Variant-selective stereopure oligonucleotides protect against pathologies associated with C9orf72-repeat expansion in preclinical models.

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.

Systemic epothilone D improves hindlimb function after spinal cord contusion injury in rats.

Following a spinal cord injury (SCI) a growth aversive environment forms, consisting of a fibroglial scar and inhibitory factors, further restricting the already low intrinsic growth potential of injured adult central nervous system (CNS) neurons. Previous studies have shown that local administration of the microtubule-stabilizing drug paclitaxel or epothilone B (Epo B) reduce fibrotic scar formation and axonal dieback as well as induce axonal growth/sprouting after SCI. Likewise, systemic administration of Epo B promoted functional recovery. In this study, we investigated the effects of epothilone D (Epo D), an analog of Epo B with a possible greater therapeutic index, on fibrotic scarring, axonal sprouting and functional recovery after SCI. Delayed systemic administration of Epo D after a moderate contusion injury (150 kDyn) in female Fischer 344 rats resulted in a reduced number of footfalls when crossing a horizontal ladder at 4 and 8 weeks post-injury. Hindlimb motor function assessed with the BBB open field locomotor rating scale and Catwalk gait analysis were not significantly altered. Moreover, formation of laminin positive fibrotic scar tissue and 5-HT positive serotonergic fiber length caudal to the lesion site were not altered after treatment with Epo D. These findings recapitulate a functional benefit after systemic administration of a microtubule-stabilizing drug in rat contusion SCI.

Systemic administration of epothilone D improves functional recovery of walking after rat spinal cord contusion injury.

Central nervous system (CNS) injuries cause permanent impairments of sensorimotor functions as mature neurons fail to regenerate their severed axons. The poor intrinsic growth capacity of adult CNS neurons and the formation of an inhibitory lesion scar are key impediments to axon regeneration. Systemic administration of the microtubule stabilizing agent epothilone B promotes axon regeneration and recovery of motor function by activating the intrinsic axonal growth machinery and by reducing the inhibitory fibrotic lesion scar. Thus, epothilones hold clinical promise as potential therapeutics for spinal cord injury. Here we tested the efficacy of epothilone D, an epothilone B analog with a superior safety profile. By using liquid chromatography and mass spectrometry (LC/MS), we found adequate CNS penetration and distribution of epothilone D after systemic administration, confirming the suitability of the drug for non-invasive CNS treatment. Systemic administration of epothilone D reduced inhibitory fibrotic scarring, promoted regrowth of injured raphespinal fibers and improved walking function after mid-thoracic spinal cord contusion injury in adult rats. These results confirm that systemic administration of epothilones is a valuable therapeutic strategy for CNS regeneration and repair after injury and provides a further advance for potential clinical translation.

Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury.

After central nervous system (CNS) injury, inhibitory factors in the lesion scar and poor axon growth potential prevent axon regeneration. Microtubule stabilization reduces scarring and promotes axon growth. However, the cellular mechanisms of this dual effect remain unclear. Here, delayed systemic administration of a blood-brain barrier-permeable microtubule-stabilizing drug, epothilone B (epoB), decreased scarring after rodent spinal cord injury (SCI) by abrogating polarization and directed migration of scar-forming fibroblasts. Conversely, epothilone B reactivated neuronal polarization by inducing concerted microtubule polymerization into the axon tip, which propelled axon growth through an inhibitory environment. Together, these drug-elicited effects promoted axon regeneration and improved motor function after SCI. With recent clinical approval, epothilones hold promise for clinical use after CNS injury.

Sleep disturbances in highly stress reactive mice: modeling endophenotypes of major depression.

BACKGROUND: Neuronal mechanisms underlying affective disorders such as major depression (MD) are still poorly understood. By selectively breeding mice for high (HR), intermediate (IR), or low (LR) reactivity of the hypothalamic-pituitary-adrenocortical (HPA) axis, we recently established a new genetic animal model of extremes in stress reactivity (SR). Studies characterizing this SR mouse model on the behavioral, endocrine, and neurobiological levels revealed several similarities with key endophenotypes observed in MD patients. HR mice were shown to have changes in rhythmicity and sleep measures such as rapid eye movement sleep (REMS) and non-REM sleep (NREMS) as well as in slow wave activity, indicative of reduced sleep efficacy and increased REMS. In the present study we were interested in how far a detailed spectral analysis of several electroencephalogram (EEG) parameters, including relevant frequency bands, could reveal further alterations of sleep architecture in this animal model. Eight adult males of each of the three breeding lines were equipped with epidural EEG and intramuscular electromyogram (EMG) electrodes. After recovery, EEG and EMG recordings were performed for two days. RESULTS: Differences in the amount of REMS and wakefulness and in the number of transitions between vigilance states were found in HR mice, when compared with IR and LR animals. Increased frequencies of transitions from NREMS to REMS and from REMS to wakefulness in HR animals were robust across the light-dark cycle. Detailed statistical analyses of spectral EEG parameters showed that especially during NREMS the power of the theta (6-9 Hz), alpha (10-15 Hz) and eta (16-22.75 Hz) bands was significantly different between the three breeding lines. Well defined distributions of significant power differences could be assigned to different times during the light and the dark phase. Especially during NREMS, group differences were robust and could be continuously monitored across the light-dark cycle. CONCLUSIONS: The HR mice, i.e. those animals that have a genetic predisposition to hyper-activating their HPA axis in response to stressors, showed disturbed patterns in sleep architecture, similar to what is known from depressed patients. Significant alterations in several frequency bands of the EEG, which also seem to at least partly mimic clinical observations, suggest the SR mouse lines as a promising animal model for basic research of mechanisms underlying sleep impairments in MD.

Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury.

Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents through various cellular mechanisms, including dampening of transforming growth factor-ß signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth-competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow.

Electrical activity suppresses axon growth through Ca(v)1.2 channels in adult primary sensory neurons.

BACKGROUND: Primary sensory neurons of the dorsal root ganglia (DRG) regenerate their spinal cord axon if the peripheral nerve axon has previously been cut. This conditioning lesion confers axon growth competence to the neurons. However, the signal that is sensed by the cell upon peripheral lesion to initiate the regenerative response remains elusive. RESULTS: We show here that loss of electrical activity following peripheral deafferentiation is an important signal to trigger axon regrowth. We first verified that firing in sensory fibers, as recorded from dorsal roots in vivo, declined after peripheral lesioning but was not altered after central lesioning. We found that electrical activity strongly inhibited axon outgrowth in cultured adult sensory neurons. The inhibitory effect depended on the L-type voltage-gated Ca(2+) channel current and involved transcriptional changes. After a peripheral lesion, the L-type current was consistently diminished and the L-type pore-forming subunit, Ca(v)1.2, was downregulated. Genetic ablation of Ca(v)1.2 in the nervous system caused an increase in axon outgrowth from dissociated DRG neurons and enhanced peripheral nerve regeneration in vivo. CONCLUSIONS: Our data indicate that cessation of electrical activity after peripheral lesion contributes to the regenerative response observed upon conditioning and might be necessary to promote regeneration after central nervous system injury.

Rhythmicity in mice selected for extremes in stress reactivity: behavioural, endocrine and sleep changes resembling endophenotypes of major depression.

BACKGROUND: Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, including hyper- or hypo-activity of the stress hormone system, plays a critical role in the pathophysiology of mood disorders such as major depression (MD). Further biological hallmarks of MD are disturbances in circadian rhythms and sleep architecture. Applying a translational approach, an animal model has recently been developed, focusing on the deviation in sensitivity to stressful encounters. This so-called 'stress reactivity' (SR) mouse model consists of three separate breeding lines selected for either high (HR), intermediate (IR), or low (LR) corticosterone increase in response to stressors. METHODOLOGY/PRINCIPLE FINDINGS: In order to contribute to the validation of the SR mouse model, our study combined the analysis of behavioural and HPA axis rhythmicity with sleep-EEG recordings in the HR/IR/LR mouse lines. We found that hyper-responsiveness to stressors was associated with psychomotor alterations (increased locomotor activity and exploration towards the end of the resting period), resembling symptoms like restlessness, sleep continuity disturbances and early awakenings that are commonly observed in melancholic depression. Additionally, HR mice also showed neuroendocrine abnormalities similar to symptoms of MD patients such as reduced amplitude of the circadian glucocorticoid rhythm and elevated trough levels. The sleep-EEG analyses, furthermore, revealed changes in rapid eye movement (REM) and non-REM sleep as well as slow wave activity, indicative of reduced sleep efficacy and REM sleep disinhibition in HR mice. CONCLUSION/SIGNIFICANCE: Thus, we could show that by selectively breeding mice for extremes in stress reactivity, clinically relevant endophenotypes of MD can be modelled. Given the importance of rhythmicity and sleep disturbances as biomarkers of MD, both animal and clinical studies on the interaction of behavioural, neuroendocrine and sleep parameters may reveal molecular pathways that ultimately lead to the discovery of new targets for antidepressant drugs tailored to match specific pathologies within MD.