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After complete spinal cord injuries (SCI), spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. However, it is unknown whether selective manipulation of these circuits can restore locomotor function in the absence of brain-derived inputs. By taking advantage of the compromised blood-spinal cord barrier following SCI, we optimized a set of procedures in which AAV9 vectors administered via the tail vein efficiently transduce neurons in lesion-adjacent spinal segments after a thoracic crush injury in adult mice. With this method, we used chemogenetic actuators to alter the excitability of propriospinal neurons in the thoracic cord of the adult mice with a complete thoracic crush injury. We showed that activating these thoracic neurons enables consistent and significant hindlimb stepping improvement, whereas direct manipulations of the neurons in the lumbar spinal cord led to muscle spasms without meaningful locomotion. Strikingly, manipulating either excitatory or inhibitory propriospinal neurons in the thoracic levels leads to distinct behavioural outcomes, with preferential effects on standing or stepping, two key elements of the locomotor function. These results demonstrate a strategy of engaging thoracic propriospinal neurons to improve hindlimb function and provide insights into optimizing neuromodulation-based strategies for treating SCI., After complete spinal cord injury, spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. Here, the authors show that neurons in these circuits can be chemogenetically modulated to improve locomotor function in mice after spinal cord injury.

Myelination facilitates rapid axonal conduction thereby enabling efficient communication across different parts of the nervous system. Here we examined mechanisms controlling myelination after injury and during axon regeneration in the central nervous system (CNS). Previously we discovered multiple molecular pathways and strategies that could promote robust axon regrowth after optic nerve injury. However, regenerated axons remain unmyelinated and the underlying mechanisms were elusive. In this study, we found that in injured optic nerves, oligodendrocyte precursor cells (OPCs) undergo transient proliferation, but fail to differentiate into mature myelination-competent oligodendrocytes, reminiscent of what is observed in human progressive multiple sclerosis. Mechanistically, we showed that both OPC-intrinsic GPR17 signaling and sustained activation of microglia inhibit different stages of OPC differentiation. Importantly, co-manipulation of GPR17 and microglia led to extensive myelination of regenerated axons. The regulatory mechanisms of stage-dependent OPC differentiation uncovered here suggest a translatable strategy for efficient de novo myelination after CNS injury.

Graphical abstract, Excessive glutamate release following traumatic spinal cord injury (SCI) has been associated with exacerbating the extent of SCI. However, the mechanism behind sustained high levels of extracellular glutamate is unclear. Spinal cord segments mounted in a sucrose double gap recording chamber are an established model for traumatic spinal cord injury. We have developed a method to record, with micro-scale printed glutamate biosensors, glutamate release from ex vivo rat spinal cord segments following injury. This protocol would work equally well for similar glutamate biosensors.

Lipid desaturation plays important roles in biological processes and the disease states. Here, we report a simple but efficient method for mapping unsaturated phospholipids including the spatial distribution of lipid C=C location isomers in animal organs by coupling the C=C specific derivatization with direct analysis mass spectrometry (MS). Lipids are sampled directly by a stainless-steel wire from rat brain or kidney, extracted, and derivatized via the Paterno-Buchi reaction in a glass emitter of the nanoelectrospray ionization (nanoESI) source. Subsequent analysis by nanoESI-tandem mass spectrometry reveals C=C locations and relative quantities of lipid C=C location isomers. Unsaturated lipids, such as phospholipids and free fatty acids, have been identified with ion intensities spanning two orders of magnitude in rat brain. Typical sample consumption is less than 10 μg/measurement and the time for each analysis is about 3 min. This method should serve as a complementary method to high spatial resolution mass spectrometry imaging techniques, because it offers a streamlined experimental workflow for rapid profiling of lipids with C=C specificity to enable such applications as point-of-care disease diagnostics.

Brown adipose tissues (BAT) burn lipids to generate heat through uncoupled respiration, thus representing a powerful target to counteract lipid accumulation and obesity. The tumor suppressor liver kinase b1 (Lkb1) is a key regulator of cellular energy metabolism; and adipocyte-specific knockout of Lkb1 (Ad-Lkb1 KO) leads to the expansion of BAT, improvements in systemic metabolism and resistance to obesity in young mice. Here we report the unexpected finding that the Ad-Lkb1 KO mice develop hindlimb paralysis at mid-age. Gene expression analyses indicate that Lkb1 KO upregulates the expression of inflammatory cytokines in interscapular BAT and epineurial brown adipocytes surrounding the sciatic nerve. This is followed by peripheral neuropathy characterized by infiltration of macrophages into the sciatic nerve, axon degeneration, reduced nerve conductance, and hindlimb paralysis. Mechanistically, Lkb1 KO reduces AMPK phosphorylation and amplifies mammalian target-of-rapamycin (mTOR)-dependent inflammatory signaling specifically in BAT but not WAT. Importantly, pharmacological or genetic inhibition of mTOR ameliorates inflammation and prevents paralysis. These results demonstrate that BAT inflammation is linked to peripheral neuropathy., Highlights • Adipocyte specific knockout Lkb1 mice develop late onset hindlimb paralysis despite better metabolism in young animals. • Lkb1 KO in brown adipocytes promotes expression of proinflammatory cytokines that are linked to sciatic nerve neuropathy. • Rapamycin or genetic inhibition of mTOR ameliorates BAT inflammation and prevents paralysis. Brown adipose tissues (BAT) burn lipids to dissipate heat, expansion of BAT is thus considered as an attractive strategy to counteract obesity. Here, we use a mouse model in which the Lkb1 gene is knockout in adipocytes to show that the knockout mice develop enlarged BAT, resulting in better systemic metabolism. However, the knockout mice later develop peripheral neuropathy and hindlimb paralysis due to inflammation of the enlarged BAT. Inhibition of a signaling cascade known as mTOR effectively blunted the inflammation of BAT and prevented hindlimb paralysis. Therefore, even though BAT is known to promote metabolic health in the context of obesity, chronic inflammation of BAT is linked to devastating peripheral neuropathy. This study further suggests that blockage of mTOR is an effective strategy to treat inflammation-induced peripheral neuropathy.

Glutamate excitotoxicity is a pathology in which excessive glutamate can cause neuronal damage and degeneration. It has also been linked to secondary injury mechanisms in traumatic spinal cord injury. Conventional bioanalytical techniques used to characterize glutamate levels in vivo, such as microdialysis, have low spatiotemporal resolution, which has impeded our understanding of this dynamic event. In this study, we present an amperometric biosensor fabricated using a simple direct ink writing technique for the purpose of in vivo glutamate monitoring. The biosensor is fabricated by immobilizing glutamate oxidase on nanocomposite electrodes made of platinum nanoparticles, multi-walled carbon nanotubes, and a conductive polymer on a flexible substrate. The sensor is designed to measure extracellular dynamics of glutamate and other potential biomarkers during a traumatic spinal cord injury event. Here we demonstrate good sensitivity and selectivity of these rapidly prototyped implantable biosensors that can be inserted into a spinal cord and measure extracellular glutamate concentration. We show that our biosensors exhibit good flexibility, linear range, repeatability, and stability that are suitable for future in vivo evaluation.

The combination of direct sampling ionization and miniature mass spectrometer presents a promising technical pathway of point-of-care analysis in clinical applications. In this work, a miniature mass spectrometry system was used for analysis of tissue samples. Direct tissue sampling coupled with extraction spray ionization was used with a home-built miniature mass spectrometer, Mini 12. Lipid species in tissue samples were well profiled in rat brain, kidney, and liver in a couple of minutes. By incorporating a photochemical (Paterno-Buchi) reaction, fast identification of lipid C═C location was realized. Relative quantitation of the lipid C═C isomer was performed by calculating the intensity ratio C═C diagnostic product ions, by which FA 18:1 (Δ9)/FA 18:1 (Δ11) was found to change significantly in mouse cancerous breast tissue samples. Accumulation of 2-hydroxylglutarate in human glioma samples, not in normal brains, can also be easily identified for rapid diagnosis.

Myelin damage is a hallmark of spinal cord injury (SCI), and potassium channel blocker (PCB) is proven effective to restore axonal conduction and regain neurological function. Aiming to improve this therapy beyond the U.S. Food and Drug Administration-approved 4-aminopyridine (4-AP), we have developed multiple new PCBs, with 4-aminopyridine-3-methanol (4-AP-3-MeOH) being the most potent and effective. The current study evaluated two PCBs, 4-AP-3-MeOH and 4-AP, in parallel in both ex vivo and in vivo rat mechanical SCI models. Specifically, 4-AP-3-MeOH induced significantly greater augmentation of axonal conduction than 4-AP in both acute and chronic injury. 4-AP-3-MeOH had no negative influence on the electrical responsiveness of rescued axons whereas 4-AP-recruited axons displayed a reduced ability to follow multiple stimuli. In addition, 4-AP-3-MeOH can be applied intraperitoneally at a dose that is at least 5 times higher (5 mg/kg) than that of 4-AP (1 mg/kg) in vivo. Further, 5 mg/kg of 4-AP-3-MeOH significantly improved motor function whereas both 4-AP-3-MeOH (1 and 5 mg/kg) and, to a lesser degree, 4-AP (1 mg/kg) alleviated neuropathic pain-like behavior when applied in rats 2 weeks post-SCI. Based on these and other findings, we conclude that 4-AP-3-MeOH appears to be more advantageous over 4-AP in restoring axonal conduction because of the combination of its higher efficacy in enhancing the amplitude of compound action potential, lesser negative effect on axonal responsiveness to multiple stimuli, and wider therapeutic range in both ex vivo and in vivo application. As a result, 4-AP-3-MeOH has emerged as a strong alternative to 4-AP that can complement the effectiveness, and even partially overcome the shortcomings, of 4-AP in the treatment of neurotrauma and degenerative diseases where myelin damage is implicated.

Acrolein-mediated myelin damage is thought to be a critical mechanism leading to conduction failure following neurotrauma and neurodegenerative diseases. The exposure and activation of juxtaparanodal voltage-gated K+ channels due to myelin damage leads to conduction block, and K+ channel blockers have long been studied as a means for restoring axonal conduction in spinal cord injury (SCI) and multiple sclerosis (MS). In this study, we have found that 100 μM K+ channel blockers 4-aminopyridine-3-methanol (4-AP-3-MeOH), and to a lesser degree 4-aminopyridine (4-AP), can significantly restore compound action potential (CAP) conduction in spinal cord tissue following acrolein-mediated myelin damage using a well-established ex vivo SCI model. In addition, 4-AP-3-MeOH can effectively restore CAP conduction in acrolein-damaged axons with a range of concentrations from 0.1 to 100 μM. We have also shown that while both compounds at 100 μM showed no preference of small- and large-caliber axons when restoring CAP conduction, 4-AP-3-MeOH, unlike 4-AP, is able to augment CAP amplitude while causing little change in axonal responsiveness measured in refractory periods and response to repetitive stimuli. In a prior study, we show that 4-AP-3-MeOH was able to functionally rescue mechanically injured axons. In this investigation, we conclude that 4-AP-3-MeOH is an effective K+ channel blocker in restoring axonal conduction following both primary (physical) and secondary (chemical) insults. These findings also suggest that 4-AP-3-MeOH is a viable alternative of 4-AP for treating myelin damage and improving function following central nervous system trauma and neurodegenerative diseases.

Cd²⁺ is an industrial pollutant that can cause cytotoxicity in multiple organs. We examined the effects of extracellular Cd²⁺ on permeation and gating of Ca(v)3.1 (α1G) channels stably transfected in HEK293 cells, by using whole-cell recording. With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, Cd²⁺ rapidly blocked currents with 2 mM Ca²⁺ in a voltage-dependent manner. The block caused by Cd²⁺ was relieved at more-hyperpolarized potentials, which suggests that Cd²⁺ can permeate through the selectivity filter of the channel into the cytosol. In the absence of other permeant ions (Ca²⁺ and Na⁺ replaced by N-methyl-d-glucamine), Cd²⁺ carried sizable inward currents through Ca(v)3.1 channels (210 ± 20 pA at -60 mV with 2 mM Cd²⁺). Ca(v)3.1 channels have a significant "window current" at that voltage (open probability, ∼1%), which makes them a candidate pathway for Cd²⁺ entry into cells during Cd²⁺ exposure. Incubation with radiolabeled ¹⁰⁹Cd²⁺ confirmed uptake of Cd²⁺ into cells with Ca(v)3.1 channels.

N-methyl-d-aspartate (NMDA) receptor activation involves a dynamic series of structural rearrangements initiated by glutamate binding to glycine-loaded receptors and culminates with the clearing of the permeation pathway, which allows ionic flux. Along this sequence, three rate-limiting transitions can be quantified with kinetic analyses of single-channel currents, even though the structural determinants of these critical steps are unknown. In inactive receptors, the major permeation barrier resides at the intersection of four M3 transmembrane helices, two from each GluN1 and GluN2 subunits, at the level of the invariant SYTANLAAF sequence, known as the lurcher motif. Because the A7 but not A8 residues in this region display agonist-dependent accessibility to extracellular solutes, they were hypothesized to form the glutamate-sensitive gate. We tested this premise by examining the reaction mechanisms of receptors with substitutions in the lurcher motifs of GluN1 or GluN2A subunits. We found that, consistent with their locations relative to the proposed activation gate, A8Y decreased open-state stability, whereas A7Y dramatically stabilized open states, primarily by preventing gate closure; the equilibrium distribution of A7Y receptors was strongly shifted toward active states and resulted in slower microscopic association and dissociation rate constants for glutamate. In addition, for both A8- and A7-substituted receptors, we noticed patterns of kinetic changes that were specific to GluN1 or GluN2 locations. This may be a first indication that the sequence of discernible kinetic transitions during NMDA receptor activation may reflect subunit-dependent movements of M3 helices. Testing this hypothesis may afford insight into the activation mechanism of NMDA receptors.

Myelin is a critical component of the nervous system facilitating efficient propagation of electrical signals and thus communication between the central and peripheral nervous systems and the organ systems that they innervate throughout the body. In instances of neurotrauma and neurodegenerative disease, injury to myelin is a prominent pathological feature responsible for conduction deficits, and leaves axons vulnerable to damage from noxious compounds. Although the pathological mechanisms underlying myelin loss have yet to be fully characterized, oxidative stress (OS) appears to play a prominent role. Specifically, acrolein, a neurotoxic aldehyde that is both a product and an instigator of OS, has been observed in studies to elicit demyelination through calcium-independent and -dependent mechanisms and also by affecting glutamate uptake and promoting excitotoxicity. Furthermore, pharmacological scavenging of acrolein has demonstrated a neuroprotective effect in animal disease models, by conserving myelin's structural integrity and alleviating functional deficits. This evidence indicates that acrolein may be a key culprit of myelin damage while acrolein scavenging could potentially be a promising therapeutic approach for patients suffering from nervous system trauma and disease.

Cd²⁺ is an industrial pollutant that can cause cytotoxicity in multiple organs. We examined the effects of extracellular Cd²⁺ on permeation and gating of Ca(v)3.1 (α1G) channels stably transfected in HEK293 cells, by using whole-cell recording. With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, Cd²⁺ rapidly blocked currents with 2 mM Ca²⁺ in a voltage-dependent manner. The block caused by Cd²⁺ was relieved at more-hyperpolarized potentials, which suggests that Cd²⁺ can permeate through the selectivity filter of the channel into the cytosol. In the absence of other permeant ions (Ca²⁺ and Na⁺ replaced by N-methyl-d-glucamine), Cd²⁺ carried sizable inward currents through Ca(v)3.1 channels (210 ± 20 pA at -60 mV with 2 mM Cd²⁺). Ca(v)3.1 channels have a significant "window current" at that voltage (open probability, ∼1%), which makes them a candidate pathway for Cd²⁺ entry into cells during Cd²⁺ exposure. Incubation with radiolabeled ¹⁰⁹Cd²⁺ confirmed uptake of Cd²⁺ into cells with Ca(v)3.1 channels.

Myelin damage in the central nervous system (CNS) plays an important role in motor and sensory dysfunction (Shi and Sun, 2011). This neuropathology is observed widely in neurotrauma such as spinal cord injuries (SCI) and is also a distinguishing feature of many neurological diseases such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) (Shi and Sun, 2011). Damage to myelin structure leads to severe consequences at both a macromolecular and systems levels, which remains as one of the most challenging medical problems for both basic researchers and clinicians (Shi and Sun, 2011). Consequently, few successful therapeutic strategies exist today that can reverse or even slow the progression of this CNS pathology. Understanding the mechanisms underlying myelin damage is crucial for designing new, more effective treatments to mitigate symptoms and bring hope to millions of victims.

In all glutamate-activated channels, the outward portion of the M3 transmembrane helix consists of a conserved nine amino acid residue stretch (lurcher motif): SYTANALAAF. Substitution of position 8 alanine with threonine (A8T, the lurcher position) in a wide range of glutamate receptors result in constitutively active channels. Scanning mutagenesis and cysteine modification experiments, implicated the position 7 alanine (A7) of GluN1 and GluN2 subunits as critical elements in gating, perhaps the equivalent of the ‘bundle crossing’ in potassium-selective channels. To investigate how A7 and A8 residues affect NMDA receptor gating, we used single channel recordings and kinetic models of receptor activity from HEK 293 cells. We found that substitutions at these positions in GluN1 or GluN2A resulted indeed in channels with modified gating. Compared to wild-type receptors (PO = 0.51 ± 0.05, MOT = 8.6 ± 1 ms, MCT = 7.5 ± 0.7 ms), A7C and A8T resulted in distinct gating patterns depending on the subunit in which they were introduced. GluN1(A7C) had shorter openings (Po = 0.02 ± 0.01, MOT= 3.2 ± 0.5 ms, MCT = 143 ± 17 ms) but GluN2A A7C) had significantly prolonged openings (PO = 0.17 ± 0.02, MOT = 13 ± 1 ms, MCT = 65 ± 7 ms). Further, A8T prolonged openings in GluN1 (PO = 0.41 ± 0.07, MOT = 21 ± 4 ms, MCT = 30 ± 6.4 ms) but had no effect on gating when introduced in GluN2A (PO = 0.57 ± 0.08, MOT = 11.2 ± 1 ms, MCT = 9.5 ± 2.6 ms). The implication is that the A7 and A8 positions play distinct gating roles in GluN1 vs. GluN2A subunits, supporting the view that in NMDA receptors, subunits are staggered along M3 into the channel's external vestibule.