Regulatory T cells promote remyelination in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis following human neural stem cell transplant.

Multiple sclerosis (MS) is a chronic, inflammatory autoimmune disease that affects the central nervous system (CNS) for which there is no cure. In MS, encephalitogenic T cells infiltrate the CNS causing demyelination and neuroinflammation; however, little is known about the role of regulatory T cells (Tregs) in CNS tissue repair. Transplantation of neural stem and progenitor cells (NSCs and NPCs) is a promising therapeutic strategy to promote repair through cell replacement, although recent findings suggest transplanted NSCs also instruct endogenous repair mechanisms. We have recently described that dampened neuroinflammation and increased remyelination is correlated with emergence of Tregs following human NPC transplantation in a murine viral model of immune-mediated demyelination. In the current study we utilized the prototypic murine autoimmune model of demyelination experimental autoimmune encephalomyelitis (EAE) to test the efficacy of hNSC transplantation. Eight-week-old, male EAE mice receiving an intraspinal transplant of hNSCs during the chronic phase of disease displayed remyelination, dampened neuroinflammation, and an increase in CNS CD4+CD25+FoxP3+ regulatory T cells (Tregs). Importantly, ablation of Tregs abrogated histopathological improvement. Tregs are essential for maintenance of T cell homeostasis and prevention of autoimmunity, and an emerging role for Tregs in maintenance of tissue homeostasis through interactions with stem and progenitor cells has recently been suggested. The data presented here provide direct evidence for collaboration between CNS Tregs and hNSCs promoting remyelination.

Synaptic ultrastructure changes in trigeminocervical complex posttrigeminal nerve injury.

Trigeminal nerves collecting sensory information from the orofacial area synapse on second-order neurons in the dorsal horn of subnucleus caudalis and cervical C1/C2 spinal cord (Vc/C2, or trigeminocervical complex), which is critical for sensory information processing. Injury to the trigeminal nerves may cause maladaptive changes in synaptic connectivity that plays an important role in chronic pain development. Here we examined whether injury to the infraorbital nerve, a branch of the trigeminal nerves, led to synaptic ultrastructural changes when the injured animals have developed neuropathic pain states. Transmission electron microscopy was used to examine synaptic profiles in Vc/C2 at 3 weeks postinjury, corresponding to the time of peak behavioral hypersensitivity following chronic constriction injury to the infraorbital nerve (CCI-ION). Using established criteria, synaptic profiles were classified as associated with excitatory (R-), inhibitory (F-), and primary afferent (C-) terminals. Each type was counted within the superficial dorsal horn of the Vc/C2 and the means from each rat were compared between sham and injured animals; synaptic contact length was also measured. The overall analysis indicates that rats with orofacial pain states had increased numbers and decreased mean synaptic length of R-profiles within the Vc/C2 superficial dorsal horn (lamina I) 3 weeks post-CCI-ION. Increases in the number of excitatory synapses in the superficial dorsal horn of Vc/C2 could lead to enhanced activation of nociceptive pathways, contributing to the development of orofacial pain states.

Sphingosine-1-phosphate receptor antagonism enhances proliferation and migration of engrafted neural progenitor cells in a model of viral-induced demyelination.

The oral drug FTY720 affects sphingosine-1-phosphate (S1P) signaling on targeted cells that bear the S1P receptors S1P1, S1P3, S1P4, and S1P5. We examined the effect of FTY720 treatment on the biology of mouse neural progenitor cells (NPCs) after transplantation in a viral model of demyelination. Intracerebral infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in an acute encephalomyelitis, followed by demyelination similar in pathology to the human demyelinating disease, multiple sclerosis. We have previously reported that intraspinal transplantation of mouse NPCs into JHMV-infected animals resulted in selective colonization of demyelinated lesions, preferential differentiation into oligodendroglia accompanied by axonal preservation, and increased remyelination. Cultured NPCs expressed transcripts for S1P receptors S1P1, S1P2, S1P3, S1P4, and S1P5. FTY720 treatment of cultured NPCs resulted in increased mitogen-activated protein kinase phosphorylation and migration after exposure to the chemokine CXCL12. Administration of FTY720 to JHMV-infected mice resulted in enhanced migration and increased proliferation of transplanted NPCs after spinal cord engraftment. FTY720 treatment did not improve clinical disease, diminish neuroinflammation or the severity of demyelination, nor increase remyelination. These findings argue that FTY720 treatment selectively increases NPC proliferation and migration but does not either improve clinical outcome or enhance remyelination after transplantation into animals in which immune-mediated demyelination is initiated by the viral infection of the central nervous system.

Human neural precursor cells promote neurologic recovery in a viral model of multiple sclerosis.

Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-ß1 and TGF-ß2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments.

Rapamycin and interleukin-1ß impair brain-derived neurotrophic factor-dependent neuron survival by modulating autophagy.

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF's support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF's suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1ß impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.

Immunological demyelination triggers macrophage/microglial cells activation without inducing astrogliosis.

The glial scar formed by reactive astrocytes and axon growth inhibitors associated with myelin play important roles in the failure of axonal regeneration following central nervous system (CNS) injury. Our laboratory has previously demonstrated that immunological demyelination of the CNS facilitates regeneration of severed axons following spinal cord injury. In the present study, we evaluate whether immunological demyelination is accompanied with astrogliosis. We compared the astrogliosis and macrophage/microglial cell responses 7 days after either immunological demyelination or a stab injury to the dorsal funiculus. Both lesions induced a strong activated macrophage/microglial cells response which was significantly higher within regions of immunological demyelination. However, immunological demyelination regions were not accompanied by astrogliosis compared to stab injury that induced astrogliosis which extended several millimeters above and below the lesions, evidenced by astroglial hypertrophy, formation of a glial scar, and upregulation of intermediate filaments glial fibrillary acidic protein (GFAP). Moreover, a stab or a hemisection lesion directly within immunological demyelination regions did not induced astrogliosis within the immunological demyelination region. These results suggest that immunological demyelination creates a unique environment in which astrocytes do not form a glial scar and provides a unique model to understand the putative interaction between astrocytes and activated macrophage/microglial cells.

PTEN deletion enhances the regenerative ability of adult corticospinal neurons.

Despite the essential role of the corticospinal tract (CST) in controlling voluntary movements, successful regeneration of large numbers of injured CST axons beyond a spinal cord lesion has never been achieved. We found that PTEN/mTOR are critical for controlling the regenerative capacity of mouse corticospinal neurons. After development, the regrowth potential of CST axons was lost and this was accompanied by a downregulation of mTOR activity in corticospinal neurons. Axonal injury further diminished neuronal mTOR activity in these neurons. Forced upregulation of mTOR activity in corticospinal neurons by conditional deletion of Pten, a negative regulator of mTOR, enhanced compensatory sprouting of uninjured CST axons and enabled successful regeneration of a cohort of injured CST axons past a spinal cord lesion. Furthermore, these regenerating CST axons possessed the ability to reform synapses in spinal segments distal to the injury. Thus, modulating neuronal intrinsic PTEN/mTOR activity represents a potential therapeutic strategy for promoting axon regeneration and functional repair after adult spinal cord injury.

Unexpected survival of neurons of origin of the pyramidal tract after spinal cord injury.

There is continuing controversy about whether the cells of origin of the corticospinal tract (CST) undergo retrograde cell death after spinal cord injury (SCI). All previous attempts to assess this have used imaging and/or histological techniques to assess upper motoneurons in the cerebral cortex. Here, we address the question in a novel way by assessing Wallerian degeneration and axon numbers in the medullary pyramid of Sprague Dawley rats after both acute SCI, either at cervical level 5 (C5) or thoracic level 9 (T9), and chronic SCI at T9. Our findings demonstrate that only a fraction of a percentage of the total axons in the medullary pyramid exhibit any sign of degeneration at any time after SCI--no more so than in uninjured control rats. Moreover, design-based counts of myelinated axons revealed no decrease in axon number in the medullary pyramid after SCI, regardless of injury level, severity, or time after injury. Spinal cord-injured rats had fewer myelinated axons in the medullary pyramid at 1 year after injury than aged matched controls, suggesting that injury may affect ongoing myelination of axons during aging. We conclude that SCI does not cause death of the CST cell bodies in the cortex; therefore, therapeutic strategies aimed at promoting axon regeneration of the CST in the spinal cord do not require a separate intervention to prevent retrograde degeneration of upper motoneurons in the cortex.