Wobbler Spinal Cord Research Articles

The progesterone receptor agonist Nestorone holds back proinflammatory mediators and neuropathology in the wobbler mouse model of motoneuron degeneration

Wobbler mutant mice suffer from progressive motoneuron degeneration and glial cell reactivity in the spinal cord. To prevent development of these abnormalities, we employed Nestorone, a high-affinity progesterone receptor agonist endowed with neuroprotective, promyelinating and anti-inflammatory activities in experimental brain ischemia, preventing neuroinflammation and chemical degeneration. Five-month-old Wobbler mice (wr−/wr−) received s.c. injections of 200μg/day/mouse of Nestorone in vegetable oil or vehicle for 10days. Control NFR/NFR mice (background strain for Wobbler) received vehicle only. Vehicle-treated Wobblers showed typical spinal cord abnormalities, such as vacuolated motoneurons, decreased immunoreactive choline-acetyltransferase, decreased expression of glutamine synthase (GS), increased glial fibrillary acidic protein-positive (GFAP) astrogliosis and curved digits in forelimbs. These cell-specific abnormalities were normalized in Nestorone-treated Wobblers. In addition, vehicle-treated Wobblers showed Iba1+ microgliosis, high expression of the microglial marker CD11b mRNA and up-regulation of the proinflammatory markers TNFα and iNOS mRNAs. In Nestorone-treated Wobblers, Iba1+ microgliosis subsided, whereas CD11b, TNFα and iNOS mRNAs were down-regulated. NFκB mRNA was increased in Wobbler spinal cord and decreased by Nestorone, whereas expression of its inhibitor IκBα was increased in Nestorone-treated Wobblers compared to control mice and vehicle-treated Wobblers. In conclusion, our results showed that Nestorone restraining effects on proinflammatory mediators, microgliosis and astrogliosis may support neurons in their resistance against degenerative processes.

The ESCRT-deubiquitinating enzyme USP8 in the cervical spinal cord of wild-type and Vps54-recessive (wobbler) mutant mice

Usp8 is a deubiquitinating enzyme that works as regulator of endosomal trafficking and is involved in cell proliferation. "In vivo" USP8 is predominantly expressed in the central nervous system and testis, two organs with highly polarized cells. Considering that neuronal cell functionality is strictly dependent on vesicular traffic and ubiquitin-mediated sorting of the endocytosed cargo, it could be of relevance to investigate about USP8 in neuronal cells, in particular motor neurons. In this study, we found that USP8 is expressed in the gray and white matter of the spinal cord, labeling neuronal cell bodies, axonal microtubules and synaptic terminals. The glia component is essentially USP8-immunonegative. The partial colocalization of USP8 with EEA1 in motor neurons indicates that USP8 is involved in early endosomal trafficking while that with Vps54 suggests an involvement in the retrograde traffic. The variant Vps54(L967Q) is responsible for the wobbler phenotype, a disorder characterized by motor neuron degeneration. We searched for USP8/Vps54 in wobbler spinal cord. The most worth-mention result was that wobbler oligodendrocytes, in contrast to the wild-type, are heavily USP8-immunoreactive; no significant modification was appreciated about the cellular expression of mutated Vps54. On the other hand, as to the neuronal intracellular localization, both USP8 and Vps54(L967Q) did not show the typical spot-like distribution, but seemed to accumulate in proteinaceous aggregates. Collectively, our study suggests that in neuronal cells USP8 could be involved in endosomal trafficking, retrograde transport and synaptic plasticity. In disorders leading to neurodegeneration USP8 is upregulated and could influence the neuron-oligodendrocyte interactions.

Wobbler mice modeling motor neuron disease display elevated transactive response DNA binding protein

Wobbler mice model motor neuron disease with a substantial decline in motor neurons. TDP-43 is a nucleic acid binding protein that accumulates, along with ubiquitin, in the cytoplasm of amyotrophic lateral sclerosis (ALS) motor neurons. Recently, it was reported that Cu/Zn superoxide dismutase type 1 (SOD1) familial amyotrophic lateral sclerosis (fALS) model mice do not mimic the TDP-43 changes seen in sporadic ALS, although they share a large number of other properties with the human disorder. We examined ubiquitin inclusions and TDP-43 expression in wobbler mice. TDP-43 mRNA, measured by quantitative reverse transcription–coupled PCR, was elevated in the wobbler spinal cord. Immunohistochemistry revealed intracellular ubiquitin inclusions and abnormal distribution of TDP-43 into the cytoplasm in wobblers similar to the staining reported in ALS. Finally, nuclear and cytoplasmic fractions, examined by Western immunoblotting, confirmed a delocalization of TDP-43 in the neurodegenerative wobbler. These observations indicate that wobbler mice, which suffer motor neuron loss at 21 days, undergo TDP-43 and ubiquitin changes characteristic of sporadic ALS.

Aberrant δPKC activation in the spinal cord of Wobbler mouse: a model of motor neuron disease

Protein kinase C (PKC) was suggested to play a role in the pathology of amyotrophic lateral sclerosis (ALS) patients. Activation of PKC delta (deltaPKC) modulates mitochondrially induced apoptosis. The goal of the present study was to define whether deltaPKC activation occurs in Wobbler mouse spinal cord (a model of motor neuron disease). The level of deltaPKC in the soluble fraction was significantly decreased in the spinal cord of Wobbler mice, which was associated with a significant increase in deltaPKC cleavage. Since caspase-3 is known to cleave deltaPKC, we determined caspase-3 activation in the Wobbler mice spinal cord, immunohistochemically. The results demonstrated intense immunoreactivity for activated caspase-3 in corticospinal tract motor neurons of Wobbler mice spinal cord. We hypothesize from these results that caspase-3 activation cleaves deltaPKC, which in turn promotes an aberrant signal transduction pathway in the Wobbler spinal cord.

Early mitochondrial dysfunction occurs in motor cortex and spinal cord at the onset of disease in the Wobbler mouse

The Wobbler mouse is recognized as an animal model for motoneuron disease that exhibits motoneuron pathology. We have recently demonstrated the occurrence of mitochondrial dysfunction in the Wobbler mouse brain. The aim of the present study was to evaluate whether mitochondrial dysfunction occurred at an early age at the time where disease symptoms appear, and whether it was more pronounced in the motor cortex or in the spinal cord. We report here a significant decrease in mitochondrial state 3 and 4 respiration rates at an early age in the Wobbler spinal cord. In addition, there was a pronounced decrease in oxidative phosphorylation in mitochondria isolated from both spinal cord and motor cortex in both age groups. This mitochondrial dysfunction was accompanied by impairment of complex I activity in mitochondria isolated from spinal cord at an early age. Decreases in complex III and IV activities were observed only in mitochondria isolated from the motor cortex at an early age, but impairment of complex III activity prevailed until later in the disease. We conclude that mitochondrial dysfunction ensues at an early stage of the disease and is more pronounced in the spinal cord, which correlates with previous studies that reported degeneration of spinal cord motorneurons.

Early defect in the expression of mouse sperm DnaJ 1, a member of the DNAJ/heat shock protein 40 chaperone protein family, in the spinal cord of the wobbler mouse, a murine model of motoneuronal degeneration

Prevention of protein misfolding is ensured by chaperone proteins, including the heat shock proteins (HSP) of the DNAJ/HSP40 family. Detection of abnormal protein aggregates in various neurodegenerative diseases has led to the proposal that altered chaperone activity contributes to neurodegeneration. Msj-1, a DNAJ/HSP40 protein located around the spermatozoa acrosome, was recently found to be down-regulated in the testis of wobbler mutant mice. Wobbler is an unidentified recessive mutation which triggers progressive motoneuron degeneration with abnormal intracellular protein accumulations, and defective spermatozoa maturation. Here, we examined Msj-1 expression in the spinal cord of the mutants and their controls. Msj-1 transcripts were amplified by reverse transcription-polymerase chain reaction from mutant and wild-type spinal cord RNA. Sequencing of Msj-1 coding region revealed no change in the mutant. In contrast, decreased Msj-1 mRNA levels were observed in five to six-week-old wobbler mice spinal cord, when motoneuron degeneration is at its apex, as compared to controls. A similar decrease was observed in two-week-old wobbler spinal cord, when the number of motoneurons is still unaltered, indicating that the decreased mRNA content is intrinsic to the mutant and not simply related to the loss of cells expressing Msj-1. Assays of Msj-1 protein levels yielded similar results. Immunofluorescent labeling revealed numerous Msj-1-ir motoneurons in five-week-old control spinal cord while no signal was observed in age-matched wobbler. Our results show, therefore, that Msj-1 expression is down-regulated in both organs affected by the wobbler mutation, the CNS and the testis, and that this defect precedes the first histological signs of motoneuron degeneration. These results provide the first example of an association between transcriptional repression of a chaperone protein and a neurodegenerative process.

Specific features of chronic astrocyte gliosis after experimental central nervous system (CNS) xenografting and in Wobbler neurological mutant CNS

This study sets out to compare and contrast the astrocyte reaction in two unrelated experimental designs both resulting in marked chronic astrogliosis and natural motoneuron death in the wobbler mutant mouse and brain damage in the context of transplantation of xenogeneic embryonic CNS tissue into the striatum of newborn mice. The combined use of GFAP-labeling and confocal imaging allows the morphological comparison between these two different types of astrogliosis. Our findings demonstrate that, in mice, after tissue transplantation in the striatum, gliosis is not restricted to the regions of damage: it occurs not only near the site of transplantation, the striatum, but also in more distant regions of the CNS and particularly in the spinal cord. In the wobbler mutant mouse, a strong gliosis is observed in the spinal cord, site of motoneuronal cell loss. However, moderate astrocytic reaction (increased GFAP-immunoreactivity) can also be found in other wobbler CNS regions, remote from the spinal cord. In the wobbler ventral horn, where neurons degenerate, the hypertrophied reactive astrocytes exhibit a dramatic increase of glial fibrils and surround the motoneuron cell bodies, occupying most of the motoneuron environment. The striking and specific presence of hypertrophic astrocytes in wobbler mice accompanied by a dramatic increase of glial fibrils located in the vicinity of motoneuron cell bodies suggests that short astrogliosis fills the space left by degenerating motoneurons and interferes with their survival. In the spinal cord of xenografted mice, chronic astrogliosis is also observed, but only glial processes without hypertrophied cell bodies are found in the neuronal micro-environment. It is tempting to speculate that gliosis in the wobbler spinal cord, the local accumulation of astrocyte cell bodies, and high density of astrocytic processes may interfere with the diffusion of neuroactive substances in gliotic tissue, some of which are neurotoxic, and cooperate or even trigger neuronal death.

Cellular basis of steroid neuroprotection in the wobbler mouse, a genetic model of motoneuron disease.

1. The Wobbler mouse suffers an autosomal recessive mutation producing severe motoneuron degeneration and astrogliosis in the spinal cord. It has been considered a suitable model of human motoneuron disease, including the sporadic form of amyotrophic lateral sclerosis (ALS). 2. Evidences exist demonstrating increased oxidative stress in the spinal cord of Wobbler mice, whereas antioxidant therapy delayed neurodegeneration and improved muscle trophism. 21-Aminosteroids are glucocorticoid-derived hydrophobic compounds with antioxidant potency 3 times higher than vitamin E and 100 times higher than methylprednisolone. They do not bind to intracellular receptors, and prevent lipid peroxidation by insertion into membrane lipid bilayers. 3. In common with the spinal cord of ALS patients, Wobbler mice present astrocytosis with hyperexpression of glial fibrillary acidic protein (GFAP), and increased expression of nitric oxide synthase (NOS) and growth-associated protein (GAP-43) in motoneurons. Here, we review our studies on the effects of a 21-aminosteroid on GFAP, NOS, and GAP-43. 4. First, we showed that 21-aminosteroid treatment further increased GFAP-expressing astrocytes in gray matter of the Wobbler spinal cord. This effect may provide neuroprotection if one considers a trophic and beneficial function of astrocytes during the course of degeneration. Other neuroprotectans used in Wobbler mice (T-588) also increased pre-existing astrocytosis. 5. Second, histochemical determination of NADPH-diaphorase, a parameter indicative of neuronal NOS activity, showed that the 21-aminosteroid down-regulated the high activity of this enzyme in ventral horn motoneurons. Therefore, suppression of nitric oxide by decreasing NADPH-diaphorase (NOS) activity may provide neuroprotection considering that excess NO is highly toxic to motoneurons. 6. Finally, 21-aminosteroid treatment significantly attenuated the aberrant expression of both GAP-43 protein and mRNA in Wobbler motoneurons. Hyperexpression of GAP-43 possibly indicated abnormal synaptogenesis, denervation, and muscle atrophy, parameters which may return to normal following antioxidant steroid treatment. 7. Besides 21-aminosteroids, other steroids also behave as neuroprotectans. In this regard, degenerative diseases may constitute potential targets of these hormones, based on the fact that the spinal cord expresses in a regional and cell-specific fashion, receptors for androgens. progesterone, adrenal steroids, and estrogens.

Excess extracellular and low intracellular glutamate in poorly differentiating wobbler astrocytes and astrocyte recovery in glutamine-depleted culture medium.

The wobbler mouse develops an inherited motoneuronal degeneration of unknown origin in the spinal cord. Primary cultures of adult wobbler spinal cord astrocytes display abnormal morphological characteristics with fewer processes and paucity of cell-cell contacts. We have searched for a possible involvement of glutamate and glutamine intra- and extracellular accumulations in vitro in the abnormal differentiation of mutant astrocytes. We have found significantly higher glutamate and glutamine concentrations in the culture media of mutant astrocytes over a 3-day period compared with normal control astrocytes. Moreover, intracellular glutamate concentrations decreased substantially in mutant astrocytes, but intracellular glutamine concentrations remained unchanged. Furthermore, decreasing initial glutamine concentrations in the culture medium (glutamine-depleted medium) led to the recovery of normal extra- and intracellular concentrations of glutamate and recovery of quasi-normal morphological differentiation and increased cell-cell contacts, leading to an essentially normal looking astrocyte network after 3 days of culture. Under these conditions, which lead to recovery, the only remaining abnormality was the higher glutamine extracellular concentration attained in the originally depleted glutamine media. These findings suggest that mechanisms regulating glutamate/glutamine synthesis and/or influx/efflux are defective in wobbler astrocytes, leading to metabolic imbalance and possible cytotoxic effects characterized by disturbed intercellular networks and poor differentiation.

Astrocytosis in wobbler mouse spinal cord involves a population of astrocytes which is glutamine synthetase-negative

Mice affected by the wobbler mutation are characterized by a muscular atrophy associated with motoneuron degeneration. As soon as the first clinical signs of the disease appear, reactive astrocytes, strongly glial fibrillary acidic protein (GFAP)-positive, are observed in the spinal cord grey matter. They become prevalent at all levels with disease progression. Immunostaining of glutamine synthetase (GS) shows that these reactive astrocytes are never GS-positive. The activity and protein amounts of GS remain normal in wobbler spinal cord although astrocytosis develops. Thus, gliosis in the wobbler mouse seems to involve a subpopulation of astrocytes, which is strongly GFAP-positive but GS-negative.

Quantitative autoradiographic distribution of glutamate receptors in the cervical segment of the spinal cord of the wobbler mouse

The reduction of glutamate content has been observed in the spinal cord of the wobbler mouse, a purported model of amyotrophic lateral sclerosis (ALS). To elucidate glutamate receptors in the wobbler spinal cord, we measured densities of N-methyl- d-aspartate (NMDA), kainate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate (mGlu) binding sites using in vitro autoradiography. In wobbler mice, NMDA, kainate, and AMPA binding sites were increased in the dorsal horn and kainate binding sites were also increased in the intermediate zone. However, mGlu binding was unchanged. These results disagree with those observed in ALS spinal cords, in which NMDA and kinate binding sites are decreased. The wobbler mouse may have the glutamate dysfunction, but in a different way from ALS.

Abnormal astrocyte differentiation and defective cellular interactions in wobbler mouse spinal cord

The wobbler mutation is inherited as an autosomal recessive trait and displays a muscular atrophy associated with motoneuron degeneration in early postnatal development. It has been shown that the level of glial fibrillary acidic protein (GFAP) is greatly increased in the spinal cord of wobbler mice. We performed immunocytochemical analyses combined with confocal microscopy to study the developmental distribution of GFAP-positive astrocytes in the spinal cord of wobbler mice during the course of the disease, and in primary cultures of adult wobbler spinal cord astrocytes. Many changes in the number and distribution of astrocytes were observed in the wobbler mice from 1-10 months post-partum. Strongly GFAP-positive astrocytes are present in small number in the anterior horn by 1 month. They increase in number and are observed in the entire spinal cord grey and white matters by 2-10 months. These reactive astrocytes have thick, short, extensively branched processes which contrast with the long, unbranched processes observed in control mice. The wobbler astrocyte processes are oriented perpendicular to the surface of the spinal cord, which contrasts with the normal parallel, concentric orientation. No expansion of astrocyte processes exit from the white matter towards the grey matter. Moreover, the surface of the wobbler spinal cord beneath the meninges displays a dramatic decrease of interdigitating processes, end feet and flattened cell bodies of astrocytes that form a disorganized layer. In vitro, mutant astrocytes have morphological characteristics similar to those in vivo and, in particular, develop short, thick, branched processes. These mutant astrocytes in cultures do not contact one another, whereas normal mature cultures show an increased incidence of cell-cell contacts between long processes. The increase of astrocyte reactivity associated with these modifications in astrocytic process arrangement may reflect an important primary event in the course of the wobbler disease rather than a non-specific response to motoneuronal death.

Alteration in the levels of thyrotropin releasing hormone, substance p and enkephalins in the spinal cord, brainstem, hypothalamus and midbrain of the wobbler mouse at different stages of the motoneuron disease

The present study was undertaken to quantify selected neuropeptides (thyrotropin releasing hormone, substance P, methionine and leucine enkephalin) in the cervical spinal cord and other regions of the central nervous system of Wobbler mice by radioimmunoassays during several stages of the motoneuron disease compared with age- and sex-matched normal phenotype littermates. In Wobbler spinal cord, thyrotropin releasing hormone is higher early in the disease, whereas in the brainstem it is higher at a later stage. Substance P in spinal cord is also higher late in the disease. Leucine enkephalin levels are greater at all stages in diseased spinal cord and brainstem, but methionine enkephalin increases only late in the disease. Highly significant increases of the peptides (except thyrotropin releasing hormone) appear in hypothalamus and midbrain only late in the motoneuron disease. Regression analyses show that thyrotropin releasing hormone in spinal cord and brainstem decreases normally with age in the control mice and at a faster rate related to the extent of motor impairment in Wobbler mice. Thyrotropin releasing hormone and methionine enkephalin in the Wobbler brainstem correlate ( P < 0.05) with the progress of the motoneuron disease. Methionine enkephalin increases faster in Wobbler brainstem and decreases faster in control spinal cord with age. The increase of leucine enkephlin in the Wobbler spinal cord correlates significantly with age and with the progress of the disease, but leucine enkephalin declines slightly with age in the controls. The changes of substance P in spinal cord and brainstem do not correlate significantly with the progress of the disease. In the hypothalamus, increasing values for substance P in control specimens and enkephalins in Wobbler specimens are significantly correlated with age. However, in the midbrain, higher methionine and leucine enkephalin levels are significantly associated with age only in the control mice. Alterations of neuropeptides in the Wobbler mouse spinal cord and brainstem may result from the degeneration of bulbospinal raphe neurons projecting to the ventral spinal cord, or from primary afferent or interneuronal nerve terminals. The data imply that the neuronal degeneration process in the Wobbler motoneuron disease is not limited to motoneurons. In the spinal cord, the data support our previous hypothesis that neuronal sprouting presynaptic to the motoneurons may account for increased neuropeptide concentrations. Alternatively, synthesis and/or degradation of these peptides may be altered. In addition, it is proposed that enkephalinergic neurons may develop abnormally in Wobbler mice. The early increase of leucine enkephalin in the Wobbler spinal cord possibly indicates its importance in the etiology of the motoneuron disease.