Cuprizone-induced Demyelination in Mouse Brain is not due to Depletion of Copper.

SUMMARY STATEMENT: The demyelinating effects of CPZ are not due to Cu deficiency but are instead consistent with acute toxicity of a CPZ + Cu complex.

Traumatic Injury Reduces Amyloid Plaque Burden in the Transgenic 5xFAD Alzheimer's Mouse Spinal Cord.

BACKGROUND: Axonal injury has been implicated in the development of amyloid-ß in experimental brain injuries and clinical cases. The anatomy of the spinal cord provides a tractable model for examining the effects of trauma on amyloid deposition. OBJECTIVE: Our goal was to examine the effects of axonal injury on plaque formation and clearance using wild type and 5xFAD transgenic Alzheimer's disease mice. METHODS: We contused the spinal cord at the T12 spinal level at 10 weeks, an age at which no amyloid plaques spontaneously accumulate in 5xFAD mice. We then explored plaque clearance by impacting spinal cords in 27-week-old 5xFAD mice where amyloid deposition is already well established. We also examined the cellular expression of one of the most prominent amyloid-ß degradation enzymes, neprilysin, at the lesion site. RESULTS: No plaques were found in wild type animals at any time points examined. Injury in 5xFAD prevented plaque deposition rostral and caudal to the lesion when the cords were examined at 2 and 4 months after the impact, whereas age-matched naïve 5xFAD mice showed extensive amyloid plaque deposition. A massive reduction in the number of plaques around the lesion was found as early as 7 days after the impact, preceded by neprilysin upregulation in astrocytes at 3 days after injury. At 7 days after injury, the majority of amyloid was found inside microglia/macrophages. CONCLUSION: These observations suggest that the efficient amyloid clearance after injury in the cord may be driven by the orchestrated efforts of astroglial and immune cells.

The triple monoamine re-uptake inhibitor DOV 216,303 promotes functional recovery after spinal cord contusion injury in mice.

Serotonin, noradrenaline and dopamine are important neuromodulators for locomotion in the spinal cord. Disruption of descending axons after spinal cord injury resulted in reduction of excitatory and neuromodulatory inputs to spinal neurons for locomotion. Receptor agonists or reuptake inhibitors for these neuromodulators have been shown to be beneficial in incomplete spinal cord injury. In this study, we tested a triple re-uptake inhibitor, DOV 216,303, for its ability to affect motor function recovery after spinal cord injury in mice. We impacted C57 mouse spinal cord at the T11 vertebral level and administered vehicle or DOV 216,303 at 10 mg/kg, b.i.d via intraperitoneal injections for 7 days. We monitored motor function with the Basso Mouse Scale for locomotion for 4 weeks. Spinal cords were harvested and histological examinations were performed to assess tissue sparing and lesion severity. Results showed that DOV 216,303-treated mice recovered significantly better than vehicle treated mice starting at 14 days post injury until the end of the survival period. Lesion size of the DOV 216,303 treated mice was also smaller compared to that of vehicle treated mice. This study suggests DOV 216,303 as a potential therapeutic after spinal cord injury warrants further investigation.

Axonal and myelinic pathology in 5xFAD Alzheimer's mouse spinal cord.

As an extension of the brain, the spinal cord has unique properties which could allow us to gain a better understanding of CNS pathology. The brain and cord share the same cellular components, yet the latter is simpler in cytoarchitecture and connectivity. In Alzheimer's research, virtually all focus is on brain pathology, however it has been shown that transgenic Alzheimer's mouse models accumulate beta amyloid plaques in spinal cord, suggesting that the cord possesses the same molecular machinery and conditions for plaque formation. Here we report a spatial-temporal map of plaque load in 5xFAD mouse spinal cord. We found that plaques started to appear at 11 weeks, then exhibited a time dependent increase and differential distribution along the cord. More plaques were found in cervical than other spinal levels at all time points examined. Despite heavy plaque load at 6 months, the number of cervical motor neurons in 5xFAD mice is comparable to wild type littermates. On detailed microscopic examination, fine beta amyloid-containing and beta sheet-rich thread-like structures were found in the peri-axonal space of many axons. Importantly, these novel structures appear before any plaque deposits are visible in young mice spinal cord and they co-localize with axonal swellings at later stages, suggesting that these thread-like structures might represent the initial stages of plaque formation, and could play a role in axonal damage. Additionally, we were able to demonstrate increasing myelinopathy in aged 5xFAD mouse spinal cord using the lipid probe Nile Red with high resolution. Collectively, we found significant amyloid pathology in grey and white matter of the 5xFAD mouse spinal cord which indicates that this structure maybe a useful platform to study mechanisms of Alzheimer's pathology and disease progression.

The molecular physiology of the axo-myelinic synapse.

Myelinated axons efficiently transmit information over long distances. The apposed myelin sheath confers favorable electrical properties, but restricts access of the axon to its extracellular milieu. Therefore, axonal metabolic support may require specific axo-myelinic communication. Here we explored activity-dependent glutamate-mediated signaling from axon to myelin. 2-Photon microscopy was used to image Ca(2+) changes in myelin in response to electrical stimulation of optic nerve axons ex vivo. We show that optic nerve myelin responds to axonal action potentials by a rise in Ca(2+) levels mediated by GluN2D and GluN3A-containing NMDA receptors. Glutamate is released from axons in a vesicular manner that is tetanus toxin-sensitive. The Ca(2+) source for vesicular fusion is provided by ryanodine receptors on axonal Ca(2+) stores, controlled by L-type Ca(2+) channels that sense depolarization of the internodal axolemma. Genetic ablation of GluN2D and GluN3A subunits results in greater lability of the compact myelin. Our results support the existence of a novel synapse between the axon and its myelin, suggesting a means by which traversing action potentials can signal the overlying myelin sheath. This may be an important physiological mechanism by which an axon can signal companion glia for metabolic support or adjust properties of its myelin in a dynamic manner. The axo-myelinic synapse may contribute to learning, while its disturbances may play a role in the pathophysiology of central nervous system disorders such as schizophrenia, where subtle abnormalities of myelinated white matter tracts have been shown in the human, or to frank demyelinating disorders such as multiple sclerosis.

Fluorescent Phosphorus Dendrimer as a Spectral Nanosensor for Macrophage Polarization and Fate Tracking in Spinal Cord Injury.

Dendrimers and dendriplexes, highly branched synthetic macromolecules, have gained popularity as new tools for a variety of nanomedicine strategies due to their unique structure and properties. We show that fluorescent phosphorus dendrimers are well retained by bone marrow-derived macrophages and exhibit robust spectral shift in its emission in response to polarization conditions. Fluorescence properties of this marker can also assist in identifying macrophage presence and phenotype status at different time points after spinal cord injury. Potential use of a single dendrimer compound as a drug/siRNA carrier and phenotype-specific cell tracer offers new avenues for enhanced cell therapies combined with monitoring of cell fate and function in spinal cord injury.

Axoplasmic reticulum Ca(2+) release causes secondary degeneration of spinal axons.

OBJECTIVE: Transected axons of the central nervous system fail to regenerate and instead die back away from the lesion site, resulting in permanent disability. Although both intrinsic (eg, microtubule instability, calpain activation) and extrinsic (ie, macrophages) processes are implicated in axonal dieback, the underlying mechanisms remain uncertain. Furthermore, the precise mechanisms that cause delayed "bystander" loss of spinal axons, that is, ones that were not directly damaged by the initial insult, but succumbed to secondary degeneration, remain unclear. Our goal was to evaluate the role of intra-axonal Ca(2+) stores in secondary axonal degeneration following spinal cord injury. METHODS: We developed a 2-photon laser-induced spinal cord injury model to follow morphological and Ca(2+) changes in live myelinated spinal axons acutely following injury. RESULTS: Transected axons "died back" within swollen myelin or underwent synchronous pan-fragmentation associated with robust Ca(2+) increases. Spared fibers underwent delayed secondary bystander degeneration. Reducing Ca(2+) release from axonal stores mediated by ryanodine and inositol triphosphate receptors significantly decreased axonal dieback and bystander injury. Conversely, a gain-of-function ryanodine receptor 2 mutant or pharmacological treatments that promote axonal store Ca(2+) release worsened these events. INTERPRETATION: Ca(2+) release from intra-axonal Ca(2+) stores, distributed along the length of the axon, contributes significantly to secondary degeneration of axons. This refocuses our approach to protecting spinal white matter tracts, where emphasis has been placed on limiting Ca(2+) entry from the extracellular space across cell membranes, and emphasizes that modulation of axonal Ca(2+) stores may be a key pharmacotherapeutic goal in spinal cord injury.

Toll-like receptor 2-mediated alternative activation of microglia is protective after spinal cord injury.

Improving neurological outcome after spinal cord injury is a major clinical challenge because axons, once severed, do not regenerate but 'dieback' from the lesion site. Although microglia, the immunocompetent cells of the brain and spinal cord respond rapidly to spinal cord injury, their role in subsequent injury or repair remains unclear. To assess the role of microglia in spinal cord white matter injury we used time-lapse two-photon and spectral confocal imaging of green fluorescent protein-labelled microglia, yellow fluorescent protein-labelled axons, and Nile Red-labelled myelin of living murine spinal cord and revealed dynamic changes in white matter elements after laser-induced spinal cord injury in real time. Importantly, our model of acute axonal injury closely mimics the axonopathy described in well-characterized clinically relevant models of spinal cord injury including contusive-, compressive- and transection-based models. Time-lapse recordings revealed that microglia were associated with some acute pathophysiological changes in axons and myelin acutely after laser-induced spinal cord injury. These pathophysiological changes included myelin and axonal spheroid formation, spectral shifts in Nile Red emission spectra in axonal endbulbs detected with spectral microscopy, and 'bystander' degeneration of axons that survived the initial injury, but then succumbed to secondary degeneration. Surprisingly, modulation of microglial-mediated release of neurotoxic molecules failed to protect axons and myelin. In contrast, sterile stimulation of microglia with the specific toll-like receptor 2 agonist Pam2CSK4 robustly increased the microglial response to ablation, reduced secondary degeneration of central myelinated fibres, and induced an alternative (mixed M1:M2) microglial activation profile. Conversely, Tlr2 knock out: Thy1 yellow fluorescent protein double transgenic mice experienced greater axonal dieback than littermate controls. Thus, promoting an alternative microglial response through Pam2CSK4 treatment is neuroprotective acutely following laser-induced spinal cord injury. Therefore, anti-inflammatory treatments that target microglial activation may be counterintuitive after spinal cord injury.