Early and progressive impairment of spinal blood flow-glucose metabolism coupling in motor neuron degeneration of ALS model mice

The exact mechanism of selective motor neuron death in amyotrophic lateral sclerosis (ALS) remains still unclear. In the present study, we performed in vivo capillary imaging, directly measured spinal blood flow (SBF) and glucose metabolism, and analyzed whether if a possible flow-metabolism coupling is disturbed in motor neuron degeneration of ALS model mice. In vivo capillary imaging showed progressive decrease of capillary diameter, capillary density, and red blood cell speed during the disease course. Spinal blood flow was progressively decreased in the anterior gray matter (GM) from presymptomatic stage to 0.80-fold of wild-type (WT) mice, 0.61 at early-symptomatic, and 0.49 at end stage of the disease. Local spinal glucose utilization (LSGU) was transiently increased to 1.19-fold in anterior GM at presymptomatic stage, which in turn progressively decreased to 0.84 and 0.60 at early-symptomatic and end stage of the disease. The LSGU/SBF ratio representing flow-metabolism uncoupling (FMU) preceded the sequential pathological changes in the spinal cord of ALS mice and was preferentially found in the affected region of ALS. The present study suggests that this early and progressive FMU could profoundly involve in the whole disease process as a vascular factor of ALS pathology, and could also be a potential target for therapeutic intervention of ALS.     

薄束轴索变性小鼠的轴索变性和Ubiquitin的相互关系

目的 探索ＧＡＤ小鼠中枢神经系统的轴索变性和Ｕｂｉｑｕｔｉｎ的相互关系。方法 用免疫组织化学染色方法。结果 在ＧＡＤ小鼠中枢神经系统的轴索变性部位,出现了多数Ｕｂｉｑｕｉｔｉｎ阳性的点状构造物。这种ＤＳ从９周龄开始,首先在构成薄束路和脊髓小脑后路的上行纤维的远端区域－薄束核区和小脑白质出现,逐渐按照延髓,颈髓,胸髓,腰髓的顺序,向细胞体方向逆行性地进展。１８周龄时波及到锥体路,３２周龄时进一步波及     

Uncoupling of neuroinflammation from axonal degeneration in mice lacking the myelin protein tetraspanin‐2

Deficiency of the major constituent of central nervous system (CNS) myelin, proteolipid protein (PLP), causes axonal pathology in spastic paraplegia type‐2 patients and in Plp1^null‐mice but is compatible with almost normal myelination. These observations led us to speculate that PLP's role in myelination may be partly compensated for by other tetraspan proteins. Here, we demonstrate that the abundance of the structurally related tetraspanin‐2 (TSPAN2) is highly increased in CNS myelin of Plp1^null‐mice. Unexpectedly, Tspan2^null‐mutant mice generated by homologous recombination in embryonic stem cells displayed low‐grade activation of astrocytes and microglia in white matter tracts while they were fully myelinated and showed no signs of axonal degeneration. To determine overlapping functions of TSPAN2 and PLP, Tspan2^null*Plp1^null double‐mutant mice were generated. Strikingly, the activation of astrocytes and microglia was strongly enhanced in Tspan2^null*Plp1^null double‐mutants compared with either single‐mutant, but the levels of dysmyelination and axonal degeneration were not increased. In this model, glial activation is thus unlikely to be caused by axonal pathology, and vice versa does not potentiate axonal degeneration. Our results support the concept that multiple myelin proteins have distinct roles in the long‐term preservation of a healthy CNS, rather than in myelination per se. GLIA 2013;61:1832–1847     

Mechanisms of axonal degeneration in EAE--lessons from CNTF and MHC I knockout mice

The major pathological hallmarks of multiple sclerosis (MS) comprise inflammation, demyelination with associated gliosis and axonal damage, which most likely correlates with persisting disability. Axonal damage can occur by several mechanisms. This article focuses on myelin disintegration and direct immune attack on axons by CD8-positive T-cells as two possible scenarios for axonal injury. As protoypic models, we investigated experimental autoimmune encephalomyelitis (EAE) in ciliary neurotrophic factor gene knockout mice (CNTF-/- mice) with severe myelin pathology and EAE in beta-2 microglobulin gene knockout mice (beta2m-/- mice) lacking CD8-positive T-cells. The results from these studies indicate that the trigger attack for axonal injury even in a well-defined experimental design can be multi-faceted. No single factor seems to be absolutely necessary for the initiation of the process, but they rather act in concert and orchestrate tissue destruction, inflammation and regeneration. Some mechanisms of primary or secondary axonal damage may be shared between inflammatory and degenerative diseases of the nervous system, thereby establishing a link which might be of importance for future therapeutic strategies.     

Progressive behavioral deficits in DJ-1-deficient mice are associated with normal nigrostriatal function

Loss-of-function mutations in the DJ-1 gene account for an autosomal recessive form of Parkinson's disease (PD). To investigate the physiological functions of DJ-1 in vivo, we generated DJ-1 knockout (DJ-1^−/−) mice. Younger (< 1 year) DJ-1^−/− mice were hypoactive and had mild gait abnormalities. Older DJ-1^−/−, however, showed decreased body weight and grip strength and more severe gait irregularities compared to wild-type littermates. The basal level of extracellular dopamine, evoked dopamine release and dopamine receptor D2 sensitivity appeared normal in the striatum of DJ-1^−/− mice, which was consistent with similar results between DJ-1^−/− and controls in behavioral paradigms specific for the dopaminergic system. An examination of spinal cord, nerve and muscle tissues failed to identify any pathological changes that were consistent with the noted motor deficits. Taken together, our findings suggest that loss of DJ-1 leads to progressive behavioral changes without significant alterations in nigrostriatal dopaminergic and spinal motor systems.     

Oxidative damage to mitochondrial DNA in spinal motoneurons of transgenic ALS mice

In order to clarify a possible role of oxidative stress in motoneuron death in amyotrophic lateral sclerosis (ALS), we examined a presence of 8-hydroxy-2-deoxyguanosine (8-OHdG), one of the best markers of the oxidative DNA damage, in the spinal cord of transgenic mice harboring a mutant Cu/Zn superoxide dismutase (SOD1) gene. Immunocytochemistry showed a progressive accumulation of 8-OHdG in ventral horn neurons from early and presymptomatic stage (25 weeks) before significant loss of ventral horn neurons, while no detectable 8-OHdG in non-transgenic control mice. At the late and symptomatic stage (35 weeks), the 8-OHdG-like immunoreactivity spread over the posterior horn of spinal cord in Tg mice. The immunoreactivity for 8-OHdG was not localized in the nucleus but in cytoplasm with small granular pattern. These data suggest that an oxidative damage to mitochondrial DNA is happening in spinal motoneurons of the Tg mice from very early stage of the disease, and may be involved in the mechanism of the subsequent motoneuron death in this model.     

Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice

We investigated the usefulness of YFP-H transgenic mice [Neuron 28 (2000) 41] which express yellow fluorescent protein (YFP) in a restricted subset of neurons to study Wallerian degeneration in the PNS. Quantification of YFP positive axons and myelin basic protein (MBP) immunocytochemistry revealed that YFP was randomly distributed to approximately 3% of myelinated motor and sensory fibres. Axotomy-induced Wallerian degeneration appeared as fragmentation of fluorescent signals in individual YFP positive axons with a morphology and timing similar to Wallerian degeneration observed by more traditional methods. In YFP-H transgenic mice co-expressing a high dosage of WldS, a chimeric gene that protects from Wallerian degeneration [Nat Neurosci. 4 (2001) 1199], axonal fragmentation in distal tibial nerves after sciatic nerve axotomy was approximately 10 times delayed. Considerable retardations of Wallerian degeneration using the same transgenic expression system were also observed in cultures of nerve explants, enabling in vitro real-time imaging of axonal fragmentation. Remarkably, single YFP-labelled axons could be traced in peripheral nerves for unusually long distances of up to 2.9 cm exploiting confocal fluorescence imaging. Altogether transgenic YFP-H mice prove to be a valuable tool to study mechanisms of Wallerian degeneration in vivo and in vitro.     

Demyelination can proceed independently of axonal degradation during Wallerian degeneration in wlds mice

Peripheral nerve injury induces axonal degeneration and demyelination, which are collectively referred to as Wallerian degeneration. It is generally assumed that axonal degeneration is a trigger for the subsequent demyelination processes such as myelin destruction and de-differentiation of Schwann cells, but the detailed sequence of events that occurs during this initial phase of demyelination following axonal degeneration remains unclear. Here we performed a morphological analysis of injured sciatic nerves of wlds mice, a naturally occurring mutant mouse in which Wallerian degeneration shows a significant delay. The slow Wallerian degerenation phenotype of the wlds mutant mice would enable us to dissect the events that take place during the initial phase of demyelination. Ultrastrucural analysis using electron microscopy showed that the initial process of myelin destruction was activated in injured nerves of wlds mice even though they exhibit morphologically complete protection of axons against nerve injury. We also found that some intact axons were completely demyelinated in degenerating nerves of wlds mice. Furthermore, we observed that de-differentiation of myelinating Schwann cells gradually proceeded even though the axons remained morphologically intact. These data suggest that initiation and progression of demyelination in injured peripheral nerves is, at least in part, independent of axonal degeneration.     

Apoptosis in oligodendrocytes is associated with axonal degeneration in P301L tau mice

Transgenic mice overexpressing human tau with the P301L mutation develop neurofibrillary tangles, extensive gliosis, adult-onset motor abnormalities, and neuronal loss in affected brain regions. We investigated the mechanism of neuronal cell death in this model of tauopathy. There was no evidence of neuronal apoptosis at any age; however, a population of oligodendorocytes was immunopositive for TUNEL and activated caspase-3. EM confirmed that these oligodendrocytes were undergoing apoptosis. These data suggest that classical apoptosis is not a major mechanism of neuronal cell death associated with the tau dysfunction in this mouse model; however, prominent white matter pathology in the spinal cord suggests that axonal degeneration in dying neurons causes oligodendrocytes to undergo apoptosis. It is unknown if loss of oligodendrocytes either through apoptosis or through the formation of intracellular tau lesions further contributes to the neurodegeneration seen in these mice.     

Immunization with neurofilament light protein induces spastic paresis and axonal degeneration in Biozzi ABH mice

Axonal damage is the major cause of irreversible neurologic disability in patients with multiple sclerosis. Although axonal damage correlates with antibodies against neurofilament light (NF-L) protein, a major component of the axonal cytoskeleton, the possible pathogenic role of autoimmunity to axonal antigens such as NF-L has so far been ignored. Here we show that Biozzi ABH mice immunized with NF-L protein develop neurologic disease characterized by spastic paresis and paralysis concomitant with axonal degeneration and inflammation primarily in the dorsal column of the spinal cord. The inflammatory central nervous system lesions were dominated by F4/80+ macrophages/microglia and relatively low numbers of CD4+ and CD8+ T-cells. In splenocyte cultures, proliferation to NF-L was observed in CD4+ T-cells accompanied by the production of the proinflammatory cytokine interferon-gamma. Elevated levels of circulating antibodies recognizing recombinant mouse NF-L were present in the serum, and immunoglobulin deposits were observed within axons in spinal cord lesions of mice exhibiting clinical disease. These data provide evidence that autoimmunity to NF-L protein induces axonal degeneration and clinical neurologic disease in mice, indicating that autoimmunity to axonal antigens, as described in multiple sclerosis, may be pathogenic rather than acting merely as a surrogate marker for axonal degeneration.     

Neurological deficits in mice with profound biotinidase deficiency are associated with demylination and axonal degeneration

Biotinidase deficiency is an autosomal recessively inherited disorder characterized by neurological and cutaneous abnormalities. We have developed a transgenic knock-out mouse with biotinidase deficiency to better understand aspects of pathophysiology and natural history of the disorder in humans. Neurological deficits observed in symptomatic mice with biotinidase deficiency are similar to those seen in symptomatic children with the disorder. Using a battery of functional neurological assessment tests, the symptomatic mice performed poorly compared to wild-type mice. Demyelination, axonal degeneration, ventriculomegaly, and corpus callosum compression were found in the brains of untreated, symptomatic enzyme-deficient mice. With biotin treatment, the symptomatic mice improved neurologically and the white matter abnormalities resolved. These functional and anatomical findings and their reversal with biotin therapy are similar to those observed in untreated, symptomatic and treated individuals with biotinidase deficiency. The mouse with biotinidase deficiency appears to be an appropriate animal model in which to study the neurological abnormalities and the effects of treatment of the disorder.     

Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration

Alterations in mitochondrial dynamics (fission, fusion, and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimer's disease. However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood. Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species. By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration. Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction. We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease. These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration.     

Progressive morphological abnormalities observed in rat spinal motor neurons at extended intervals after axonal regeneration.

It is generally accepted that mammalian spinal motor neurons return to normal after axotomy if their regenerated axons successfully reinnervate appropriate peripheral targets. However, morphological abnormalities, recently observed in spinal motor neurons examined 1 year after nerve crush injury, raise the possibility that delayed perikaryal changes occur after regeneration is complete. In order to distinguish between chronic and progressive alterations in neurons with long-term regenerated axons, rat spinal motor neurons and dorsal root ganglion cells were examined at 5 and 10 months following unilateral sciatic nerve crush. Neurons with regenerated axons were identified by retrograde labelling with horseradish peroxidase. The structural properties of neurons ipsilateral to nerve injury were compared to those of neurons from the spinal cord and dorsal root ganglia on the contralateral side and from age-matched control rats. At 5 months postcrush, the morphology of motor and sensory neurons ipsilateral to injury was comparable to that of control cells. However, several features of the motor neurons with regenerated axons distinguished them from control motor neurons at 10 months postcrush. Mean perikaryal area of ipsilateral spinal motor neurons was larger than the means for control motor neurons (p less than .001). Ipsilateral spinal motor neurons also appeared clustered within the spinal cord and had thicker dendrites. Dorsal root ganglion cells with regenerated axons were slightly larger than control cells at 10 months postcrush but they exhibited no other morphological changes. The present findings indicate that spinal motor neurons are progressively altered after their regenerated axons have reestablished functional synapses with their peripheral targets.     

Imaging neuronal and axonal degeneration in multiple sclerosis

Neurological Sciences - Neuronal and axonal damage has become an important issue in multiple sclerosis. This has been emphasised by recent magnetic resonance imaging (MRI) studies that have shown...     

White-matter astrocytes, axonal energy metabolism, and axonal degeneration in multiple sclerosis

In patients with multiple sclerosis (MS), a diffuse axonal degeneration occurring throughout the white matter of the central nervous system causes progressive neurologic disability. The underlying mechanism is unclear. This review describes a number of pathways by which dysfunctional astrocytes in MS might lead to axonal degeneration. White-matter astrocytes in MS show a reduced metabolism of adenosine triphosphate-generating phosphocreatine, which may impair the astrocytic sodium potassium pump and lead to a reduced sodium-dependent glutamate uptake. Astrocytes in MS white matter appear to be deficient in β(2) adrenergic receptors, which are involved in stimulating glycogenolysis and suppressing inducible nitric oxide synthase (NOS2). Glutamate toxicity, reduced astrocytic glycogenolysis leading to reduced lactate and glutamine production, and enhanced nitric oxide (NO) levels may all impair axonal mitochondrial metabolism, leading to axonal degeneration. In addition, glutamate-mediated oligodendrocyte damage and impaired myelination caused by a decreased production of N-acetylaspartate by axonal mitochondria might also contribute to axonal loss. White-matter astrocytes may be considered as a potential target for neuroprotective MS therapies.     

Recruitment of activated microglia cells in the spinal cord of mice by ALS IgG

Mice were injected i.p. with IgG samples of different patients to test whether IgG from amyotrophic lateral sclerosis (ALS) can initiate an immune/inflammatory reaction targeting motor neurons. All IgG samples of five ALS patients and none of the disease controls recruited activated microglia cells in the ventral horn of the spinal cord. CD3 lymphocytes were not accumulated in the same tissue. Similar reaction was evoked by injection of IgG from guinea pigs with experimental autoimmune gray matter disease (EAGMD) induced by immunization with the homogenate of the ventral horn of bovine spinal cord. The results indicate that ALS IgG and anti-motoneuron IgG induce microglia reaction targeting motor neurons without initiating T cell response in the recipient mice.