BIN1 modulation in vivo rescues dynamin-related myopathy

The 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 Dnm2RW/+ and Dnm2RW/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 Dnm2RW/+ mice and rescued the perinatal lethality and survival of Dnm2RW/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

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

In Vivo-Matured Langerhans Cells Continue to Take Up and Process Native Proteins Unlike In Vitro-Matured Counterparts

Abstract We have been able to identify the cell subset derived from Langerhans cells in the total dendritic cell population of the peripheral lymph node and hence to follow their trafficking under normal physiological conditions as well as upon skin irritation. As expected, the rapid mobilization of Langerhans cells triggered by inflammatory signals into the draining lymph node correlated with an up-regulation of costimulatory molecules and with an enhanced immunostimulatory capacity. Surprisingly, however, these cells, instead of shutting down, maintain the capacity to capture and process protein Ags during the couple of days they stay alive in stark contrast to in vitro-matured dendritic cells.</jats:p

Different in vivo impact of Dynamin 2 mutations implicated in Charcot-Marie-Tooth neuropathy or Centronuclear Myopathy

Abstract Dynamin 2 (DNM2) is a ubiquitously expressed GTPase implicated in many cellular functions such as membrane trafficking and cytoskeleton regulation. Dominant mutations in DNM2 result in tissue specific diseases affecting peripheral nerves (Charcot-Marie-Tooth neuropathy, CMT) or skeletal muscles (Centronuclear myopathy, CNM). However, the reason for this tissue specificity is unknown, and it remains unclear if these diseases share a common pathomechanism. To compare the disease pathophysiological mechanisms in skeletal muscle, we exogenously expressed wild-type DNM2 (WT-DNM2), the DNM2-CMT mutation K562E, or DNM2-CNM mutations R465W and S619L causing adult and neonatal forms respectively, by intramuscular adeno-associated virus (AAV) injections. All muscles expressing exogenous WT-DNM2 and CNM or CMT mutations exhibited reduced muscle force. However, only expression of CNM mutations and WT-DNM2 correlated with CNM-like histopathological hallmarks of nuclei centralization and reduced fiber size. The extent of alterations correlated with clinical severity in patients. Ultrastructural and immunofluorescence analyses highlighted defects of the triads, mitochondria and costameres as major causes of the CNM phenotype. Despite the reduction in force upon expression of the DNM2-CMT mutation, muscle histology and ultrastructure were almost normal. However, the neuromuscular junction was affected in all DNM2 injected muscles, with the DNM2-CMT mutation inducing the most severe alterations, potentially explaining the reduction in force observed with this mutant. In conclusion, expression of WT and CNM mutants recreate a CNM-like phenotype, suggesting CNM mutations are gain-of-function. Histological, ultrastructural and molecular analyses pointed to key pathways uncovering the different pathomechanisms involved in centronuclear myopathy or Charcot-Marie-Tooth neuropathy linked to DNM2 mutations.</jats:p

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

Effects of mGlu4 knock-down in the nucleus accumbens and basolateral amygdala of wild-type mice

Autism Spectrum Disorders (ASD) are neuro-developmental pathologies characterized by social communication and interaction deficiency, and repetitive and restricted behaviors. The social motivation network, notably the nucleus accumbens (NAc), is dysfunctional in these disorders. We previously showed that pharmacological treatment facilitating the activity of the mGlu4 glutamate receptor relieves autistic symptoms in the Oprm1 -/- mouse model of autism. Interestingly, we also evidenced that mGlu4 can modulate the activity of the amygdala, a critical brain region for emotional responses and social behavior. Aim: Our hypothesis is mGlu4 in the NAc and the basolateral amygdala (BLA) plays a critical role in modulating autism-sensitive symptoms, namely social motivation and stereotyped behaviors. Methods : In order to test this, we performed the knockdown of the gene coding for mGlu4, Grm4, in the nucleus accumbens or the basolateral amygdala of wild-type mice using bilateral stereotaxic injections of shRNA-expressing AAVs. We tested them in several behavioral tests to evaluate the presence or not of autistic symptoms. Results: Knocking down (KD) of Grm4 expression in NAc neurons induces autistic-like symptoms such as impaired social interactions and social preference Conclusions : These results suggest that mGlu4 receptors expressed in the NAc and BLA neurons play a major role in modulating sociability in mice.</jats:p

Effects of mGlu4 knock-down in the nucleus accumbens and basolateral amygdala of wild-type mice

Autism Spectrum Disorders (ASD) are neuro-developmental pathologies characterized by social communication and interaction deficiency, and repetitive and restricted behaviors. The social motivation network, notably the nucleus accumbens (NAc), is dysfunctional in these disorders. We previously showed that pharmacological treatment facilitating the activity of the mGlu4 glutamate receptor relieves autistic symptoms in the Oprm1 -/- mouse model of autism. Interestingly, we also evidenced that mGlu4 can modulate the activity of the amygdala, a critical brain region for emotional responses and social behavior. Aim: Our hypothesis is mGlu4 in the NAc and the basolateral amygdala (BLA) plays a critical role in modulating autism-sensitive symptoms, namely social motivation and stereotyped behaviors. Methods : In order to test this, we performed the knockdown of the gene coding for mGlu4, Grm4, in the nucleus accumbens or the basolateral amygdala of wild-type mice using bilateral stereotaxic injections of shRNA-expressing AAVs. We tested them in several behavioral tests to evaluate the presence or not of autistic symptoms. Results: Knocking down (KD) of Grm4 expression in NAc neurons induces autistic-like symptoms such as impaired social interactions and social preference Conclusions : These results suggest that mGlu4 receptors expressed in the NAc and BLA neurons play a major role in modulating sociability in mice.</jats:p

RSK2 Signaling in Medial Habenula Contributes to Acute Morphine Analgesia

It has been established that mu opioid receptors activate the ERK1/2 signaling cascade both in vitro and in vivo. The Ser/Thr kinase RSK2 is a direct downstream effector of ERK1/2 and has a role in cellular signaling, cell survival growth, and differentiation; however, its role in biological processes in vivo is less well known. Here we determined whether RSK2 contributes to mu-mediated signaling in vivo. Knockout mice for the rsk2 gene were tested for main morphine effects, including analgesia, tolerance to analgesia, locomotor activation, and sensitization to this effect, as well as morphine withdrawal. The deletion of RSK2 reduced acute morphine analgesia in the tail immersion test, indicating a role for this kinase in mu receptor-mediated nociceptive processing. All other morphine effects and adaptations to chronic morphine were unchanged. Because the mu opioid receptor and RSK2 both show high density in the habenula, we specifically downregulated RSK2 in this brain metastructure using an adeno-associated-virally mediated shRNA approach. Remarkably, morphine analgesia was significantly reduced, as observed in the total knockout animals. Together, these data indicate that RSK2 has a role in nociception, and strongly suggest that a mu opioid receptor–RSK2 signaling mechanism contributes to morphine analgesia at the level of habenula. This study opens novel perspectives for both our understanding of opioid analgesia, and the identification of signaling pathways operating in the habenular complex