Cerebrospinal fluid immunoglobulins in primary progressive multiple sclerosis are pathogenic.

Multiple sclerosis is clinically characterized by relapses and remissions (relapsing-remitting multiple sclerosis) that over time may evolve to a progressive course (secondary progressive multiple sclerosis) or as having a progressive course from disease onset (primary progressive multiple sclerosis). At present, it is not definitively known whether these clinical entities constitute a single pathological disease or whether these manifestations represent two distinct disease entities sharing inflammatory demyelination as a pathological feature. Here we show using a novel mouse model that CSF of primary progressive multiple sclerosis patients is unique in its capacity to induce motor disability and spinal cord pathology including demyelination, impaired remyelination, reactive astrogliosis and axonal damage. Notably, removal of immunoglobulin G from primary progressive multiple sclerosis CSF via filtration or immunodepletion attenuates its pathogenic capacity. Furthermore, injection of recombinant antibodies derived from primary progressive multiple sclerosis CSF recapitulates the pathology. Our findings suggest that the clinical and pathological features of primary progressive multiple sclerosis are antibody-mediated and pathogenically distinct from relapsing-remitting and secondary progressive multiple sclerosis. Our study has potentially important implications for the development of specific therapies for patients with primary progressive multiple sclerosis.

Apolipoprotein B-100-mediated motor neuron degeneration in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by motor neuron degeneration. Approximately 90% of cases occur sporadically with no known cause while 10% are familial cases arising from known inherited genetic mutations. In vivo studies have predominantly utilized transgenic models harbouring amyotrophic lateral sclerosis-associated gene mutations, which have not hitherto elucidated mechanisms underlying motor neuron death or identified therapeutic targets specific to sporadic amyotrophic lateral sclerosis. Here we provide evidence demonstrating pathogenic differences in CSF from patients with sporadic amyotrophic lateral sclerosis and familial amyotrophic lateral sclerosis patients with mutations in SOD1, C9orf72 and TARDBP. Using a novel CSF-mediated animal model, we show that intrathecal delivery of sporadic amyotrophic lateral sclerosis patient-derived CSF into the cervical subarachnoid space in adult wild-type mice induces permanent motor disability which is associated with hallmark pathological features of amyotrophic lateral sclerosis including motor neuron loss, cytoplasmic TDP-43 translocation, reactive astrogliosis and microglial activation. Motor impairments are not induced by SOD1, C9orf72 or TARDBP CSF, although a moderate degree of histopathological change occurs in C9orf72 and TARDBP CSF-injected mice. By conducting a series of CSF filtration studies and global proteomic analysis of CSF, we identified apolipoprotein B-100 in sporadic amyotrophic lateral sclerosis CSF as the putative agent responsible for inducing motor disability, motor neuron degeneration and pathological translocation of TDP-43. Apolipoprotein B-100 alone is sufficient to recapitulate clinical and pathological outcomes in vivo and induce death of human induced pluripotent stem cell-derived motor neurons in vitro. Targeted removal of apolipoprotein B-100 from sporadic amyotrophic lateral sclerosis CSF via filtration or immunodepletion successfully attenuated the neurotoxic capacity of sporadic amyotrophic lateral sclerosis CSF to induce motor disability, motor neuron death, and TDP-43 translocation. This study presents apolipoprotein B-100 as a novel therapeutic target specific for the predominant sporadic form of amyotrophic lateral sclerosis and establishes proof-of-concept to support CSF pheresis as a therapeutic strategy for mitigating neurotoxicity in sporadic amyotrophic lateral sclerosis.

Murine cytomegalovirus infection exacerbates complex IV deficiency in a model of mitochondrial disease.

The influence of environmental insults on the onset and progression of mitochondrial diseases is unknown. To evaluate the effects of infection on mitochondrial disease we used a mouse model of Leigh Syndrome, where a missense mutation in the Taco1 gene results in the loss of the translation activator of cytochrome c oxidase subunit I (TACO1) protein. The mutation leads to an isolated complex IV deficiency that mimics the disease pathology observed in human patients with TACO1 mutations. We infected Taco1 mutant and wild-type mice with a murine cytomegalovirus and show that a common viral infection exacerbates the complex IV deficiency in a tissue-specific manner. We identified changes in neuromuscular morphology and tissue-specific regulation of the mammalian target of rapamycin pathway in response to viral infection. Taken together, we report for the first time that a common stress condition, such as viral infection, can exacerbate mitochondrial dysfunction in a genetic model of mitochondrial disease.

Author Correction: HDAC3 inhibition ameliorates spinal cord injury by immunomodulation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

HDAC3 inhibition ameliorates spinal cord injury by immunomodulation.

Following spinal cord injury (SCI), the innate immune response of microglia and infiltrating macrophages clears up cellular debris and promotes tissue repair, but it also inflicts secondary injury from inflammatory responses. Immunomodulation aimed at maximizing the beneficial effects while minimizing the detrimental roles of the innate immunity may aid functional recovery after SCI. However, intracellular drivers of global reprogramming of the inflammatory gene networks in the innate immune cells are poorly understood. Here we show that SCI resulted in an upregulation of histone deacetylase 3 (HDAC3) in the innate immune cells at the injury site. Remarkably, blocking HDAC3 with a selective small molecule inhibitor shifted microglia/macrophage responses towards inflammatory suppression, resulting in neuroprotective phenotypes and improved functional recovery in SCI model. Mechanistically, HDAC3 activity is largely responsible for histone deacetylation and inflammatory responses of primary microglia to classic inflammatory stimuli. Our results reveal a novel function of HDAC3 inhibitor in promoting functional recovery after SCI by dampening inflammatory cytokines, thus pointing towards a new direction of immunomodulation for SCI repair.

Attenuation of Cerebral Ischemic Injury in Smad1 Deficient Mice.

Stroke results in brain tissue damage from ischemia and oxidative stress. Molecular regulators of the protective versus deleterious cellular responses after cerebral ischemia remain to be identified. Here, we show that deletion of Smad1, a conserved transcription factor that mediates canonical bone morphogenetic protein (BMP) signaling, results in neuroprotection in an ischemia-reperfusion (I/R) stroke model. Uninjured mice with conditional deletion of Smad1 in the CNS (Smad1 cKO) displayed upregulation of the reactive astrocyte marker GFAP and hypertrophic morphological changes in astrocytes compared to littermate controls. Additionally, cultured Smad1(-/-) astrocytes exhibited an enhanced antioxidant capacity. When subjected to I/R injury by transient middle cerebral artery occlusion (tMCAO), Smad1 cKO mice showed enhanced neuronal survival and improved neurological recovery at 7 days post-stroke. This neuroprotective phenotype is associated with attenuated reactive astrocytosis and neuroinflammation, along with reductions in oxidative stress, p53 induction, and apoptosis. Our data suggest that Smad1-mediated signaling pathway is involved in stroke pathophysiology and may present a new potential target for stroke therapy.

B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.

Activation of intrinsic growth programs that promote developmental axon growth may also facilitate axon regeneration in injured adult neurons. Here, we demonstrate that conditional activation of B-RAF kinase alone in mouse embryonic neurons is sufficient to drive the growth of long-range peripheral sensory axon projections in vivo in the absence of upstream neurotrophin signaling. We further show that activated B-RAF signaling enables robust regenerative growth of sensory axons into the spinal cord after a dorsal root crush as well as substantial axon regrowth in the crush-lesioned optic nerve. Finally, the combination of B-RAF gain-of-function and PTEN loss-of-function promotes optic nerve axon extension beyond what would be predicted for a simple additive effect. We conclude that cell-intrinsic RAF signaling is a crucial pathway promoting developmental and regenerative axon growth in the peripheral and central nervous systems.

Reshaping the chromatin landscape after spinal cord injury.

The pathophysiology underlying spinal cord injury is complex. Mechanistic understanding of the adaptive responses to injury is critical for targeted therapy aimed at reestablishing lost connections between proximal and distal neurons. After injury, cell-type specific gene transcription programs govern distinct cellular behaviors, and chromatin regulators play a central role in shaping the chromatin landscape to adjust transcriptional profiles in a context-dependent manner. In this review, we summarize recent progress on the pleiotropic roles of chromatin regulators in mediating the diverse adaptive behaviors of neurons and glial cells after spinal cord injury, and wherever possible, discuss the underlying mechanisms and genomic targets. We specifically draw attention to the perspective that takes into consideration the impact of epigenetic modulation on axon growth potential, together with its effect on wound-healing properties of glial cells. Epigenetic modulation of chromatin state represents an emerging therapeutic direction to promote neural repair and axon regeneration after spinal cord injury.

Epigenetic regulation of sensory axon regeneration after spinal cord injury.

Axon regeneration is hindered by a decline of intrinsic axon growth capability in mature neurons. Reversing this decline is associated with the induction of a large repertoire of regeneration-associated genes (RAGs), but the underlying regulatory mechanisms of the transcriptional changes are largely unknown. Here, we establish a correlation between diminished axon growth potential and histone 4 (H4) hypoacetylation. When neurons are triggered into a growth state, as in the conditioning lesion paradigm, H4 acetylation is restored, and RAG transcription is initiated. We have identified a set of target genes of Smad1, a proregenerative transcription factor, in conditioned DRG neurons. We also show that, during the epigenetic reprogramming process, histone-modifying enzymes work together with Smad1 to facilitate transcriptional regulation of RAGs. Importantly, targeted pharmacological modulation of the activity of histone-modifying enzymes, such as histone deacetylases, leads to induction of multiple RAGs and promotion of sensory axon regeneration in a mouse model of spinal cord injury. Our findings suggest epigenetic modulation as a potential therapeutic strategy to enhance axon regeneration.

Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain.

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration.

One day of motor training with amphetamine impairs motor recovery following spinal cord injury.

It has previously been reported that a single dose of amphetamine paired with training on a beam walking task can enhance locomotor recovery following brain injury (Feeney et al., 1982). Here, we investigated whether this same drug/training regimen could enhance functional recovery following either thoracic (T9) or cervical (C5) spinal cord injury. Different groups of female Sprague-Dawley rats were trained on a beam walking task, and in a straight alley for assessment of hindlimb locomotor recovery using the BBB locomotor scale. For rats that received C5 hemisections, forelimb grip strength was assessed using a grip strength meter. Three separate experiments assessed the consequences of training rats on the beam walking task 24 h following a thoracic lateral hemisection with administration of either amphetamine or saline. Beginning 1 h following drug administration, rats either received additional testing/retraining on the beam hourly for 6 h, or they were returned to their home cages without further testing/retraining. Rats with thoracic spinal cord injuries that received amphetamine in conjunction with testing/retraining on the beam at 1 day post injury (DPI) exhibited significantly impaired recovery on the beam walking task and BBB. Rats with cervical spinal cord injuries that received training with amphetamine also exhibited significant impairments in beam walking and locomotion, as well as impairments in gripping and reaching abilities. Even when administered at 14 DPI, the drug/training regimen significantly impaired reaching ability in cervical spinal cord injured rats. Impairments were not seen in rats that received amphetamine without training. Histological analyses revealed that rats that received training with amphetamine had significantly larger lesions than saline controls. These data indicate that an amphetamine/training regimen that improves recovery after cortical injury has the opposite effect of impairing recovery following spinal cord injury because early training with amphetamine increases lesion severity.

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.

A straight alley version of the BBB locomotor scale.

We describe here an alternative procedure for assessing hindlimb locomotor function after spinal cord injury that uses the BBB scale, but tests animals in a reward-baited straight alley rather than an open field. Rats were trained to ambulate in a straight alley and habituated to the open field typically used for BBB open field testing. Three groups of rats were tested. Sprague-Dawley rats received either 200 kD (n=19) or 300 kD contusions (n=9) at T9 with the Infinite Horizon device. Fisher rats (n=8) received moderate contusions (12.5 mm) at T8 with the NYU impactor. BBB scores were assessed at different post-injury intervals in the open field and the straight alley, and scores were compared by correlation analyses. BBB scores in the open field vs. the straight alley were highly correlated (r=0.90), validating the use of the straight alley for locomotor assessment. Rats exhibited a larger number of bouts of continuous steps in the straight alley vs. the open field (termed passes), providing more opportunities to score hindlimb use and coordination over the 4 min testing interval. Comparisons of scores across days revealed higher day-to-day correlations in the straight alley vs. the open field (r(2) values of 0.90 and 0.74 for the straight alley and open field respectively), revealing that the straight alley yielded more reliable scores.

Chronic nicotine-induced switch in Src-family kinase signaling for long-term potentiation induction in hippocampal CA1 pyramidal cells.

Here, we show that chronic nicotine exposure induces changes in Src signaling for the modulation of N-methyl-D-aspartate receptor (NMDAR) function and LTP induction in CA1 pyramidal cells. Activation of muscarinic receptors normally potentiates NMDAR responses in pyramidal cells via a Gq/protein kinase C (PKC)/proline-rich tyrosine kinase 2/Src signaling cascade. However, muscarinic, PKC and Src stimulation had no effect on NMDAR responses after chronic nicotine treatment. The lack of effect was apparently due to enhanced tyrosine phosphorylation, and therefore further stimulation of the signaling cascade caused no effect on NMDAR responses. Interestingly, another Src-family kinase potentiated NMDAR responses after, but not before, chronic nicotine treatment. In control pyramidal cells, Src inhibitor peptides prevented tetanus-induced long-term potentiation (LTP). Conversely, in chronic nicotine-exposed cells, the inhibitor was ineffective in blocking tetanus-induced LTP. Furthermore, in control pyramidal cells, applying exogenous Src and administration of an endogenous Src-family kinase activator increased alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR)-mediated responses. This increase was blocked by Src inhibitor peptides and occluded tetanus-induced LTP, as reported previously. In contrast, in chronic nicotine-treated pyramidal cells, applying exogenous Src had no effect on AMPAR-mediated responses and a tetanus-induced LTP. Interestingly, however, administration of an endogenous Src-family kinase activator enhanced AMPAR-mediated responses, which occluded tetanus-induced LTP. This enhancement was not prevented by co-application of Src inhibitor peptides. Thus, it appears that chronic nicotine exposure recruits another member of the Src-family for the regulation of NMDAR function and LTP induction. The nicotine-induced distinct signaling cascades may be involved in long-lasting memories of nicotine misuse.

Comparison of alpha2 nicotinic acetylcholine receptor subunit mRNA expression in the central nervous system of rats and mice.

The nicotinic acetylcholine receptor (nAChR) alpha2 subunit was the first neuronal nAChR to be cloned. However, data for the distribution of alpha2 mRNA in the rodent exists in only a few studies. Therefore, we investigated the expression of alpha2 mRNA in the rat and mouse central nervous systems using nonradioactive in situ hybridization histochemistry. We detected strong hybridization signals in cell bodies located in the internal plexiform layer of the olfactory bulb, the interpeduncular nucleus of the midbrain, the ventral and dorsal tegmental nuclei, the median raphe nucleus of the pons, the ventral part of the medullary reticular nucleus, the ventral horn in the spinal cord of both rats and mice, and in a few Purkinje cells of rats, but not of mice. Cells that moderately express alpha2 mRNA were localized to the cerebral cortex layers V and VI, the subiculum, the oriens layer of CA1, the medial septum, the diagonal band complex, the substantia innominata, and the amygdala of both animals. They were also located in a few midbrain nuclei of rats, whereas in mice they were either few or absent in these areas. However, in the upper medulla oblongata alpha2 mRNA was expressed in several large neurons of the gigantocellular reticular nucleus and the raphe magnus nucleus of mice, but not of rats. The data obtained show that a similar pattern of alpha2 mRNA expression exists in both rats and mice, with the exception of a few regions, and provide the basis for cellular level analysis.