Testosterone Reduces Myelin Abnormalities in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron degenerative disease that is associated with demyelination. The Wobbler (WR) mouse exhibits motoneuron degeneration, gliosis and myelin deterioration in the cervical spinal cord. Since male WRs display low testosterone (T) levels in the nervous system, we investigated if T modified myelin-relative parameters in WRs in the absence or presence of the aromatase inhibitor, anastrozole (A). We studied myelin by using luxol-fast-blue (LFB) staining, semithin sections, electron microscopy and myelin protein expression, density of IBA1+ microglia and mRNA expression of inflammatory factors, and the glutamatergic parameters glutamine synthetase (GS) and the transporter GLT1. Controls and WR + T showed higher LFB, MBP and PLP staining, lower g-ratios and compact myelin than WRs and WR + T + A, and groups showing the rupture of myelin lamellae. WRs showed increased IBA1+ cells and mRNA for CD11b and inflammatory factors (IL-18, TLR4, TNFαR1 and P2Y12R) vs. controls or WR + T. IBA1+ cells, and CD11b were not reduced in WR + T + A, but inflammatory factors' mRNA remained low. A reduction of GS+ cells and GLT-1 immunoreactivity was observed in WRs and WR + T + A vs. controls and WR + T. Clinically, WR + T but not WR + T + A showed enhanced muscle mass, grip strength and reduced paw abnormalities. Therefore, T effects involve myelin protection, a finding of potential clinical translation.

Progesterone and Allopregnanolone Neuroprotective Effects in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

Progesterone regulates a number of processes in neurons and glial cells not directly involved in reproduction or sex behavior. Several neuroprotective effects are better observed under pathological conditions, as shown in the Wobbler mouse model of amyotrophic laterals sclerosis (ALS). Wobbler mice are characterized by forelimb atrophy due to motoneuron degeneration in the spinal cord, and include microgliosis and astrogliosis. Here we summarized current evidence on progesterone reversal of Wobbler neuropathology. We demonstrated that progesterone decreased motoneuron vacuolization with preservation of mitochondrial respiratory complex I activity, decreased mitochondrial expression and activity of nitric oxide synthase, increased Mn-dependent superoxide dismutase, stimulated brain-derived neurotrophic factor, increased the cholinergic phenotype of motoneurons, and enhanced survival with a concomitant decrease of death-related pathways. Progesterone also showed differential effects on glial cells, including increased oligodendrocyte density and downregulation of astrogliosis and microgliosis. These changes associate with reduced anti-inflammatory markers. The enhanced neurochemical parameters were accompanied by longer survival and increased muscle strength in tests of motor behavior. Because progesterone is locally metabolized to allopregnanolone (ALLO) in nervous tissues, we also studied neuroprotection by this derivative. Treatment of Wobbler mice with ALLO decreased oxidative stress and glial pathology, increased motoneuron viability and clinical outcome in a progesterone-like manner, suggesting that ALLO could mediate some progesterone effects in the spinal cord. In conclusion, the beneficial effects observed in different parameters support the versatile properties of progesterone and ALLO in a mouse model of motoneuron degeneration. The studies foresee future therapeutic opportunities with neuroactive steroids for deadly diseases like ALS.

Neuroprotective Effects of Testosterone in Male Wobbler Mouse, a Model of Amyotrophic Lateral Sclerosis.

Patients suffering of amyotrophic lateral sclerosis (ALS) present motoneuron degeneration leading to muscle atrophy, dysphagia, and dysarthria. The Wobbler mouse, an animal model of ALS, shows a selective loss of motoneurons, astrocytosis, and microgliosis in the spinal cord. The incidence of ALS is greater in men; however, it increases in women after menopause, suggesting a role of sex steroids in ALS. Testosterone is a complex steroid that exerts its effects directly via androgen (AR) or Sigma-1 receptors and indirectly via estrogen receptors (ER) after aromatization into estradiol. Its reduced-metabolite 5α-dihydrotestosterone acts via AR. This study analyzed the effects of testosterone in male symptomatic Wobblers. Controls or Wobblers received empty or testosterone-filled silastic tubes for 2 months. The cervical spinal cord from testosterone-treated Wobblers showed (1) similar androgen levels to untreated control and (2) increased levels of testosterone, and its 5α-reduced metabolites, 5α- dihydrotestosterone, and 3ß-androstanediol, but (3) undetectable levels of estradiol compared to untreated Wobblers. Testosterone-treated controls showed comparable steroid concentrations to its untreated counterpart. In testosterone- treated Wobblers a reduction of AR, ERα, and aromatase and high levels of Sigma-1 receptor mRNAs was demonstrated. Testosterone treatment increased ChAT immunoreactivity and the antiinflammatory mediator TGFß, while it lessened vacuolated motoneurons, GFAP+ astrogliosis, the density of IBA1+ microgliosis, proinflammatory mediators, and oxidative/nitrosative stress. Clinically, testosterone treatment in Wobblers slowed the progression of paw atrophy and improved rotarod performance. Collectively, our findings indicate an antiinflammatory and protective effect of testosterone in the degenerating spinal cord. These results coincided with a high concentration of androgen-reduced derivatives after testosterone treatment suggesting that the steroid profile may have a beneficial role on disease progression.

Insights into the Therapeutic Potential of Glucocorticoid Receptor Modulators for Neurodegenerative Diseases.

Glucocorticoids are crucial for stress-coping, resilience, and adaptation. However, if the stress hormones become dysregulated, the vulnerability to stress-related diseases is enhanced. In this brief review, we discuss the role of glucocorticoids in the pathogenesis of neurodegenerative disorders in both human and animal models, and focus in particular on amyotrophic lateral sclerosis (ALS). For this purpose, we used the Wobbler animal model, which mimics much of the pathology of ALS including a dysfunctional hypothalamic-pituitary-adrenal axis. We discuss recent studies that demonstrated that the pathological cascade characteristic for motoneuron degeneration of ALS is mimicked in the genetically selected Wobbler mouse and can be attenuated by treatment with the selective glucocorticoid receptor antagonist (GRA) CORT113176. In long-term treatment (3 weeks) GRA attenuated progression of the behavioral, inflammatory, excitatory, and cell-death-signaling pathways while increasing the survival signal of serine-threonine kinase (pAkt). The action mechanism of the GRA may be either by interfering with GR deactivation or by restoring the balance between pro- and anti-inflammatory signaling pathways driven by the complementary mineralocorticoid receptor (MR)- and GR-mediated actions of corticosterone. Accordingly, GR antagonism may have clinical relevance for the treatment of neurodegenerative diseases.

Long-term effects of the glucocorticoid receptor modulator CORT113176 in murine motoneuron degeneration.

The Wobbler mouse spinal cord shows vacuolated motoneurons, glial reaction, inflammation and abnormal glutamatergic parameters. Wobblers also show deficits of motor performance. These conditions resemble amyotrophic lateral sclerosis (ALS). Wobbler mice also show high levels of corticosterone in blood, adrenals and brain plus adrenal hypertrophy, suggesting that chronically elevated glucocorticoids prime spinal cord neuroinflammation. Therefore, we analyzed if treatment of Wobbler mice with the glucocorticoid receptor (GR) antagonist CORT113176 mitigated the mentioned abnormalities. 30 mg/kg CORT113176 given daily for 3 weeks reduced motoneuron vacuolation, decreased astro and microgliosis, lowered the inflammatory mediators high mobility group box 1 protein (HMGB1), toll-like receptor 4, myeloid differentiation primary response 88 (MyD88), p50 subunit of nuclear factor kappa B (NFκB), tumor necrosis factor (TNF) receptor, and interleukin 18 (IL18) compared to untreated Wobblers. CORT113176 increased the survival signal pAKT (serine-threonine kinase) and decreased the death signal phosphorylated Junk-N-terminal kinase (pJNK), symptomatic of antiapoptosis. There was a moderate positive effect on glutamine synthase and astrocyte glutamate transporters, suggesting decreased glutamate excitotoxicity. In this pre-clinical study, Wobblers receiving CORT113176 showed enhanced resistance to fatigue in the rota rod test and lower forelimb atrophy at weeks 2-3. Therefore, long-term treatment with CORT113176 attenuated degeneration and inflammation, increased motor performance and decreased paw deformity. Antagonism of the GR may be of potential therapeutic value for neurodegenerative diseases.

Comparative effects of progesterone and the synthetic progestin norethindrone on neuroprotection in a model of spontaneous motoneuron degeneration.

The Wobbler mouse has been proposed as an experimental model of the sporadic form of amyotrophic lateral sclerosis (ALS). The administration of natural progesterone (PROG) to Wobbler mice attenuates neuropathology, inhibits oxidative stress, enhances the expression of genes involved in motoneuron function, increases survival and restores axonal transport. However, current pharmacological treatments for ALS patients are still partially effective. This encouraged us to investigate if the synthetic progestin norethindrone (NOR), showing higher potency than PROG and used for birth control and hormone therapy might also afford neuroprotection. Two-month-old Wobbler mice (wr/wr) were left untreated or received either a 20 mg pellet of PROG or a 1 mg pellet of NOR for 18 days. Untreated control NFR/NFR mice (background strain for Wobbler) were also employed. Wobblers showed typical clinical and spinal cord abnormalities, while these abnormalities were normalized with PROG treatment. Surprisingly, we found that NOR did not increase immunoreactivity and gene expression for choline-acetyltransferase, drastically decreased GFAP + astrogliosis, favored proinflammatory mediators, promoted the inflammatory phenotype of IBA1+ microglia, increased the receptor for advanced glycation end products (RAGE) mRNA and protein expression and the activity of nitric oxide synthase (NOS)/NADPH diaphorase in the cervical spinal cord. Additionally, NOR treatment produced atrophy of the thymus. The combined negative effects of NOR on clinical assessments (forelimb atrophy and rotarod performance) suggest a detrimental effect on muscle trophism and motor function. These findings reinforce the evidence that the type of progestin used for contraception, endometriosis or replacement therapy, may condition the outcome of preclinical and clinical studies targeting neurodegenerative diseases.

Protective effects of the neurosteroid allopregnanolone in a mouse model of spontaneous motoneuron degeneration.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by progressive death of motoneurons. The Wobbler (WR) mouse is a preclinical model sharing neuropathological similarities with human ALS. We have shown that progesterone (PROG) prevents the progression of motoneuron degeneration. We now studied if allopregnanolone (ALLO), a reduced metabolite of PROG endowed with gabaergic activity, also prevents WR neuropathology. Sixty-day old WRs remained untreated or received two steroid treatment regimens in order to evaluate the response of several parameters during early or prolonged steroid administration. ALLO was administered s.c. daily for 5days (4mg/kg) or every other day for 32days (3, 3mg/kg), while another group of WRs received a 20mg PROG pellet s.c. for 18 or 60days. ALLO administration to WRs increased ALLO serum levels without changing PROG and 5 alpha dihydroprogesterone (5α-DHP), whereas PROG treatment increased PROG, 5α-DHP and ALLO. Untreated WRs showed higher basal levels of serum 5α-DHP than controls. In the cervical spinal cord we studied markers of oxidative stress or associated to trophic responses. These included nitric oxide synthase (NOS) activity, motoneuron vacuolation, MnSOD immunoreactivity (IR), brain derived neurotrophic factor (BDNF) and TrkB mRNAs, p75 neurotrophin receptor (p75NTR) and, cell survival or death signals such as pAKT and the stress activated kinase JNK. Untreated WRs showed a reduction of MnSOD-IR and BDNF/TrkB mRNAs, associated to high p75NTR in motoneurons, neuronal and glial NOS hyperactivity and neuronal vacuolation. Also, low pAKT, mainly in young WRs, and a high pJNK in the old stage characterized WRs spinal cord. Except for MnSOD and BDNF, these alterations were prevented by an acute ALLO treatment, while short-term PROG elevated MnSOD. Moreover, after chronic administration both steroids enhanced MnSOD-IR and BDNF mRNA, while attenuated pJNK and NOS in glial cells. Long-term PROG also increased pAKT and reduced neuronal NOS, parameters not modulated by chronic ALLO. Clinically, both steroids improved muscle performance. Thus, ALLO was able to reduce neuropathology in this model. Since high oxidative stress activates p75NTR and pJNK in neurodegeneration, steroid reduction of these molecules may provide adequate neuroprotection. These data yield the first evidence that ALLO, a gabaergic neuroactive steroid, brings neuroprotection in a model of motoneuron degeneration.

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.

Progesterone protective effects in neurodegeneration and neuroinflammation.

Progesterone is a neuroprotective, promyelinating and anti-inflammatory factor for the nervous system. Here, we review the effects of progesterone in models of motoneurone degeneration and neuroinflammation. In neurodegeneration of the Wobbler mouse, a subset of spinal cord motoneurones showed increased activity of nitric oxide synthase (NOS), increased intramitochondrial NOS, decreased activity of respiratory chain complexes, and decreased activity and protein expression of Mn-superoxide dismutase type 2 (MnSOD2). Clinically, Wobblers suffered several degrees of motor impairment. Progesterone treatment restored the expression of neuronal markers, decreased the activity of NOS and enhanced complex I respiratory activity and MnSOD2. Long-term treatment with progesterone increased muscle strength, biceps weight and survival. Collectively, these data suggest that progesterone prevented neurodegeneration. To study the effects of progesterone in neuroinflammation, we employed mice with experimental autoimmune encephalomyelitis (EAE). EAE mice spinal cord showed increased mRNA levels of the inflammatory mediators tumour necrosis factor (TNF)α and its receptor TNFR1, the microglial marker CD11b, inducible NOS and the toll-like receptor 4. Progesterone pretreatment of EAE mice blocked the proinflammatory mediators, decreased Iba1+ microglial cells and attenuated clinical signs of EAE. Therefore, reactive glial cells became targets of progesterone anti-inflammatory effects. These results represent a starting point for testing the usefulness of neuroactive steroids in neurological disorders.