chronic administration of the calcium channel blocker verapamil rescued survival and improved force generation, myotonia, and respiratory function. These results suggest that Ca2+/Cl– bi-channelopathy contributes to muscle impairment in DM1 and is potentially mitigated by common clinically available calcium channel blockers.
Lily A. Cisco, Matthew T. Sipple, Katherine M. Edwards, Charles A. Thornton, John D. Lueck
Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell–enhancing target. In murine CD8+ T cell–therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.
Michael R. Schlabach, Sharon Lin, Zachary R. Collester, Christopher Wrocklage, Sol Shenker, Conor Calnan, Tianlei Xu, Hugh S. Gannon, Leila J. Williams, Frank Thompson, Paul R. Dunbar, Robert A. LaMothe, Tracy E. Garrett, Nicholas Colletti, Anja F. Hohmann, Noah J. Tubo, Caroline P. Bullock, Isabelle Le Mercier, Katri Sofjan, Jason J. Merkin, Sean Keegan, Gregory V. Kryukov, Caroline Dugopolski, Frank Stegmeier, Karrie Wong, Fiona A. Sharp, Louise Cadzow, Micah J. Benson
Glycogen storage disease type III (GSDIII) is a rare inborn error of metabolism affecting liver, skeletal muscle, and heart due to mutations of the AGL gene encoding for the glycogen debranching enzyme (GDE). No curative treatment exists for GSDIII. The 4.6 kb GDE cDNA represents the major technical challenge toward the development of a single recombinant adeno-associated virus (rAAV)-derived vector gene therapy strategy. Using information on GDE structure and molecular modeling, we generated multiple truncated GDEs retaining activity. Among them, an N-terminal-truncated mutant ∆Nter2-GDE had a similar efficacy in vivo compared to the full-size enzyme. A rAAV vector expressing ∆Nter2-GDE allowed significant glycogen reduction in heart and muscle of Agl–/– mice three months after intravenous injection, as well as normalization of histology features and restoration of muscle strength. Similarly, glycogen accumulation and histological features were corrected in a recently generated Agl–/– rat model. Finally, transduction with rAAV vectors encoding ∆Nter2-GDE corrected glycogen accumulation in an in vitro human skeletal muscle cellular model of GSDIII. In conclusion, our results demonstrated the ability of a single rAAV vector expressing a functional mini-GDE transgene to correct the muscle and heart phenotype in multiple models of GSDIII, supporting its clinical translation to GSDIII patients.
Antoine Gardin, Jérémy Rouillon, Valle Montalvo-Romeral, Lucille Rossiaud, Patrice Vidal, Romain Launay, Mallaury Vie, Youssef Krimi Benchekroun, Jérémie Cosette, Bérangère Bertin, Tiziana La Bella, Guillaume Dubreuil, Justine Nozi, Louisa Jauze, Romain Fragnoud, Nathalie F. Daniele, Laetitia Van Wittenberghe, Jérémy Esque, Isabelle André, Xavier Nissan, Lucile Hoch, Giuseppe Ronzitti
Half of all men with advanced prostate cancer (PCa) inherit at least 1 copy of an adrenal-permissive HSD3B1 (1245C) allele, which increases levels of 3β-hydroxysteroid dehydrogenase 1 (3βHSD1) and promotes intracellular androgen biosynthesis. Germline inheritance of the adrenally permissive allele confers worse outcomes in men with advanced PCa. We investigated whether HSD3B1 (1245C) drives resistance to combined androgen deprivation and radiotherapy. Adrenally permissive 3βHSD1 enhanced resistance to radiotherapy in PCa cell lines and xenograft models engineered to mimic the human adrenal/gonadal axis during androgen deprivation. The allele-specific effects on radiosensitivity were dependent on availability of DHEA, the substrate for 3βHSD1. In lines expressing the HSD3B1 (1245C) allele, enhanced expression of DNA damage response (DDR) genes and more rapid DNA double-strand break (DSB) resolution were observed. A correlation between androgen receptor (AR) expression and increased DDR gene expression was confirmed in 680 radical prostatectomy specimens. Treatment with the nonsteroidal antiandrogen enzalutamide reversed the resistant phenotype of HSD3B1 (1245C) PCa in vitro and in vivo. In conclusion, 3βHSD1 promotes prostate cancer resistance to combined androgen deprivation and radiotherapy by upregulating DNA DSB repair. This work supports prospective validation of early combined androgen blockade for high-risk men harboring the HSD3B1 (1245C) allele.
Shinjini Ganguly, Zaeem Lone, Andrew Muskara, Jarrell Imamura, Aimalie Hardaway, Mona Patel, Mike Berk, Timothy D. Smile, Elai Davicioni, Kevin L. Stephans, Jay Ciezki, Christopher J. Weight, Shilpa Gupta, Chandana A. Reddy, Rahul D. Tendulkar, Abhishek A. Chakraborty, Eric A. Klein, Nima Sharifi, Omar Y. Mian
The PI3K/AKT/mTOR pathway is commonly dysregulated in cancer. Rapalogs exhibit modest clinical benefit, likely owing to their lack of effects on 4EBP1. We hypothesized that bi-steric mTORC1-selective inhibitors would have greater potential for clinical benefit than rapalogs in tumors with mTORC1 dysfunction. We assessed this hypothesis in tumor models with high mTORC1 activity both in vitro and in vivo. Bi-steric inhibitors had strong growth inhibition, eliminated phosphorylated 4EBP1, and induced more apoptosis than rapamycin or MLN0128. Multiomics analysis showed extensive effects of the bi-steric inhibitors in comparison with rapamycin. De novo purine synthesis was selectively inhibited by bi-sterics through reduction in JUN and its downstream target PRPS1 and appeared to be the cause of apoptosis. Hence, bi-steric mTORC1-selective inhibitors are a therapeutic strategy to treat tumors driven by mTORC1 hyperactivation.
Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John M. Asara, Kwok-Kin Wong, Elizabeth P. Henske, Mallika Singh, David J. Kwiatkowski
Several poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are approved by FDA treat cancer with BRCA mutations. BRCA mutation is considered to fuel PARPi killing effect by inducing apoptosis. However, resistance to PARPi is frequently observed in clinic due to incomplete understanding on the molecular basis of PARPi function and lack of good markers to predict response in addition to BRCA mutations. Here we show that gasdermin C (GSDMC) sensitized tumor cells to PARPi in vitro and in immunocompetent mice and caused durable tumor regression in an immune-dependent manner. High expression level of GSDMC predicted better response to PARPi treatment in triple-negative breast cancer (TNBC) patients. PARPi treatment triggered GSDMC/caspase-8-mediated cancer cell pyroptosis (CCP) that enhanced PARPi killing of tumor cells. GSDMC-mediated CCP increased memory CD8+ T cell population in lymph node (LN), spleen, tumor, and thus promoted cytotoxic CD8+ T cell infiltration in tumor microenvironment. T cell-derived granzyme B (GZMB) activated caspase-6, which subsequently cleaved GSDMC to induce pyroptosis. Interestingly, IFN-γ induced GSDMC expression, which in turn enhanced the cytotoxicity of PARPi and T cell. Importantly, GSDMC promoted tumor clearance independent of BRCA deficiency in multiple cancer types with PARPi treatment. This study identifies a general marker and target for PARPi therapy and offers new insights into the mechanism of PARPi function.
Shuanglian Wang, Chiung-Wen Chang, Juan Huang, Shan Zeng, Xin Zhang, Mien-Chie Hung, Junwei Hou
Garth T. Whiteside, Donald J. Kyle, Ram P. Kapil, Alessandra Cipriano, Ellie He, Mingyan Zhou, Manjunath S. Shet, Michele Hummel, Terri Knappenberger, Kazuya Fukumura, Yoshiyuki Matsuo, Masahiro Uehira, Shuichi Hiroyama, Nozomi Takai, Sandra K. Willsie, Stephen C. Harris
Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-alpha (G6Pase-α, encoded by G6PC), primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves the life expectancy, patients still suffer from intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a, but potentially require complicated glycemic management throughout life. Here, we present a oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. A generated mouse strain with homozygous knock-in of this variant well reflected the pathophysiology of GSD1a patients. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b exhibited long-lasting efficacy for more than 12 weeks in the mice with a single dose and favorable pharmacokinetics and tolerability in mice and monkeys. Taking these findings together, this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.
Kentaro Ito, Go Tajima, Chikako Kamisato, Miyuki Tsumura, Mitsuhiro Iwamoto, Yukiko Sekiguchi, Yukinobu Numata, Kyoko Watanabe, Yoshiyuki Yabe, Satomi Kanki, Yusuke Fujieda, Koichi Goto, Yoshitaka Sogawa, Masataka Oitate, Hiroyuki Nagase, Shinnosuke Tsuji, Tomohiro Nishizawa, Masayo Kakuta, Takeshi Masuda, Yoshiyuki Onishi, Makoto Koizumi, Hidefumi Nakamura, Satoshi Okada, Masafumi Matsuo, Kiyosumi Takaishi
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