Neonatal finasteride administration alters hippocampal α4 and δ GABAAR subunits expression and behavioural responses to progesterone in adult rats.

Allopregnanolone is a neurosteroid that has been reported to fluctuate during early developmental stages. Previous experiments reported the importance of neonatal endogenous allopregnanolone levels for the maturation of the central nervous system and particularly for the hippocampus. Changes in neonatal allopregnanolone levels have been related to altered adult behaviour and with psychopathological susceptibility, including anxiety disorders, schizophrenia and drug abuse. However, the mechanism underlying these changes remains to be elucidated. In the present study we assessed changes in hippocampal expression of α4 and δ GABAA receptor (GABAAR) subunits as a consequence of neonatal finasteride (a 5-α reductase inhibitor) administration during early development (PD6 to PD15) in male rats. We observed that the treatment altered the temporal window of the natural peak in the expression of these subunits during development. Additionally, the level of these subunits were higher than in non-handled and control animals in the adult hippocampus. We observed that in adulthood, neonatal finasteride-treated animals presented an anxiogenic-like profile in response to progesterone administration which was absent in the rest of the groups. In conclusion, these results corroborate the relevance of neonatal maintenance of neurosteroid levels for behavioural anxiety responses in the adult, and point to some of the mechanisms involved in this alterations.

The C-terminal domain of tetanus toxin protects motoneurons against acute excitotoxic damage on spinal cord organotypic cultures.

The C-terminal domain of tetanus toxin (Hc-TeTx) has been suggested to act as a neuroprotective agent by activating signaling pathways related to neurotrophins and also to exert anti-apoptotic effects. Here, we show the beneficial properties of the recombinant protein Hc-TeTx to protect spinal motoneurons against excitotoxic damage. In vitro spinal cord organotypic cultures were used to assess acute glutamate excitotoxic damage. Our results indicate that Hc-TeTx treatment improves motoneuron survival within a short therapeutical window (the first 2 h post-injury). Within this interval, we found that p44/p42 MAP kinase (ERK1/2) and glycogen synthase kinase-3 (GSK3ß) signaling pathways play a crucial role in the neuroprotective effect. Moreover, we demonstrated that Hc-TeTx treatment initiate autophagy which is ERK1/2- and GSK3ß-dependent. These findings suggest a possible therapeutical tool to improve motoneuron survival immediately after excitotoxic insults or during the secondary injury phase that occurs after spinal cord trauma.

Effect of genetic background on onset and disease progression in the SOD1-G93A model of amyotrophic lateral sclerosis.

Knowledge of the potential effect of genetic background in disease models is important. The SOD1-G93A transgenic mouse is the most widely used model in amyotrophic lateral sclerosis (ALS). Since these animals show considerable variability both in the onset and the progression of the disease, this study aimed to characterize the potential differences between the two most widely used strains, C56BL/6 (B6) and B6SJL. A rotarod test was carried out to assess strength and motor coordination, while electrophysiology tests were performed to evaluate the function of upper and lower motor neurons. Survival of the animals and motor neuron loss were also studied. The results did not show any background effect regarding the rotarod test, despite the differences in the pattern of decline in central and peripheral motor conduction. The onset of motor neuron abnormalities was later in B6SJL mice, but progressed more rapidly. Lifespan was longer for B6 than for B6SJL animals. In conclusion, background differences in disease onset and progression are important. The characteristics of the strain should be taken into account in experimental design of therapeutic studies.

Endogenous Mechanisms of Neuroprotection: To Boost or Not to Boost.

Postmitotic cells, like neurons, must live through a lifetime. For this reason, organisms/cells have evolved with self-repair mechanisms that allow them to have a long life. The discovery workflow of neuroprotectors during the last years has focused on blocking the pathophysiological mechanisms that lead to neuronal loss in neurodegeneration. Unfortunately, only a few strategies from these studies were able to slow down or prevent neurodegeneration. There is compelling evidence demonstrating that endorsing the self-healing mechanisms that organisms/cells endogenously have, commonly referred to as cellular resilience, can arm neurons and promote their self-healing. Although enhancing these mechanisms has not yet received sufficient attention, these pathways open up new therapeutic avenues to prevent neuronal death and ameliorate neurodegeneration. Here, we highlight the main endogenous mechanisms of protection and describe their role in promoting neuron survival during neurodegeneration.

GRP78 Overexpression Triggers PINK1-IP3R-Mediated Neuroprotective Mitophagy.

An experimental model of spinal root avulsion (RA) is useful to study causal molecular programs that drive retrograde neurodegeneration after neuron-target disconnection. This neurodegenerative process shares common characteristics with neuronal disease-related processes such as the presence of endoplasmic reticulum (ER) stress and autophagy flux blockage. We previously found that the overexpression of GRP78 promoted motoneuronal neuroprotection after RA. After that, we aimed to unravel the underlying mechanism by carrying out a comparative unbiased proteomic analysis and pharmacological and genetic interventions. Unexpectedly, mitochondrial factors turned out to be most altered when GRP78 was overexpressed, and the abundance of engulfed mitochondria, a hallmark of mitophagy, was also observed by electronic microscopy in RA-injured motoneurons after GRP78 overexpression. In addition, GRP78 overexpression increased LC3-mitochondria tagging, promoted PINK1 translocation, mitophagy induction, and recovered mitochondrial function in ER-stressed cells. Lastly, we found that GRP78-promoted pro-survival mitophagy was mediated by PINK1 and IP3R in our in vitro model of motoneuronal death. This data indicates a novel relationship between the GRP78 chaperone and mitophagy, opening novel therapeutical options for drug design to achieve neuroprotection.

NeuroHeal Treatment Alleviates Neuropathic Pain and Enhances Sensory Axon Regeneration.

Peripheral nerve injury (PNI) leads to the loss of motor, sensory, and autonomic functions, and often triggers neuropathic pain. During the last years, many efforts have focused on finding new therapies to increase axonal regeneration or to alleviate painful conditions. Still only a few of them have targeted both phenomena. Incipient or aberrant sensory axon regeneration is related to abnormal unpleasant sensations, such as hyperalgesia or allodynia. We recently have discovered NeuroHeal, a combination of two repurposed drugs; Acamprosate and Ribavirin. NeuroHeal is a neuroprotective agent that also enhances motor axon regeneration after PNI. In this work, we investigated its effect on sensory fiber regeneration and PNI-induced painful sensations in a rat model of spare nerve injury and nerve crush. The follow up of the animals showed that NeuroHeal treatment reduced the signs of neuropathic pain in both models. Besides, the treatment favored sensory axon regeneration, as observed in dorsal root ganglion explants. Mechanistically, the effects observed in vivo may improve the resolution of cell-protective autophagy. Additionally, NeuroHeal treatment modulated the P2X4-BDNF-KCC2 axis, which is an essential driver of neuropathic pain. These data open a new therapeutic avenue based on autophagic modulation to foster endogenous regenerative mechanisms and reduce the appearance of neuropathic pain in PNI.

SIRT2 Inhibition Improves Functional Motor Recovery After Peripheral Nerve Injury.

Sirtuin-2 (Sirt2) is a member of the NAD (+)-dependent protein deacetylase family involved in neuroprotection, cellular metabolism, homeostasis, and stress responses after injury of the nervous system. So far, no data have been published describing the role of SIRT2 in motor functional recovery after damage. We found that SIRT2 expression and deacetylase activity were increased within motoneurons after axotomy. To shed light onto the biological relevance of this change, we combined in vitro and in vivo models with pharmacological and genetic ablation approaches. We found that SIRT2 KO (knockout) mice exhibited improved functional recovery after sciatic nerve crush. SIRT2 activity blockage, using AK7, increased neurite outgrowth and length in organotypic spinal cord cultures and human cell line models. SIRT2 blockage enhanced the acetyltransferase activity of p300, which in turn increased the levels of an acetylated form of p53 (Ac-p53 k373), histone 3 (Ac-H3K9), and expression of GAP43, a downstream marker of regeneration. Lastly, we verified that p300 acetyltransferase activity is essential for these effects. Our results suggest that bolstering an epigenetic shift that promotes SIRT2 inhibition can be an effective therapy to increase functional recovery after peripheral nerve injury.

Is it the time of autophagy fine-tuners for neuroprotection?

Cells and organisms are intrinsically prepared to effectively deal with damage caused by insults and heal themselves by triggering a plethora of stress responses including macroautophagy/autophagy. However, autophagy may become malfunctional during aging, neurodegeneration, and neurotrauma. We aimed to overcome autophagy dysfunction by refining therapeutics using multi-target approaches. Thus, we have demonstrated that modulation of autophagy with the multitarget drug NeuroHeal is neuroprotective in several neurodegeneration models in which previous autophagy modulators have failed. The key element of success is the coordinated activation of opposing forces that modulate autophagy with NeuroHeal, probably leading to the autophagy-dependent degradation of death executors such as PARP1. The precise tuning of autophagy thus allows the neuron to adapt to insults, survive and repair itself. These findings support the advent a new era of neuroprotectants that counteract neuronal damage by targeting in unison different pathways of the self-repair process, including autophagy.

Neurotrophic Properties of C-Terminal Domain of the Heavy Chain of Tetanus Toxin on Motor Neuron Disease.

The carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) exerts a neuroprotective effect in neurodegenerative diseases via the activation of signaling pathways related to neurotrophins, and also through inhibiting apoptotic cell death. Here, we demonstrate that Hc-TeTx preserves motoneurons from chronic excitotoxicity in an in vitro model of amyotrophic lateral sclerosis. Furthermore, we found that PI3-K/Akt pathway, but not p21ras/MAPK pathway, is involved in their beneficial effects under chronic excitotoxicity. Moreover, we corroborate the capacity of the Hc-TeTx to be transported retrogradely into the spinal motor neurons and also its capacity to bind to the motoneuron-like cell line NSC-34. These findings suggest a possible therapeutic tool to improve motoneuron preservation in neurodegenerative diseases such as amyotrophic lateral sclerosis.

NeuroHeal Improves Muscle Regeneration after Injury.

Musculoskeletal injuries represent a challenging medical problem. Although the skeletal muscle is able to regenerate and recover after injury, the process engaged with conservative therapy can be inefficient, leading to a high re-injury rate. In addition, the formation of scar tissue implies an alteration of mechanical properties in muscle. There is still a need for new treatments of the injured muscle. NeuroHeal may be one option. Published studies demonstrated that it reduces muscle atrophy due to denervation and disuse. The main objective of the present work was to assess the potential of NeuroHeal to improve muscle regeneration after traumatic injury. Secondary objectives included characterizing the effect of NeuroHeal treatment on satellite cell biology. We used a rat model of sport-induced injury in the gastrocnemius and analyzed the effects of NeuroHeal on functional recovery by means of electrophysiology and tetanic force analysis. These studies were accompanied by immunohistochemistry of the injured muscle to analyze fibrosis, satellite cell state, and fiber type. In addition, we used an in vitro model to determine the effect of NeuroHeal on myoblast biology and partially decipher its mechanism of action. The results showed that NeuroHeal treatment advanced muscle fiber recovery after injury in a preclinical model of muscle injury, and significantly reduced the formation of scar tissue. In vitro, we observed that NeuroHeal accelerated the formation of myotubes. The results pave the way for novel therapeutic avenues for muscle/tendinous disorders.

Novel neuroprotective therapy with NeuroHeal by autophagy induction for damaged neonatal motoneurons.

Rationale: Protective mechanisms allow healthy neurons to cope with diverse stresses. Excessive damage as well as aging can lead to defective functioning of these mechanisms. We recently designed NeuroHeal using artificial intelligence with the goal of bolstering endogenous neuroprotective mechanisms. Understanding the key nodes involved in neuroprotection will allow us to identify even more effective strategies for treatment of neurodegenerative diseases. Methods: We used a model of peripheral nerve axotomy in rat pups, that induces retrograde apoptotic death of motoneurons. Nourishing mothers received treatment with vehicle, NeuroHeal or NeuroHeal plus nicotinamide, an inhibitor of sirtuins, and analysis of the pups were performed by immunohistochemistry, electron microscopy, and immunoblotting. In vitro, the post-translational status of proteins of interest was detailed using organotypic spinal cord cultures and genetic modifications in cell lines to unravel the neuroprotective mechanisms involved. Results: We found that the concomitant activation of the NAD+-dependent deacetylase SIRT1 and the PI3K/AKT signaling pathway converge to increase the presence of deacetylated and phosphorylated FOXO3a, a transcription factor, in the nucleus. This favors the activation of autophagy, a pro-survival process, and prevents pro-apoptotic PARP1/2 cleavage. Major conclusion: NeuroHeal is a neuroprotective agent for neonatal motoneurons that fine-tunes autophagy on by converging SIRT1/AKT/FOXO3a axis. NeuroHeal is a combo of repurposed drugs that allow its readiness for prospective pediatric use.

NeuroHeal Reduces Muscle Atrophy and Modulates Associated Autophagy.

Muscle wasting is an unmet medical need which leads to a reduction of myofiber diameter and a negative impact on the functional performance of daily activities. We previously found that a new neuroprotective drug called NeuroHeal reduced muscle atrophy produced by transient denervation. Aiming to decipher whether NeuroHeal has a direct role in muscle biology, we used herein different models of muscle atrophy: one caused by chronic denervation, another caused by hindlimb immobilization, and lastly, an in vitro model of myotube atrophy with Tumor Necrosis Factor-α (TNFα). In all these models, we observed that NeuroHeal reduced muscle atrophy and that SIRT1 activation seems to be required for that. The treatment downregulated some critical markers of protein degradation: Muscle Ring Finger 1 (MuRF1), K48 poly-Ub chains, and p62/SQSTM1. Moreover, it seems to restore the autophagy flux associated with denervation. Hence, we envisage a prospective use of NeuroHeal at clinics for different myopathies.

Drug screening of neuroprotective agents on an organotypic-based model of spinal cord excitotoxic damage.

PURPOSE: Damage to segmental motoneurons and to spinal cord parenchyma cause denervation atrophy to the muscles, contributing to the chronic disability originated by spinal cord injury (SCI) and spinal motor neuron diseases. After SCI, damage is promoted by several underlying mechanisms, including release of glutamate and consequent over-activation of glutamate receptors, mainly NMDA receptors, that lead to neuronal death. Due to the lack of effective treatments for such conditions, new alternatives need to be explored. METHODS: In order to perform a relatively quick and high-fidelity drug screening, we optimized a postnatal rat organotypic spinal cord culture. By using a glutamate excitotoxic model of spinal cord damage on the explants, we compared the neuroprotective efficacy of four agents: methylprednisolone, erythropoietin, riluzole and rolipram. We evaluated the number of surviving ventral motor neurons stained with the SMI32 antibody and estimated the cord tissue preservation by quantifying the amount of EthD fluorescent probe incorporated into the cells. RESULTS: The best tissue protection was achieved with riluzole (98%) whereas the highest motoneuron preservation was obtained with methylprednisolone (92%). CONCLUSION: The in vitro model used may serve to initiate comparative analyses of new compounds to narrow the choice for future neuroprotective agents to be tested in vivo.

Effects of Schwann cell transplants in an experimental nerve amputee model.

PURPOSE: By using a nerve amputee model of the rat sciatic nerve (Lago and Navarro, 2007), we have tested a strategy for the long-term maintenance of regenerated axons without distal target reinnervation, by grafting Schwann cells (SCs) into a capped silicone chamber containing the ending nerve stump. METHODS: The sciatic nerve of rats was transected and repaired with a silicone tube, the distal nerve was again cut at 10 mm and inserted in a capped tube that was filled with saline or with a suspension of cultured SCs. Transplants of SCs obtained from primary cultures have been compared with those of an immortalized SC line (SCTM41) or the same line overexpressing GDNF. RESULTS: The histological results show that nerve fibers were able to regenerate through a short distal nerve segment ending into the capped chamber, and sustain distal branches without degenerating for several months. There was abundant axonal sprouting forming an ending neuroma, and the caliber of myelinated fibers remained far thinner than normal during the 9 months investigated. With a distal transplant of primary SCs there were significantly more regenerated myelinated fibers than in the control group at 9 months, indicating that the grafted cells stimulated the axonal growth response and helped to maintain survival of axon branches. In contrast, axonal regeneration was significantly reduced with grafts of SCTM41 cells, probably due to physical competition between cell proliferation and axonal growth. SCTM41 cells overexpressing GDNF improved the regenerative response with respect to the parent SCTM41 cells, although not to the same extent as the primary SCs. CONCLUSION: A graft of primary SCs in the capped chamber stimulated axonal growth response and/or maintained survival of axonal branches on the long term in the nerve amputee model.

Network-centric medicine for peripheral nerve injury: Treating the whole to boost endogenous mechanisms of neuroprotection and regeneration.

Peripheral nerve injuries caused by accidents may lead to paralysis, sensory disturbances, anaesthesia, and lack of autonomic functions. Functional recovery after disconnection of the motoneuronal soma from target tissue with proximal rupture of axons is determined by several factors: motoneuronal soma viability, proper axonal sprouting across inhibitory zones and elongation toward specific muscle, effective synapse contact rebuilding, and prevention of muscle atrophy. Therapies, such as adjuvant drugs with pleiotropic effects, that promote functional recovery after peripheral nerve injury are needed. Toward this aim, we designed a drug discovery workflow based on a network-centric molecular vision using unbiased proteomic data and neural artificial computational tools. Our focus is on boosting intrinsic capabilities of neurons for neuroprotection; this is in contrast to the common approach based on suppression of a pathobiological pathway known to be associated with disease condition. Using our workflow, we discovered neuroheal, a combination of two repurposed drugs that promotes motoneuronal soma neuroprotection, is anti-inflammatory, enhances axonal regeneration after axotomy, and reduces muscle atrophy. This drug discovery workflow has thus yielded a therapy that is close to its clinical application.

Improved Motor Nerve Regeneration by SIRT1/Hif1a-Mediated Autophagy.

Complete restoring of functional connectivity between neurons or target tissue after traumatic lesions is still an unmet medical need. Using models of nerve axotomy and compression, we investigated the effect of autophagy induction by genetic and pharmacological manipulation on motor nerve regeneration. ATG5 or NAD+-dependent deacetylase sirtuin-1 (SIRT1) overexpression on spinal motoneurons stimulates mTOR-independent autophagy and facilitates a growth-competent state improving motor axonal regeneration with better electromyographic records after nerve transection and suture. In agreement with this, using organotypic spinal cord cultures and the human cell line SH-SY5Y, we observed that the activation of SIRT1 and autophagy by NeuroHeal increased neurite outgrowth and length extension and that this was mediated by downstream HIF1a. To conclude, SIRT1/Hifα-dependent autophagy confers a more pro-regenerative phenotype to motoneurons after peripheral nerve injury. Altogether, we provide evidence showing that autophagy induction by SIRT1/Hifα activation or NeuroHeal treatment is a novel therapeutic option for improving motor nerve regeneration and functional recovery after injury.

ATG5 overexpression is neuroprotective and attenuates cytoskeletal and vesicle-trafficking alterations in axotomized motoneurons.

Injured neurons should engage endogenous mechanisms of self-protection to limit neurodegeneration. Enhancing efficacy of these mechanisms or correcting dysfunctional pathways may be a successful strategy for inducing neuroprotection. Spinal motoneurons retrogradely degenerate after proximal axotomy due to mechanical detachment (avulsion) of the nerve roots, and this limits recovery of nervous system function in patients after this type of trauma. In a previously reported proteomic analysis, we demonstrated that autophagy is a key endogenous mechanism that may allow motoneuron survival and regeneration after distal axotomy and suture of the nerve. Herein, we show that autophagy flux is dysfunctional or blocked in degenerated motoneurons after root avulsion. We also found that there were abnormalities in anterograde/retrograde motor proteins, key secretory pathway factors, and lysosome function. Further, LAMP1 protein was missorted and underglycosylated as well as the proton pump v-ATPase. In vitro modeling revealed how sequential disruptions in these systems likely lead to neurodegeneration. In vivo, we observed that cytoskeletal alterations, induced by a single injection of nocodazole, were sufficient to promote neurodegeneration of avulsed motoneurons. Besides, only pre-treatment with rapamycin, but not post-treatment, neuroprotected after nerve root avulsion. In agreement, overexpressing ATG5 in injured motoneurons led to neuroprotection and attenuation of cytoskeletal and trafficking-related abnormalities. These discoveries serve as proof of concept for autophagy-target therapy to halting the progression of neurodegenerative processes.

SIRT1 activation with neuroheal is neuroprotective but SIRT2 inhibition with AK7 is detrimental for disconnected motoneurons.

Sirtuin 1 (SIRT1) activity is neuroprotective, and we have recently demonstrated its role in the retrograde degenerative process in motoneurons (MNs) in the spinal cord of rats after peripheral nerve root avulsion (RA) injury. SIRT2 has been suggested to exert effects opposite those of SIRT1; however, its roles in neurodegeneration and neuron response after nerve injury remain unclear. Here we compared the neuroprotective potentials of SIRT1 activation and SIRT2 inhibition in a mouse model of hypoglossal nerve axotomy. This injury induced a reduction of around half MN population within the hypoglossal nucleus by a non-apoptotic neurodegenerative process triggered by endoplasmic reticulum (ER) stress that resulted in activation of the unfolded protein response mediated by IRE1α and XBP1 by 21 days post injury. Both SIRT1 activation with NeuroHeal and SIRT2 inhibition with AK7 protected NSC-34 motor neuron-like cells against ER stress in vitro. In agreement with the in vitro results, NeuroHeal treatment or SIRT1 overexpression was neuroprotective of axotomized hypoglossal MNs in a transgenic mouse model. In contrast, AK7 treatment or SIRT2 genetic depletion in mice inhibited damaged MN survival. To resolve the in vitro/in vivo discrepancies, we used an organotypic spinal cord culture system that preserves glial cells. In this system, AK7 treatment of ER-stressed organotypic cultures was detrimental for MNs and increased microglial nuclear factor-κB and the consequent transcription of cytotoxic pro-inflammatory factors similarly. The results highlight the importance of glial cells in determining the neuroprotective impact of any treatment.

Neuroprotective Drug for Nerve Trauma Revealed Using Artificial Intelligence.

Here we used a systems biology approach and artificial intelligence to identify a neuroprotective agent for the treatment of peripheral nerve root avulsion. Based on accumulated knowledge of the neurodegenerative and neuroprotective processes that occur in motoneurons after root avulsion, we built up protein networks and converted them into mathematical models. Unbiased proteomic data from our preclinical models were used for machine learning algorithms and for restrictions to be imposed on mathematical solutions. Solutions allowed us to identify combinations of repurposed drugs as potential neuroprotective agents and we validated them in our preclinical models. The best one, NeuroHeal, neuroprotected motoneurons, exerted anti-inflammatory properties and promoted functional locomotor recovery. NeuroHeal endorsed the activation of Sirtuin 1, which was essential for its neuroprotective effect. These results support the value of network-centric approaches for drug discovery and demonstrate the efficacy of NeuroHeal as adjuvant treatment with surgical repair for nervous system trauma.

Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice.

DYRK1A is the human orthologue of the Drosophila minibrain (mnb) gene, which is involved in postembryonic neurogenesis in flies. Because of its mapping position on chromosome 21 and the neurobehavioral alterations shown by mice overexpressing this gene, involvement of DYRK1A in some of the neurological defects of Down syndrome patients has been suggested. To gain insight into its physiological role, we have generated mice deficient in Dyrk1A function by gene targeting. Dyrk1A(-/-) null mutants presented a general growth delay and died during midgestation. Mice heterozygous for the mutation (Dyrk1A(+/-)) showed decreased neonatal viability and a significant body size reduction from birth to adulthood. General neurobehavioral analysis revealed preweaning developmental delay of Dyrk1A(+/-) mice and specific alterations in adults. Brains of Dyrk1A(+/-) mice were decreased in size in a region-specific manner, although the cytoarchitecture and neuronal components in most areas were not altered. Cell counts showed increased neuronal densities in some brain regions and a specific decrease in the number of neurons in the superior colliculus, which exhibited a significant size reduction. These data provide evidence about the nonredundant, vital role of Dyrk1A and suggest a conserved mode of action that determines normal growth and brain size in both mice and flies.