STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease.

INTRODUCTION: Parkinson's disease (PD) is the most prevalent disorder of the basal ganglia, propagated by the degeneration of axon terminals within the striatum and subsequent loss of dopaminergic neurons in the substantia nigra (SN). Exposure of environmental neurotoxins and mutations of several mitochondrial and proteasomal genes are primarily responsible. METHODS: To determine whether signal transducer and activator of transcription 3 (STAT3) could protect dopaminergic neurons against degeneration, we first screened it in the in vitro capacity using immortalized rat dopaminergic N27 cells under 6-OHDA neurotoxicity. We then evaluated the effectiveness of constitutively active (ca) STAT3 as a neuroprotective agent on N27 cells in a 6-hydroxydopamine (6-OHDA) induced rat model of PD and compared it to control animals or animals where AAV/caRheb was expressed in SN. Behavioral outcomes were assessed using rotational and cylinder assays and mitochondrial function using reactive oxygen species (ROS) levels. RESULTS: Using flow cytometry, the in vitro analysis determined caSTAT3 significantly decreased dopaminergic neuronal death under 6-OHDA treatment conditions. Importantly, in vivo overexpression of caSTAT3 in SN dopaminergic neurons using AAV-mediated expression demonstrated significant neuroprotection of dopaminergic neurons following 6-OHDA. Both caSTAT3 and caRheb + caSTAT3 co-injection into substantia nigra reduced D-amphetamine-induced rotational behavior and increased ipsilateral forelimb function when compared to control animals. In addition, caSTAT3 decreased mitochondrial ROS production following 6-OHDA induced neurotoxicity. CONCLUSION: caSTAT3 confers resistance against ROS production in mitochondria of susceptible SN dopaminergic neurons potentially offering a new avenue for treatment against PD.

The ventral hippocampus and nucleus accumbens as neural substrates for cocaine contextual memory reconsolidation.

Drug craving triggered by cues that were once associated with drug intoxication is a major contributor to continued drug-seeking behaviors. Addictive drugs engage molecular pathways of associative learning and memory. Reactivated memories are vulnerable to disruption by interference with the process of reconsolidation, hence targeting reconsolidation could be a strategy to reduce cue-induced drug craving and relapse. Here we examined the circuitry of cocaine contextual memory reconsolidation and explored neuroplasticity following memory reactivation. Mice underwent chemogenetic inhibition of either nucleus accumbens (NA) neurons or the glutamatergic projection neurons from the ventral hippocampus (vHPC) to NA using inhibitory designer receptors exclusively activated by designer drugs (iDREADD). Mice underwent cocaine conditioned place preference followed by reactivation of the cocaine contextual memory. Clozapine-N-oxide (CNO) was administered after memory reactivation to inhibit either NA neurons or the accumbens-projecting vHPC neurons during the reconsolidation period. When retested 3 days later, a significant reduction in the previously established preference for the cocaine context was found in both conditions. FosTRAP2-Ai14 mice were used to identify neurons activated by cocaine memory recall and to evaluate plasticity in NA medium spiny neurons (MSNs) and vHPC pyramidal neurons upon recall of cocaine memories. Results indicate a significant increase in dendritic spine density in NA MSNs activated by cocaine memory recall, particularly of the thin spine type. Sholl analysis indicated longer dendritic length and more branching of NA MSNs after cocaine memory recall than without memory reactivation. vHPC neurons showed increased spine density, with the most robust change in stubby spines. These results implicate a circuit involving glutamatergic projections from the vHPC onto NA neurons which is necessary for the reconsolidation of cocaine memories. Interruption of cocaine memory reconsolidation reduced drug-seeking behavior.

Identification of Neurocan and Phosphacan as Early Biomarkers for Open Neural Tube Defects.

Open neural tube defects (NTDs) such as myelomeningocele (MMC) are debilitating and the most common congenital defects of the central nervous system. Despite their apparent clinical importance, the existing early prenatal diagnostic options for these defects remain limited. Using a well-accepted retinoic-acid-induced model of MMC established in fetal rats, we discovered that neurocan and phosphacan, the secreted chondroitin sulfate proteoglycans of the developing nervous system, are released into the amniotic fluid (AF) of fetal rats displaying spinal cord defects. In contrast to normal controls, elevated AF levels of neurocan and phosphacan were detected in MMC fetuses early in gestation and continued to increase during MMC progression, reaching the highest level in near-term fetuses. The molecular forms of neurocan and phosphacan identified in the AF of MMC fetuses and those found in MMC spinal cords were qualitatively similar. In summary, this is the first report demonstrating the presence of neurocan and phosphacan in the AF of MMC fetuses. The identification of elevated levels of neurocan and phosphacan in the AF of MMC fetuses provides two prospective biomarkers with the potential for early prenatal diagnosis of open NTDs.

Chemogenetic modulation of sensory afferents induces locomotor changes and plasticity after spinal cord injury.

Neuromodulatory therapies for spinal cord injury (SCI) such as electrical epidural stimulation (EES) are increasingly effective at improving patient outcomes. These improvements are thought to be due, at least in part, to plasticity in neuronal circuits. Precisely which circuits are influenced and which afferent classes are most effective in stimulating change remain important open questions. Genetic tools, such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), support targeted and reversible neuromodulation as well as histological characterization of manipulated neurons. We therefore transduced and activated lumbar large diameter peripheral afferents with excitatory (hM3Dq) DREADDs, in a manner analogous to EES, in a rat hemisection model, to begin to trace plasticity and observe concomitant locomotor changes. Chronic DREADDs activation, coupled with thrice weekly treadmill training, was observed to increase afferent fluorescent labeling within motor pools and Clarke's column when compared to control animals. This plasticity may underlie kinematic differences that we observed across stages of recovery, including an increased and less variable hindquarters height in DREADDs animals, shorter step durations, a more flexed ankle joint early in recovery, a less variable ankle joint angle in swing phase, but a more variable hip joint angle. Withdrawal of DREADDs agonist, clozapine-N-oxide (CNO) left these kinematic differences largely unaffected; suggesting that DREADDs activation is not necessary for them later in recovery. However, we observed an intermittent "buckling" phenomenon in DREADDs animals without CNO activation, that did not occur with CNO re-administration. Future studies could use more refined genetic targeted of specific afferent classes, and utilize muscle recordings to find where afferent modulation is most influential in altering motor output.

Correction: Addition of angled rungs to the horizontal ladder walking task for more sensitive probing of sensorimotor changes.

[This corrects the article DOI: 10.1371/journal.pone.0246298.].

Glucocorticoid Receptor Overexpression in the Dorsal Hippocampus Attenuates Spatial Learning and Synaptic Plasticity Deficits after Pediatric Traumatic Brain Injury.

Traumatic brain injury (TBI) in children <4 years of age leads to long-term deficits in cognitive and learning abilities that can persist or even worsen as these children age into adolescence. In this study, the role of glucocorticoid receptor (GR) function in the dorsal hippocampus (DH) in hippocampal-dependent cognitive function and synaptic plasticity were assessed following injury to the 11-day-old rat. Brain injury produced significant impairments in spatial learning and memory in the Morris water maze in male and female rats at 1-month post-injury (adolescence), which was accompanied by impairments in induction and maintenance of long-term potentiation (LTP) in the CA1 region of the DH. Brain injury resulted in a significant decrease in the expression of the glucocorticoid-inducible gene, serum- and glucocorticoid-kinase 1 (sgk1), suggestive of an impairment in GR transcriptional activity within the hippocampus. Lentiviral transfection of the human GR (hGR) in the DH improved spatial learning and memory in the Morris water maze and attenuated LTP deficits following TBI. GR overexpression in the DH was also associated with a significant increase in the mRNA expression levels of sgk1, and the glutamate receptor subunits GluA1 and GluA2 within the hippocampus. Overall, these findings support an important role for dorsal hippocampal GR function in learning and memory deficits following pediatric TBI and suggest that these effects may be related to the regulation of glutamate receptor subunit expression in the DH.

Viral expression of constitutively active AKT3 induces CST axonal sprouting and regeneration, but also promotes seizures.

Increasing the intrinsic growth potential of neurons after injury has repeatedly been shown to promote some level of axonal regeneration in rodent models. One of the most studied pathways involves the activation of the PI3K/AKT/mTOR pathways, primarily by reducing the levels of PTEN, a negative regulator of PI3K. Likewise, activation of signal transducer and activator of transcription 3 (STAT3) has previously been shown to boost axonal regeneration and sprouting within the injured nervous system. Here, we examined the regeneration of the corticospinal tract (CST) after cortical expression of constitutively active (ca) Akt3 and STAT3, both separately and in combination. Overexpression of caAkt3 induced regeneration of CST axons past the injury site independent of caSTAT3 overexpression. STAT3 demonstrated improved axon sprouting compared to controls and contributed to a synergistic improvement in effects when combined with Akt3 but failed to promote axonal regeneration as an individual therapy. Despite showing impressive axonal regeneration, animals expressing Akt3 failed to show any functional improvement and deteriorated with time. During this period, we observed progressive Akt3 dose-dependent increase in behavioral seizures. Histology revealed increased phosphorylation of ribosomal S6 protein within the unilateral cortex, increased neuronal size, microglia activation and hemispheric enlargement (hemimegalencephaly).

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery.

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons. Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

Axotomy Induces Drp1-Dependent Fragmentation of Axonal Mitochondria.

It is well established that CNS axons fail to regenerate, undergo retrograde dieback, and form dystrophic growth cones due to both intrinsic and extrinsic factors. We sought to investigate the role of axonal mitochondria in the axonal response to injury. A viral vector (AAV) containing a mitochondrially targeted fluorescent protein (mitoDsRed) as well as fluorescently tagged LC3 (GFP-LC3), an autophagosomal marker, was injected into the primary motor cortex, to label the corticospinal tract (CST), of adult rats. The axons of the CST were then injured by dorsal column lesion at C4-C5. We found that mitochondria in injured CST axons near the injury site are fragmented and fragmentation of mitochondria persists for 2 weeks before returning to pre-injury lengths. Fragmented mitochondria have consistently been shown to be dysfunctional and detrimental to cellular health. Inhibition of Drp1, the GTPase responsible for mitochondrial fission, using a specific pharmacological inhibitor (mDivi-1) blocked fragmentation. Additionally, it was determined that there is increased mitophagy in CST axons following Spinal cord injury (SCI) based on increased colocalization of mitochondria and LC3. In vitro models revealed that mitochondrial divalent ion uptake is necessary for injury-induced mitochondrial fission, as inhibiting the mitochondrial calcium uniporter (MCU) using RU360 prevented injury-induced fission. This phenomenon was also observed in vivo. These studies indicate that following the injury, both in vivo and in vitro, axonal mitochondria undergo increased fission, which may contribute to the lack of regeneration seen in CNS neurons.

Quantitative Assessment of Neurite Outgrowth Over Growth Promoting or Inhibitory Substrates.

The use of sensory neurons and assessment of neurite outgrowth in vitro is an important part of understanding neuronal development and plasticity. Cultures of rat dorsal root ganglion (DRG) neurons provide quantitative results very quickly and, when grown on growth promoting or inhibitory substrates, can be utilized to study axonal growth, neurotrophic dependence, and structure and function of growth cones. Since we are interested in axon regeneration and targeting, we have sought to promote neurite outgrowth by refining the techniques of growing DRG neurons in culture. This chapter describes detailed methods for the dissection and purification of DRG neurons and quantitative assessment of neurite on promoting or inhibitory substrates.

Respiratory axon regeneration in the chronically injured spinal cord.

Promoting the combination of robust regeneration of damaged axons and synaptic reconnection of these growing axon populations with appropriate neuronal targets represents a major therapeutic goal following spinal cord injury (SCI). A key impediment to achieving this important aim includes an intrinsic inability of neurons to extend axons in adult CNS, particularly in the context of the chronically-injured spinal cord. We tested whether an inhibitory peptide directed against phosphatase and tensin homolog (PTEN: a central inhibitor of neuron-intrinsic axon growth potential) could restore inspiratory diaphragm function by reconnecting critical respiratory neural circuitry in a rat model of chronic cervical level 2 (C2) hemisection SCI. We found that systemic delivery of PTEN antagonist peptide 4 (PAP4) starting at 8 weeks after C2 hemisection promoted substantial, long-distance regeneration of injured bulbospinal rostral Ventral Respiratory Group (rVRG) axons into and through the lesion and back toward phrenic motor neurons (PhMNs) located in intact caudal C3-C5 spinal cord. Despite this robust rVRG axon regeneration, PAP4 stimulated only minimal recovery of diaphragm function. Furthermore, re-lesion through the hemisection site completely removed PAP4-induced functional improvement, demonstrating that axon regeneration through the lesion was responsible for this partial functional recovery. Interestingly, there was minimal formation of putative excitatory monosynaptic connections between regrowing rVRG axons and PhMN targets, suggesting that (1) limited rVRG-PhMN synaptic reconnectivity was responsible at least in part for the lack of a significant functional effect, (2) chronically-injured spinal cord presents an obstacle to achieving synaptogenesis between regenerating axons and post-synaptic targets, and (3) addressing this challenge is a potentially-powerful strategy to enhance therapeutic efficacy in the chronic SCI setting. In conclusion, our study demonstrates a non-invasive and transient pharmacological approach in chronic SCI to repair the critically-important neural circuitry controlling diaphragmatic respiratory function, but also sheds light on obstacles to circuit plasticity presented by the chronically-injured spinal cord.

Co-targeting myelin inhibitors and CSPGs markedly enhances regeneration of GDNF-stimulated, but not conditioning-lesioned, sensory axons into the spinal cord.

A major barrier to intraspinal regeneration after dorsal root (DR) injury is the DR entry zone (DREZ), the CNS/PNS interface. DR axons stop regenerating at the DREZ, even if regenerative capacity is increased by a nerve conditioning lesion. This potent blockade has long been attributed to myelin-associated inhibitors and (CSPGs), but incomplete lesions and conflicting reports have prevented conclusive agreement. Here, we evaluated DR regeneration in mice using novel strategies to facilitate complete lesions and analyses, selective tracing of proprioceptive and mechanoreceptive axons, and the first simultaneous targeting of Nogo/Reticulon-4, MAG, OMgp, CSPGs, and GDNF. Co-eliminating myelin inhibitors and CSPGs elicited regeneration of only a few conditioning-lesioned DR axons across the DREZ. Their absence, however, markedly and synergistically enhanced regeneration of GDNF-stimulated axons, highlighting the importance of sufficiently elevating intrinsic growth capacity. We also conclude that myelin inhibitors and CSPGs are not the primary mechanism stopping axons at the DREZ.

Addition of angled rungs to the horizontal ladder walking task for more sensitive probing of sensorimotor changes.

One method for the evaluation of sensorimotor therapeutic interventions, the horizontal ladder walking task, analyzes locomotor changes that may occur after disease, injury, or by external manipulation. Although this task is well suited for detection of large effects, it may overlook smaller changes. The inability to detect small effect sizes may be due to a neural compensatory mechanism known as "cross limb transfer", or the contribution of the contralateral limb to estimate an injured or perturbed limb's position. The robust transfer of compensation from the contralateral limb may obscure subtle locomotor outcomes that are evoked by clinically relevant therapies, in the early onset of disease, or between higher levels of recovery. Here, we propose angled rungs as a novel modification to the horizontal ladder walking task. Easily-adjustable angled rungs force rats to locomote across a different locomotion path for each hindlimb and may therefore make information from the contralateral limb less useful. Using hM3Dq (excitatory) Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) expressed in large diameter peripheral afferents of the hindlimb in the intact animal, we characterized the sensitivity of our design to detect stepping differences by comparing locomotor changes observed on angled rungs to those observed on a standard horizontal ladder. On our novel asymmetrical ladder, activation of DREADDs resulted in significant differences in rung misses (p = 0.000011) and weight-supporting events (p = 0.049). By comparison, on a standard ladder, we did not observe differences in these parameters (p = 0.86 and p = 0.98, respectively). Additionally, no locomotor differences were detected in baseline and inactivated DREADDs trials when we compared ladder types, suggesting that the angled rungs do not change animal gait behavior unless intervention or injury is introduced. Significant changes observed with angled rungs may demonstrate more sensitive probing of locomotor changes due to the decoupling of cross limb transfer.

LAR inhibitory peptide promotes recovery of diaphragm function and multiple forms of respiratory neural circuit plasticity after cervical spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the lesion after traumatic spinal cord injury (SCI), are key extracellular matrix inhibitory molecules that limit axon growth and consequent recovery of function. CSPG-mediated inhibition occurs via interactions with axonal receptors, including leukocyte common antigen- related (LAR) phosphatase. We tested the effects of a novel LAR inhibitory peptide in rats after hemisection at cervical level 2, a SCI model in which bulbospinal inspiratory neural circuitry originating in the medullary rostral ventral respiratory group (rVRG) becomes disconnected from phrenic motor neuron (PhMN) targets in cervical spinal cord, resulting in persistent partial-to-complete diaphragm paralysis. LAR peptide was delivered by a soaked gelfoam, which was placed directly over the injury site immediately after C2 hemisection and replaced at 1 week post-injury. Axotomized rVRG axons originating in ipsilateral medulla or spared rVRG fibers originating in contralateral medulla were separately assessed by anterograde tracing via AAV2-mCherry injection into rVRG. At 8 weeks post-hemisection, LAR peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. LAR peptide promoted robust regeneration of ipsilateral-originating rVRG axons into and through the lesion site and into intact caudal spinal cord to reach PhMNs located at C3-C5 levels. Furthermore, regenerating rVRG axons re-established putative monosynaptic connections with their PhMNs targets. In addition, LAR peptide stimulated robust sprouting of both modulatory serotonergic axons and contralateral-originating rVRG fibers within the PhMN pool ipsilateral/caudal to the hemisection. Our study demonstrates that targeting LAR-based axon growth inhibition promotes multiple forms of respiratory neural circuit plasticity and provides a new peptide-based therapeutic strategy to ameliorate the devastating respiratory consequences of SCI.

Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17.

After optic nerve crush (ONC), the cell bodies and distal axons of most retinal ganglion cells (RGCs) degenerate. RGC somal and distal axon degenerations were previously thought to be controlled by two parallel pathways, involving activation of the kinase dual leucine-zipper kinase (DLK) and loss of the axon survival factor nicotinamide mononucleotide adenylyltransferase-2 (NMNAT2), respectively. Here, we report that palmitoylation of both DLK and NMNAT2 by the palmitoyl acyltransferase ZDHHC17 couples these signals. ZDHHC17-dependent palmitoylation enables DLK-dependent somal degeneration after ONC and also ensures NMNAT-dependent distal axon integrity in healthy optic nerves. We provide evidence that ZDHHC17 also controls survival-versus-degeneration decisions in dorsal root ganglion (DRG) neurons, and we identify conserved motifs in NMNAT2 and DLK that govern their ZDHHC17-dependent regulation. These findings suggest that the control of somal and distal axon integrity should be considered as a single, holistic process, mediated by the concerted action of two palmitoylation-dependent pathways.

Epidural Electrical Stimulation: A Review of Plasticity Mechanisms That Are Hypothesized to Underlie Enhanced Recovery From Spinal Cord Injury With Stimulation.

Spinal cord injury (SCI) often results in life-long sensorimotor impairment. Spontaneous recovery from SCI is limited, as supraspinal fibers cannot spontaneously regenerate to form functional networks below the level of injury. Despite this, animal models and humans exhibit many motor behaviors indicative of recovery when electrical stimulation is applied epidurally to the dorsal aspect of the lumbar spinal cord. In 1976, epidural stimulation was introduced to alleviate spasticity in Multiple Sclerosis. Since then, epidural electrical stimulation (EES) has been demonstrated to improve voluntary mobility across the knee and/or ankle in several SCI patients, highlighting its utility in enhancing motor activation. The mechanisms that EES induces to drive these improvements in sensorimotor function remain largely unknown. In this review, we discuss several sensorimotor plasticity mechanisms that we hypothesize may enable epidural stimulation to promote recovery, including changes in local lumbar circuitry, propriospinal interneurons, and the internal model. Finally, we discuss genetic tools for afferent modulation as an emerging method to facilitate the search for the mechanisms of action.

Intrathecal Delivery of BDNF Into the Lumbar Cistern Re-Engages Locomotor Stepping After Spinal Cord Injury.

Delivery of neurotrophins to the spinal injury site via cellular transplants or viral vectors administration has been shown to promote recovery of locomotion in the absence of locomotor training in adult spinalized animals. These delivery methods involved risks of secondary injury to the cord and do not allow for precise and controlled dosing making them unsuitable for clinical applications. The present study was aimed at evaluating the locomotor recovery efficacy and safety of the neurotrophin BDNF delivered intrathecally to the lumbar locomotor centers using an implantable and programmable infusion mini-pump. Results showed that BDNF treated spinal cats recovered weight-bearing plantar stepping at all velocities tested (0.3-0.8 m/s). Spinal cats treated with saline did not recover stepping ability, especially at higher velocities, and dragged their hind paws on the treadmill. Histological evaluation showed minimal catheter associated trauma and tissue inflammation, underlining that intrathecal delivery by an implantable/programmable pump is a safe and effective method for delivery of a controlled BDNF dosage; it poses minimal risks to the cord and is clinically translational.

Spinal Cord Injury in Myelomeningocele: Prospects for Therapy.

Myelomeningocele (MMC) is the most common congenital defect of the central nervous system and results in devastating and lifelong disability. In MMC, the initial failure of neural tube closure early in gestation is followed by a progressive prenatal injury to the exposed spinal cord, which contributes to the deterioration of neurological function in fetuses. Prenatal strategies to control the spinal cord injury offer an appealing therapeutic approach to improve neurological function, although the definitive pathophysiological mechanisms of injury remain to be fully elucidated. A better understanding of these mechanisms at the cellular and molecular level is of paramount importance for the development of targeted prenatal MMC therapies to minimize or eliminate the effects of the injury and improve neurological function. In this review article, we discuss the pathological development of MMC with a focus on in utero injury to the exposed spinal cord. We emphasize the need for a better understanding of the causative factors in MMC spinal cord injury, pathophysiological alterations associated with the injury, and cellular and molecular mechanisms by which these alterations are induced.