Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization

Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition

Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice

Myotubular myopathy, or X-linked centronuclear myopathy, is a severe muscle disorder representing a significant burden for patients and their families. It is clinically characterized by neonatal and severe muscle weakness and atrophy. Mutations in the myotubularin (MTM1) gene cause myotubular myopathy, and no specific curative treatment is available. We previously found that dynamin 2 (DNM2) is upregulated in both Mtm1 knockout and patient muscle samples, whereas its reduction through antisense oligonucleotides rescues the clinical and histopathological features of this myopathy in mice. Here, we propose a novel approach targeting Dnm2 mRNA. We screened and validated in vitro and in vivo several short hairpin RNA (shRNA) sequences that efficiently target Dnm2 mRNA. A single intramuscular injection of AAV-shDnm2 resulted in long-term reduction of DNM2 protein level and restored muscle force, mass, histology, and myofiber ultrastructure and prevented molecular defects linked to the disease. Our results demonstrate a robust DNM2 knockdown and provide an alternative strategy based on reduction of DNM2 to treat myotubular myopathy

Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization.

Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition

Knockdown of hypocretin attenuates extended access of cocaine self-administration in rats

The hypocretin/orexin (HCRT) neuropeptide system regulates feeding, arousal state, stress responses, and reward, especially under conditions of enhanced motivational relevance. In particular, HCRT neurotransmission facilitates drug-seeking behavior in circumstances that demand increased effort and/or motivation to take the drug. The present study used a shRNA-encoding adeno-associated viral vector to knockdown Hcrt expression throughout the dorsal hypothalamus in adult rats and determine the role of HCRT in cocaine self-administration. Chronic Hcrt silencing did not impact cocaine self-administration under short-access conditions, but robustly attenuated cocaine intake under extended access conditions, a model that mimics key features of compulsive cocaine taking. In addition, Hcrt silencing decreased motivation for both cocaine and a highly palatable food reward (i.e., sweetened condensed milk; SCM) under a progressive ratio schedule of reinforcement, but did not alter responding for SCM under a fixed ratio schedule. Importantly, Hcrt silencing did not affect food or water consumption, and had no consequence for general measures of arousal and stress reactivity. At the molecular level, chronic Hcrt knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller extent melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These original findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and non-drug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides compelling evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed extended access, possibly via functional interactions with DYN and MCH signaling

Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo.

International audienceThe sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads, and Mtm1-deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated with flat membrane stacks, whereas dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Overexpression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and an in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature

The G-quadruplex Ligand Telomestatin Inhibits POT1 Binding to Telomeric Sequences In vitro and Induces GFP-POT1 Dissociation from Telomeres in Human Cells

International audienceTelomestatin is a potent G-quadruplex ligand that specifically interacts with the 3' telomeric overhang, leading to its degradation and that induces a delayed senescence and apoptosis of cancer cells. Protection of Telomere 1 (POT1) was recently identified as a specific single-stranded telomere-binding protein involved in telomere capping and T-loop maintenance. We showed here that a telomestatin treatment inhibits POT1 binding to the telomeric overhang in vitro. The treatment of human EcR293 cells by telomestatin induces a dramatic and rapid delocalization of POT1 from its normal telomere sites but does not affect the telomere localization of the double-stranded telomere-binding protein TRF2. Thus, we propose that G-quadruplex stabilization at telomeric G-overhang inactivates POT1 telomeric function, generating a telomere dysfunction in which chromosome ends are no longer properly protected

BIN1 modulation in vivo rescues dynamin-related myopathy

International audienceThe mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2 RW/+ and Dnm2 RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2 RW/+ mice and rescued the perinatal lethality and survival of Dnm2 RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations

Amphiphysin 2 modulation rescues myotubular myopathy and prevents focal adhesion defects in mice

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