Maria Claudia Gonzalez Denisellea, Juan José Lopez Costab, Susana L. Gonzaleza, Florencia Labombardaa, Laura Garaya, Rachida Guennounc, Michael Schumacherc, Alejandro F. De Nicolaa,✲
a Laboratory of Neuroendocrine Biochemistry, Department of Human Biochemistry, Faculty of Medicine, Instituto de Biologiay Medicina Experimental, University of Buenos Aires, Obligado 2490, 1428 Buenos Aires, Argentina; b Faculty of Medicine, Instituto de Biologia Celular y Neurociencias “Prof. E. De Robertis”, University of Buenos Aires, Buenos Aires, Argentina; c INSERM U488, Kremlin-Bicêtre, France.
☆ Proceedings of the 15th International Symposium of the Journal of Steroid Biochemistry and Molecular biology, “Recent Advances in Steroid Biochemistry and Molecular Biology”, Munich, Germany, 17-20 May 2002.
✲Corresponding author. Tel.: +54-11-4783-2869; fax: +54-11-4786-2564 A.F. De Nicola; E-mail: denicola@dna.uba.ar.
Abstract
Progesterone neuroprotection has been reported in experimental brain, peripheral nerve and spinal cord injury. To investigate for a similar role in neurodegeneration, we studied progesterone effects in the Wobbler mouse, a mutant presenting severe motoneuron degeneration and astrogliosis of the spinal cord. Implant of a single progesterone pellet (20 mg) during 15 days produced substantial changes in Wobbler mice spinal cord. Morphologically, motoneurons of untreated Wobbler mice showed severe vacuolation of intracellular organelles including mitochondria. In contrast, neuropathology was less pronounced in Wobbler mice receiving progesterone, together with a reduction of vacuolated cells and preservation of mitochondrial ultrastructure. Determination of mRNAs for the α3 and β1 subunits of neuronal Na, K-ATPase, showed that mRNA levels in untreated mice were significantly reduced, whereas progesterone therapy re-established the expression of both subunits. Additionally, progesterone treatment of Wobbler mice attenuated the aberrant expression of the growth-associated protein (GAP-43) mRNA which otherwise occurred in motoneurons of untreated animals. The hormone, however, was without effect on astrocytosis of Wobbler mice, determined by glial fibrillary acidic protein (GFAP)-immunostaining. Lastly, progesterone treatment of Wobbler mice enhanced grip strength and prolonged survival at the end of the 15-day observation period. Recovery of morphology and molecular motoneuron parameters of Wobbler mice receiving progesterone, suggest a new and important role for this hormone in the prevention of spinal cord neurodegenerative disorders. © 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Progesterone; Neurodegeneration; Wobbler mouse; Spinal cord.
Introduction
Functions of female steroid hormones in the central nervous system (CNS) were classically associated to repro-ductive processes and sexual behavior. However, alternative roles for these hormones were increasingly discovered, in particular effects contributing to neuroprotection by modulation of gene expression and intracellular signaling pathways in areas of the CNS other than those related to endocrine functions [1-4]. In the case of estrogens, protective and neurotrophic effects are well documented using in vitro preparations, whole animal experiments and in patients with Alzheimer’s disease [2,5-7]. Estradiol combined with progesterone also showed neuroprotective effects in an animal model of neurodegeneration [8].
In addition to estrogens, single progesterone treatment produced protective and regenerative effects in models of injury [3]. For instance, progesterone stimulated myelino-genesis of injured peripheral nerves [9,10] and prevented neuronal loss following brain contusion, ischemia and edema [11-13]. In the spinal cord, treatment of rats with progesterone increased motoneuron survival after axotomy or injury [14,15], protected cultured neurons against glu-tamate toxicity [16] and normalized defective functional parameters of injured motoneurons [17]. The presence of a progesterone receptor in peripheral nerves and central nervous system, suggested that neuroprotection may be due to transcriptional events following receptor interaction with hormone-responsive elements in DNA. However, non-genomic actions should not be disregarded. In this sense, progesterone binding to neurotransmitter receptors, to the membrane protein 25-Dx and inhibition of membrane lipid peroxidation may play a crucial role in steroid effects [12,18]. However, while the bulk of progesterone neuropro-tection was reported in central or peripheral nervous system trauma, demonstrations in neurodegeneration models are generally scarce [8].
The Wobbler mouse, an animal model of spinal cord neurodegeneration
A prototype neurodegenerative disease in humans is amy-otrophic lateral sclerosis (ALS), a devastating illness affecting motoneurons that innervate voluntary muscles. Ninety five % of ALS patients do not report a family history of the disease, for which reason they belong to the “sporadic form”, while the remaining 5% present the “familial form”, transmitted by a dominant gene. In both forms, however, surviving time is limited to about 5 years [19]. The drama imposed by ALS is due to its fatal outcome and the minor effectiveness of available treatments, making animal models useful tools to explore therapeutic alternatives for human motoneuron diseases.
The Wobbler mouse is a genetic model of spinal cord neurodegeneration. In homozygous Wobbler mice, a mutation of autosomic recessive expression (wr) produces motor neuron loss and astrocyte reactivity in the spinal cord and brain stem [20]. Although the wr gene has not been identified, it maps to chromosome 11 close to the glutamine synthetase gene [21]. Wobblers become useful models to study amyotrophic lateral sclerosis (ALS) and infantile spinal muscular atrophy (Werdenig-Hoffman disease) [19,22].
Former morphological studies of anterior horn cells of Wobbler mice spinal cord found a dramatic perikaryal vacuolar degeneration, in addition to swelling of α and γ motor neurons, interneurons and Renshaw cells [23-25]. This change was accompanied by pronounced astrocytosis [26,27] and increased density of activated microglia [28]. Whereas motoneurons in 3-4-week-old Wobbler mice are TUNEL negative [29,30] a transient massive DNA fragmen-tation of neurons and glial cells was reported by Blondet et al. [31] before the manifestation of clinical symptoms. Therefore, while apoptosis may cause cell death in the early stages, motoneuron pathology of symptomatic mice resem-bled the type II or cytoplasmic form of cell death described by Clarke [32] and attributed to increased oxidative stress. In Wobbler mice, participation of oxidative stress is supported by abnormalities of mitochondrial function [33] and the clinical, biochemical and morphological improvement caused by treatment with common antioxidants, antioxidant steroids or nitric oxide (NO) inhibitors [27,34-36]. Thus, both oxidative stress and apoptosis acting in a different time sequence may cause cell death in the Wobbler mouse and other motor neuron degeneration models including the superoxide dismutase transgenic mouse [31,37].
Therefore, the Wobbler mouse spinal cord seemed partic- ulary vulnerable to oxidative attack. In this respect, proges-terone treatment was considered a valuable therapeutic tool for several reasons: first, progesterone can diffuse and asso-ciate with phospholipids by intercalation between polyun-saturated fatty acid chains [12,38]. This mechanism caused membrane stabilization and attenuated injury-induced lipid peroxidation [12]. Second, progesterone itself can behave as a free-radical scavenger [39]. Although important, inhibition of lipid peroxidation may not be an exclusive mechanism of progesterone action in the spinal cord, since detection of an intracellular progesterone receptor capable of transcriptional regulation was reported in this tissue [40,41]. Therefore, functional multiplicity and not a single form of steroid action may account for progesterone effects in the Wobbler mouse.
In our experiments, we used 3-month-old homozygous Wobbler mice (wr/wr) (The Animal Center of the National Institutes of Health, Bethesda, MD, USA), representing stages 3-4 of the disease according to the criteria of Yung et al. [42]. All affected animals showed reduced body weight; and clinically presented tremor, ambulatory difficulty, flexion of proximal limbs, distal limb extension, positive clasp knife reflex response and diminished muscle strength. Routine histology showed motor neuron loss and astrogliosis of the spinal cord [27]. A group of Wobbler mice remained untreated, whereas another group received under the skin of the neck a 20 mg pellet of progesterone under light ether anesthesia. Animals were used 15 days after steroid pellet implantation. The efficiency of this treatment was checked by measuring progesterone levels by gas chromatography/mass-spectrometry [43], which demon-strated that progesterone pellet implantation resulted in 10-fold higher hormone levels in the cervical spinal cord compared to untreated Wobbler mice.
Parameters measured in motoneurons and astrocytes
The reported success of progesterone treatment in nervous system trauma, decided us to investigate for similar effects in Wobbler mouse motor neuron degeneration. On these premises, several objectives were pursued: first, to establish in 3 months old, clinically affected Wobbler mice if progesterone treatment modified the existing neuropathol-ogy, assessed by light and electron microscopy. Second, to explore if progesterone modulated some parameters im-portant for motoneuron function, including the mRNAs for the α3 and β1 subunits of the Na, K-ATPase and the growth-associated protein (GAP-43). The Na, K-ATPase is an essential enzyme, playing a pivotal role in neurotransmission, nutrient uptake and maintenance of membrane potential [44,45]. In addition, it showed sensitivity to progesterone treatment after spinal cord injury [17]. In contrast to the high expression of the Na, K-ATPase in mature neurons, GAP-43 is expressed only by embryonic cells [46]. Considering that GAP-43 protein and mRNA are both aberrantly expressed in Wobbler mice, its regulation might have important implications for the fate of degenerating motoneurons [46]. Third, to analyze whether progesterone treatment regulated the pre-existing spinal cord astrocytosis of Wobbler mice. This may be an important issue, in view of the fact that astrocytes have a protective role for motoneu-ron function and disease outcome [47-49]. The last aim was based on previous demonstrations of increased expression of the astrocyte glial fibrillary acidic protein (GFAP) following treatment of Wobbler mice with the neuroprotectant and antioxidant steroid U-74389F [50]. Fourth, we studied the effects of progesterone on muscle strength and survival time of Wobbler mice in order to extrapolate to the in vivo situation findings obtained at the morphological and bio-chemical level. As will be shown below, our data suggest that progesterone retarded neurodegeneration of Wobbler mice spinal cord and in this context, it may be potentially valuable for treatment of human neurodegenerative diseases.
Effects of progesterone treatment on Wobbler mice neuropathology: an electron microscopic study
Light microscopy demonstrated an increased frequency of vacuolated motor neurons in the ventral horn of Wobbler mice, in agreement with the finding of others [23-25]. The effect of progesterone was quantitatively evaluated using computerized image analysis, which demonstrated that the total number of vacuolated neurons per unit area found in untreated Wobblers ((18 ± 2.2) x 10-5 μm-2) was significantly reduced in the group of Wobbler mice receiving progesterone ((11 ± 0.8) x 10-5 μm-2, P < 0.05). Although the origin of these vacuoles was uncertain, electron microscopy suggested that vacuolization derived from de-generating mitochondria, cysternae of smooth and rough endoplasmic reticulum and Golgi apparatus. In Wobbler mice receiving progesterone, neuronal degeneration was moderate. In the perinuclear area, few vacuolae remained and rough endoplasmic reticulum was largely preserved. The shape of the nuclear membrane was partly restored and some perinuclear mitochondria regained a normal appearance.
Considering that abnormal mitochondria may be a source of free radicals which further impaired motoneuron function, we compared mitochondrial ultrastructure in untreated and progesterone-treated Wobbler mice. Fig. 1 (left-hand photomicrograph) shows a massive vacuolation suffered by a mitochondria in a Wobbler mouse motoneuron. The ul-trastructure of the organelle was highly disorganized, with rupture of the outer and inner membranes. Progesterone treatment of Wobbler mice reverted in part these abnor-malities, in that some mitochondria reassumed a normal ultrastructure. As shown in the right-hand photomicrograph of Fig. 1, the outer and inner membranes, cristae and matrix were better preserved in the motoneuron of the progesterone-treated Wobbler mice These observations sug-gested that cytoplasmic and mitochondrial vacuolation, a predominant form of cell death in Wobbler mice motoneuron disease, may be overturned by progesterone. In very few cases, such as the motoneuron of an untreated Wobbler mouse shown in Fig. 2, we detected nuclear chromatin fragmentation suggestive of apoptosis [31]. However, in contrast to the high incidence of motoneuron vacuolation of Wobbler mice, the number of cell nuclei showing chromatin cumpling was very low and similar in both treated and un-treated mutant mice. The absence of apoptotic changes was also evident using agarose gel electrophoresis. In this case, the DNA laddering profile indicative of apoptotic DNA fragmentation, was negative in both control and Wobbler mice spinal cords (Fig. 3, CTL and WR). As a positive control we used the thymus from normal mice receiving two s.c. injections of 0.5 mg dexamethasone 2 and 16 h before killing. In this case, a DNA ladder typical of apoptosis was obtained on the gels (Fig. 3, thymus + DEX).
Figure 1. Electron microscopy of Wobbler mice ventral horn motoneurons. The left-hand photomicrograph (A) shows a degenerating motoneuron from an untreated Wobbler mouse with massive vacuolation disrupting the outer, inner mitochondrial membranes and cristae. The right-hand photomicrograph (B) corresponds to a Wobbler mouse receiving progesterone, showing that mitochondrial membrane system, including the cristae, are better conserved. Magnification: 50,000 x.
Figure 2. Apoptoptic features in a motoneuron. Nuclear chromatin fragmentation, as exemplified in this photomicrograph, is observed with very low frequency in 3-month-old symptomatic Wobbler mice. In these mice, cytoplasmic and mitochondrial vacuolation of the type exemplified in Fig. 1 constituted the main ultrastructural abnormalities. Magnification: 12,000 x.
Figure 3. DNA analysis by agarose gel electrophoresis. Genomic DNA was extracted from the spinal cord of control mice (CTL), spinal cord of Wobbler mice (WR) and thymus of normal mice receiving 0.5mg dexamethasone 2 and 16h previous to killing (thymus + DEX). The bands on the left correspond to DNA markers. DNA laddering was absent from control and Wobbler mice spinal cords, but multiple bands were detected in the gel corresponding to the thymus of glucocorticoid-exposed mice run as a positive control.
Effects of progesterone on neuronal Na, K-ATPase subunit mRNA
The effects of progesterone treatment on the α3 and β1 subunit mRNAs for the neuronal NA, K-ATPase was studied in control littermates and Wobbler mice. In these exper-iments, 35S-labeled oligonucleotide probes with sequences coding for the α3 subunit and the β1 subunit mRNA were used in conjunction with in situ hybridization procedures [51,52]. The chosen oligonucleotides produced strong hybridization signals in ventral horn motoneurons without labeling of glial cells [51,52].
The hormone’s effect was determined in Wobbler mice subjected to 15-day steroid exposure as well as in untreated Wobbler mice and age-matched controls. In this experiment, neurons measuring >300 μm2 localized in Lamina IX were considered to be a motoneurons based on anatomical lo-cation and size. Control motoneurons showed a substantial grain density representing oligonucleotide probe hybridized to neuronal α3 or β1 subunits of Na, K-ATPase mRNA. This pattern contrasted with the grain depletion in motor neurons from Wobbler mice. Following 15 days of progesterone ex-posure, the hybridization signal in Wobbler mice was higher than in untreated Wobblers. The lower part of Fig. 4 shows the expression of the β1 subunit by motor neurons of control mice (A, left-hand graph), the grain depletion of untreated Wobbler mice (B, center graph) and the recovered mRNA levels in a Wobbler mouse treated with progesterone (C, right-hand photomicrograph). Quantitative analysis of ISH data (Fig. 4) further confirmed in untreated Wobbler mice a reduction in the expression of the β1 subunit mRNA for the Na, K-ATPase (41 ± 2.7 grains/100 μm2) when compared to control animals (52 ± 2.8, P < 0.05). In Wobbler mice treated with progesterone, grain density was higher than in untreated Wobblers (55 ± 3.5, P < 0.05), whereas there were non-significantly differences with controls. In addition to its effect on the β1 subunit, progesterone treatment also restored the reduction of α3 Na, K-ATPase mRNA expression shown by Wobbler mice in ventral horn motor neurons (results not shown).
Figure 4. In situ hybridization for the β1 subunit mRNA of the Na, K-ATPase in the spinal cord. Upper graph: quantitative analysis of grain density in control mice (a: open column), controls receiving progesterone (b: cross-hatched column), untreated Wobbler mice (c: filled column) and steroid-treated Wobbler mice (d: dotted column). Significance—a vs. c: P < 0.05; c vs. d: P < 0.05) (ANOVA and Newman-Keuls’s test). Lower graph: dark field photomicrographs of a control mouse (A, left-hand graph), Wobbler mouse (B, center graph) and a Wobbler mouse treated with progesterone (C, right-hand graph). Magnification: 100 x.
Attenuation of GAP-43 mRNA expression by progesterone treatment of Wobbler mice
A protein with abnormal expression in Wobbler spinal cord (as well as in patients with ALS) is the growth- associated protein or GAP-43. In the mRNA studies, a synthetic 35S-labeled oligonucleotide complementary to mouse GAP-43 mRNA [53] was hybridized to spinal cord sections. Computer-assisted image analysis was used to quantitate the number of grains per cell, a measurement proportional to the amount of cytoplasmic mRNA according to Mitsumoto and Gambetti [54]. GAP-43 mRNA was almost undetectable in control mice motoneurons, in contrast with the intense expression in Wobbler mice spinal cord, as shown in the bright field and dark field photomicrographs of Fig. 5. As also observed in Fig. 5, progesterone treatment depressed the aberrant expression of Wobbler mouse GAP-43 mRNA.
Figure 5. Dark field photomicrographs representing the low expression of GAP-43 mRNA in motoneurons of a normal mouse (A, upper graph), the high expression of this mRNA in an untreated Wobbler mouse (B, middle graph) and the attenuation of GAP-43 mRNA in a Wobbler mouse receiving progesterone (C, lower graph). Magnification 200 x.
Quantitative analysis confirmed the very low levels of GAP-43 mRNA in control mice spinal cord, which was un-affected by progesterone treatment. In Wobbler mice not receiving steroid therapy, the average number of grains per cell (85 ± 2.6) was significantly higher than controls levels (8 ± 1.4 , P < 0.001). Fifteen days after progesterone pellet implant, although GAP-43 mRNA in Wobbler mice (63±2.8) was still higher than in controls, it was significantly reduced respect of untreated Wobbler mice (P < 0.01).
Astrocytosis of Wobbler mice spinal cord was unaffected by progesterone
Wobbler mice present in common with ALS patients as- trogliosis in gray and white matters of the spinal cord, primary motor cortex and subpial regions [26]. The reactive astrocytes found in Wobbler and ALS spinal cord expressed high levels of GFAP [55]. Some authors consider this event a secondary response to neuronal illness, in which case transforming growth factor a could act as the neuronal inducer of astrogliosis [56]. Previously, we found that treatment of Wobbler mice with the antioxidant aminosteroid U-74389F enhanced the existing astrocytosis [50]. This finding encouraged us to search for progesterone effects on GFAP im- munostaining in astrocytes of control and Wobbler mice.
Four groups of animals were studied: controls, controls receiving a single progesterone pellet during 15 days, untreated Wobbler mice and Wobbler mice implanted with progesterone. GFAP expression was carried out in spinal cord slices using a rabbit anti-GFAP polyclonal antibody and routine immunohistochemical procedures [50]. Fig. 6 shows that the number of GFAP-immunoreactive astrocytes/mm2 in the ventral horn of the spinal cord was eight-fold higher in Wobbler compared to control mice (a versus c: P < 0.001). However, progesterone was unable to change the high number of GFAP positive astrocytes in Wobbler mice or modified the low number of cells found in control mice (Fig. 6—a versus b: NS; c versus d: NS).
Figure 6. Number of GFAP-immunoreactive astrocytes/mm2 in the spinal cord of control mice (a: open columns), controls receiving progesterone (b: cross-hatched columns), Wobbler mice (c: filled column), and Wobblers receiving progesterone (d: dotted column). Results represent the mean ± S.E. of n = 5-6 animals per group. Statistical significance—a vs. b: NS; a vs. c: P < 0.001; c vs. d: NS (ANOVA and post hoc Newman-Keuls’s test).
Test of muscle strength and survival of Wobbler mice receiving progesterone
As already mentioned, Wobbler mice develop forelimb paralysis and denervation muscle atrophy. These changes are secondary to degeneration and loss of spinal cord motoneu-rons. In order to study whether progesterone slowed the deterioration of muscle function at the time that prevented motoneuron degeneration, a grip strength test was used [57]. Grip strength was measured by placing mice on a vertical grid and measuring the time in seconds they remained in the grid without falling down. The results showed that untreated Wobbler mice remained in the grid for 5 ± 1.5 s only, whereas Wobbler mice receiving progesterone for 15 days tripled the time spent in the grid (16.4 ± 3.2 s, P < 0.05).
Since Wobbler mice die prematurely, we also studied if progesterone treatment prolonged survival in the mutant mice. In this study, a group of Wobbler mice received a single progesterone pellet, while an age-matched group was left untreated. Observations were prolonged for 15 days after progesterone implantation. Percent survival time in both groups was identical for the first 7 days, after which progesterone-treated Wobbler mice deviated from the un-treated group. At the end of the study period (15 days), a significant difference was measured due to increased survival of Wobbler mice receiving progesterone compared to the untreated mice (P < 0.03).
Discussion
The present experiments investigated progesterone effects in a neurodegeneration model, based on previous evidences for progesterone beneficial effects in ischemia and trauma of the central and peripheral nervous system. The resulting data demonstrated that progesterone treatment of symptomatic Wobbler mice: (1) reverted motoneuron neuropathology ac-cording to light and electron microscopic observations, (2) up-regulated the mRNAs of the α3 and β1 subunits of neu-ronal Na, K-ATPase, (3) reduced the aberrant hyperexpres-sion of GAP-43 mRNA, (4) did not affect the number of GFAP-immunostained astrocytes in the ventral horn, and (5) enhanced muscle strength and prolonged survival of clini-cally ill Wobbler mice.
The prevalent neuropathology found in symptomatic Wobbler mice consisted in motoneurons with cytoplasmic vacuolation and damaged mitochondria but with relative preservation of nuclear ultrastructure. Previous observations using Wobbler mice at stages 3-4 of the disease described similar changes [24,25,29,42,57]. At this stage of the disease, signs of overt apoptosis such as pyknotic nuclei, nuclear membrane fracture, apoptotic bodies and plasma membrane blebbing were unfrequent. In 3-month-old Wobbler mice chromatin clumping was very scarce, although it was found in some animals. In contrast, massive DNA fragmentation reportedly occurred in presymptomatic Wobbler mice [31], although DNA laddering typical of apoptosis was absent in our clinically affected Wobbler mice.
When morphological features of different types of cell death, i.e. apoptosis, necrosis and cytoplasmic cell death were confronted to the present findings, we concluded that neurodegeneration in symptomatic Wobbler mouse resem-bled the last form, also known as type II or the autolytic type [32,58]. In this process, dilatation of smooth endoplasmic reticulum cysternae and Golgi system lead to their autolytic breakdown, as already shown in clinically affected Wobbler mice [29]. Another important ultrastructural observation in untreated Wobbler mice was the presence of vacuolized mitochondria, with cristolisis, edema and lack of outer membrane integrity. At the functional level, a mitochondrial respiratory chain dysfunction was observed in Wobbler mice by Guang-Ping et al. [33]. This group reported that superoxide production, a leading cause of lipid peroxidation may cause motor neuron death. Besides, lipid peroxidation was proposed as a leading causative factor for the cytoplasmic and mitochondrial changes in neurodegeneration [59]. A cytoplasmic form of cell death may also play a role in the mutant superoxide dismutase type 1 (SOD1) transgenic model (G93A and G37R mutations), in which a toxic gain of function of the mutated enzyme led to oxygen radical-induced lipid peroxidation of intracellular membranes and mitochondrial abnormalities [60-62]. Interestingly, spinal cord neuropathology of Wobbler mice bear striking similarities to that of SOD1 transgenics, which also showed extensive vacuolation of mitochondria, Golgi apparatus and endoplasmic reticulum [61,63,64]. Similarly, while apoptotic changes and chromatin clumping were re-ported by some workers in SOD1 transgenic and Wobbler mice [31,65,66], others sustained that motor neuron death occurs primarily by massive vacuolation in the absence of apoptosis [29,67]. Thus, it is possible that at least two mechanisms—apoptosis and cytoplasmic vacuolization— caused motor neuron degeneration in the genetic and transgenic models of ALS, although with a different time course.
Cytoplasmic vacuolization suggests that free radicals produced in neurons or glial cells originated and/or ex-acerbated neurodegeneration of untreated Wobbler mice. Blockage of free radical damage to motor neurons with the antioxidants OPC-14117, n-acetylcysteine, lecythinized superoxide dismutase or T-588 corrected the motor dys-function of Wobbler mice [35,36,68,69]. Excessive pro-duction of nitric oxide is another important risk factor for neurodegeneration, considering the Wobbler mice showed increased activity of nitric oxide synthase (NOS) in ventral horn motoneurons [49]. Also, the NOS inhibitor nitroinda- zol produced beneficial effects [34]. We previously reported that Wobbler mouse spinal cord showed increased number of neurons giving strong histochemical staining for NOS-NADPH-diaphorase [49], whereas treatment with the antioxidant 21-aminosteroid U-74389F reduced this activity and, presumably, nitric oxide (NO) production. Therefore, NO toxicity may contribute to neurodegeneration, considering that after coupling with superoxide anion, NO forms peroxinitrites which damages lipids, proteins and DNA. Whether progesterone treatment interrupted NO generation is unknown but experiments are under way to study this possibility.
Another important finding of the present experiments us-ing symptomatic Wobbler mice was the pronounced reduc-tion of the α3 catalytic subunit and β1 non-catalytic subunit mRNAs of the Na, K-ATPase in motoneurons. Increased lipid peroxidation was reported to dramatically reduce the enzyme activity [70]; whether it also affects mRNA levels is unknown. Conceivably, this reduction could curtail pivotal functions of the enzyme such as Na/K exchange, maintenance of membrane potential, nutrient uptake and neurotransmission [44,45]. In connection with the last property, a reduction of Na, K-ATPase would also impair acetylcholine release at the neuromuscular plate. This might cause further ambulatory difficulty and weakness. Continuous inhibition of this enzyme, as occurred in epilepsy, ischemia, hypoglycemia and aging first caused malfunction and eventually neuronal death [41,71]. Therefore, reduction of the Na, K-ATPase may exacerbate neuropathology, because the enzyme plays an important role for motor neuron function in the spinal cord.
In this scenario, it was highly rewarding that a relatively short course of progesterone treatment partially restored motoneuron morphology and also mRNAs for the Na, K-ATPase α3 and β1 subunits in clinically affected Wobbler mice. Although our experiments did not elucidate the mechanism involved in this effect, direct modulation of the α3 or β1 subunit Na, K-ATPase genes by the progesterone receptor was not previously reported. Nevertheless, a glu-cocorticoid receptor (GR) consensus sequence responsive element was found in both the Na, K-ATPase a and p genes [72,73]. Reportedly, this DNA sequence is common to both GR and the progesterone receptor [74,75], opening the pos-sibility that the Na, K-ATPase gene may be a direct target of progesterone in the degenerating tissue. Using this or an indirect mechanism, increased expression of the mRNA for the Na, K-ATPase subunits could have rescued motor neu-rons from the devastating consequences of degeneration.
Therefore, several neuronal functions would return after re-instating the mRNA for the Na, K-ATPase subunits [44,45], including the reestablishment of cholinergic neurotransmis-sion. Indeed, synaptic transmission at the neuromuscular junction is under progesterone regulation [76].
A protein with abnormal expression in Wobbler spinal cord (as well as in patients with ALS) is the growth- associated protein or GAP-43. Physiologically, this phos- phoprotein is found in high concentrations in embryonic life, during synaptogenesis and in axons regenerating after injury [77]. Usually, the high expression of GAP-43 subsides when growing axons reach their target or myelinize [78]. In the normal adult spinal cord, moderate levels of GAP-43 are found in axon terminals of the dorsal horn, in the corticospinal tract and in the area surrounding the central canal Lamina X but is absent in motoneurons [79]. However, GAP-43 is up-regulated in motoneurons after experimental spinal cord trauma [80]. In humans, ventral horn motoneurons are normally devoid of GAP-43 but high mRNA levels appeared in ALS patients [81].
It is not unreasonable to hypothesize that down-regulation of GAP-43 mRNA and protein may be closely related to progesterone beneficial effects in Wobbler mice. A priori, increased accumulation of GAP-43 protein may be caused by slow transportation from perikaryon to the axonal growth cone [77]. However, the severe muscle atrophy typical of Wobbler mice suggests, as an alternative mechanism, that muscle denervation could originate the GAP-43 hyperex-pression of motoneurons. Degenerating motoneurons may react to denervation and synaptic loss by compensatory but useless strengthening of the remaining synapsis. Hyperex-pression of GAP-43 may be of little help, considering that abnormal synaptic contacts including collateral synaptoge- nesis and hypomyelination usually accompany motoneuron degeneration in Wobbler mice and ALS patients [77,81,82]. Actually, in these cases as well as in other neurophatologies (Alzheimer’s disease and schizophrenia) GAP-43 may be a marker for neurodegeneration.
In agreement with previous reports [25-27], we found an increased number of GFAP-immunoreactive astrocytes in Wobbler mice spinal cord. Although the biological signifi-cance of this finding and its relation to neuronal sickness of Wobbler mice is still unclear, we interpret it in terms of the known beneficial role of astrocytes on neuronal function. Thus, neuronal damage caused by ischemia, toxins, excito- toxicity, injury and neurodegeneration was first followed by astrocyte hypertrophy and then hyperplasia with GFAP hy-perexpression [83]. In this way, stimulated astrocytes would be endowed to provide neuroprotection. For a number of years, it has been realized that among other properties, as-trocytes take up excess glutamate and potassium released during neurotransmission, provide neurons with trophic fac-tors, glucose and lactate used for energy purposes and even play a role in myelination of axons [47,48]. Thus, stimulation of a preexisting astrocytosis would be advantageous, in contrast to traditional concepts which associated astrocytosis with increased pathology and inhibition of neuronal re-generation [84]. Recently, Ikeda et al. [35] treated Wobbler mice with T-588, a non-steroidal antioxidant. By the time T-588 enhanced motor function, muscle parameters and survival time of the affected animals, it also stimulated astrocytosis. These data, in conjunction with previous work using the 21-aminosteroid U-74389F [49] supported that some antioxidant neuroprotectants also up-regulate astrocyte number and/or function. However, we did not find an effect of progesterone on GFAP immunostaining of astrocytes, suggesting that in contrast to the effects of U-74389F and T-588, progesterone protection may have occurred primarily on motoneurons. However, it is still possible that progesterone can affect in astrocytes molecules other than GFAP but not considered in our investigation.
Conclusions
We would like to suggest that restoration of motoneuron morphology, up-regulation of Na, K-ATPase subunit mR- NAs, and down-regulation of aberrantly expressed GAP-43 mRNA may be linked to progesterone neuroprotection of symptomatic Wobbler mice. This assumption is supported by the increased muscle strength and prolonged survival of mutant mice receiving progesterone. The present work ex-panded to a genetic degeneration model multiple evidences of progesterone beneficial effects in experimental brain, pe-ripheral nerve and spinal cord injury [3,9-17]. As already discussed, some of these effects may be due to progesterone binding to a transcriptionally-active progesterone intra-cellular receptor. However, membrane-based non-genomic mechanisms, including inhibition of free-radical attack of intracellular membranes, may also provide clues towards the molecular basis of steroid neuroprotection. Future studies and clinical trials will unravel the importance of progesterone to evade neuronal loss and accelerate reparative responses in human degenerative diseases.
Acknowledgements
This work was supported by grants from the University of Buenos Aires (M048), National Research Council of Ar-gentina (PIP No. 02007), Beca Carrillo Onativia from the Minister of Health, FONCYT (BID 802 OC AR PICT 2000 05-08663) and a Cooperative Program between the Govern-ments of France and Argentina (ECOS/SECYT # A98SO1). The laboratory assistance of Paulina Roig, Analia Lima and Esther I. Nievas and EM assistance of Emerita Jorge Vilela and Mariana Lopez Ravasio is gratefully acknowledged.
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