C15 RESPONSE OF MICROGLIA IN A MODEL OF MOTOR NEURON PATHOLOGY
Kohsaka S
National Institute of Neuroscience, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Microglia, one type of glial cells in the CNS, are initially considered to play significant roles as immunocompetent and scavenger cells. Microglia exhibit ramified morphology in normal brain; they are activated under pathological conditions and show a number of features including morphological change, proliferation, migration and induction of various biologically active molecules. Increasing numbers of reports indicate that activated microglia initiate or facilitate inflammatory and degenerative processes by producing neurotoxic substances. Likewise, a neuroprotective role of microglia has also been suggested from studies showing the production of a variety of neurotrophic factors.
Axotomized facial motor nucleus is an advantageous in vivo model to study the functional roles of microglia on injured motor neurons in brain pathology. Transection of the facial nerve causes activation of microglia and stimulates the proliferation. The activated microglia migrate toward the injured motor neurons and wrap up the cell bodies. A number of studies from our laboratory strongly suggested that the activated microglia surrounding motor neurons following facial nerve axotomy play neuroprotective roles by secreting neurotrophic factors such as BDNF and GDNF, and also by increasing expression of glial glutamate transporter, GLT‐1. However, in order to function properly at the site of the lesion, microglia must migrate close to the injured motor neurons. Thus, cell migration is an important first stage of microglial response to ameliorate the damage to motor neurons.
We have recently shown that extracellular ATP induced membrane ruffling and chemotaxis of microglia and suggested that the effects are mediated by Gi/o‐protein‐coupled P2Y12 receptor (P2Y12R). We showed here that the ATP‐induced chemotaxis of microglia is also regulated by the ionotropic receptor, P2X4R in addition to the P2Y12R. Stimulation of G‐protein‐coupled receptors leads to activation of phospholipase C (PLC) and phosphoinositide 3‐kinase (PI3K). We examined the effect of PLC and PI3K inhibitors on the formation of membrane ruffling and the chemotaxis of microglia following the stimulation by ATP. A PLC inhibitor inhibited both membrane ruffling and chemotaxis, while PI3K inhibitors suppressed only chemotaxis without inhibiting the membrane ruffling. Phosphorylation of Akt, which is known to be a downstream target for PI3K, was enhanced by ATP stimulation. The increase in Akt phosphorylation was suppressed by chelating extracellular calcium. These results indicate that activation of PI3K pathway is modulated by the extracellular calcium influx suggesting a possibility that ionotropic P2XRs are involved in the PI3K activation. Studies by using various antagonists and short‐hairpin RNAs against P2XRs showed that suppression of P2X4R reduced the ATP‐induced chemotaxis of the cells.
These results indicate that P2X4R, in addition to P2Y12R, is involved in the ATP‐induced chemotaxis of microglia.
C16 MOLECULAR‐TARGETED THERAPEUTICS FOR SPINAL AND BULBAR MUSCULAR ATROPHY (SBMA)
Sobue G
Nagoya University School of Medicine, Nagoya, Japan
E‐mail address for correspondence: [email protected]
Spinal and bulbar muscular atrophy (SBMA) is a motor neuron disease characterized by slowly progressive muscle weakness and atrophy of bulbar, facial and limb muscles. The onset of weakness is usually between 30 and 60 years followed by a slow progression of neuromuscular symptoms. Bulbar palsy often aggravates in the advanced stage of this disease, resulting in life‐threatening respiratory tract infections and eventual early death. To date, no curative therapy has been established for SBMA.
The cause of SBMA is expansion of a trinucleotide CAG repeat, which encodes the polyglutamine tract, in the first exon of the androgen receptor (AR) gene. SBMA chiefly occurs in adult males, whereas neurological symptoms are rarely detected in females having a mutant AR gene. To elucidate the sex‐dependent neurological phenotypes of SBMA, we generated a transgenic mouse model carrying full‐length AR containing 97 CAGs driven by a chicken ß‐actin promoter. The male transgenic mice exhibited marked progressive motor impairment and nuclear accumulation of mutant AR, but neurological phenotypes were not observed or were far less severe in the females. Leuprorelin, an LHRH agonist that reduces testosterone release from the testis, suppressed nuclear accumulation of mutant AR, leading to rescue of motor dysfunction in male SBMA mice. In a randomized, double‐blind, placebo‐controlled clinical trial, leuprorelin significantly inhibited accumulation of pathogenic AR protein in the scrotal skin of patients, significantly decreased the level of serum creatine kinase, and suppressed the progression of dysphagia, suggesting that hormonal intervention with LHRH agonist is capable of interfering with the central pathogenesis of SBMA.
Our studies have also indicated several candidates of therapeutics for SBMA. Oral administration of geranylgeranylacetone (GGA) up‐regulates the levels of Hsp70 in the central nervous system and inhibits nuclear accumulation of the pathogenic AR protein, resulting in amelioration of polyglutamine‐dependent neuromuscular phenotypes of SBMA mice. On the other hand, selective inhibition of HSP facilitated degradation of pathogenic AR, leading to improvements of phenotypes in the SBMA mice. Inhibition of Hsp90 is also demonstrated to arrest the neurodegeneration in SBMA mice. Treatment with 17‐allylamino geldanamycin (17‐AAG), a potent Hsp90 inhibitor, dissociated p23 from the Hsp90‐AR complex, and thus facilitated proteasomal degradation of the pathogenic AR in cellular and mouse models of SBMA. 17‐AAG thereby inhibits nuclear accumulation of this protein, leading to marked amelioration of motor phenotypes of the SBMA mouse model without detectable toxicity. These HSP‐mediated therapies are applicable for other neurodegenerative diseases.
To date, various therapeutic strategies for SBMA have emerged from animal studies, underlining the necessity of clinical studies to verify the results from basic research. Since SBMA is a slowly progressive disorder, appropriate biomarkers would help to improve the power and cost‐effectiveness of longitudinal clinical treatment trials.
C17 LOSS OF ENDOGENOUS ANDROGEN RECEPTOR ACCELERATES MOTOR DYSFUNCTION AND ANDROGEN INSENSITIVITY IN A MOUSE MODEL OF SPINAL AND BULBAR MUSCULAR ATROPHY (SBMA)
Thomas Jr, PS1, Fraley GS2, Woodke LB1, Damien VM1, Sopher BL1, Plymate SR1, La Spada AR1
1University of Washington, Seattle, WA, USA, 2Hope College, Holland, MI, USA
E‐mail address for correspondence: [email protected]
X‐linked spinal and bulbar muscular atrophy (SBMA) is a slowly progressive motor neuronopathy that also results in mild androgen insensitivity. Evidence from SBMA and other polyglutamine repeat expansion diseases suggests that polyglutamine (PolyQ) expansion causes pathology by imparting a toxic gain of function. However, androgen insensitivity typically involves a deficit of wild‐type androgen receptor (AR) function, suggesting that the mild androgen insensitivity of SBMA involves some loss of function as well. In order to investigate the role of normal AR function in SBMA, we compared male mice carrying the human AR gene with 100 polyglutamine repeats (AR100) to mice carrying the same transgene, but lacking the endogenous androgen receptor (AR100Tfm). Both AR100 and AR100Tfm mice express the same amount of polyglutamine‐expanded AR; however, AR100Tfm mice exhibit earlier onset of weight loss, kyphosis, and hind limb atrophy compared to AR100 mice. AR100Tfm mice also performed worse on the grip‐strength test than AR100 mice. In addition to the neuromuscular phenotype, absence of AR uncovered signs of androgen insensitivity. The anogenital distance of male AR100Tfm mice is indistinguishable from a female mouse, whereas the anogenital distance of AR100 mice is normal. AR100Tfm mice also display elevated levels of luteinizing hormone (LH) (3‐fold increase, p>0.01); however, testes size in AR100Tfm mice was markedly diminished compared to AR100 animals (28% less, p<0.05). Furthermore, AR100Tfm mice show a decrease in the size of the spinal nucleus of the bulbocavernosis (SNB), which is extremely sensitive to androgen function (p>0.05). We thus hypothesized that the androgen insensitivity results from a diminished ability of polyglutamine‐expanded AR to activate transcription. Reporter gene assays using AR‐null PC‐3 cells reveal a two‐fold decrease (relative to wild‐type) in the transactivation ability of a polyglutamine expanded AR (p<0.05). These results suggest that the endogenous AR ameliorates pathology in a mouse model of SBMA, and supports a role for the loss of wild‐type function of AR in SBMA pathogenesis.
C18 MODULATION OF HSP90 FUNCTION: A MOLECULAR TARGETED THERAPY FOR NEURODEGENERATIVE DISORDERS
Waza MW, Adachi HA, Katsuno MK, Minamiyama MM, Tokui KT, Tanaka FT, Doyu MD, Sobue GS
Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan
E‐mail address for correspondence: [email protected]
Background: Hsp90 functions in a multi‐chaperone complex in folding, activating and assembling its client proteins. Androgen receptor (AR) belongs to the Hsp90 client protein family. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the AR. 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG), a potent Hsp90 inhibitor, is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity.
Objectives: 17‐AAG is now in phase II trials as a potential anti‐cancer agent because of its ability to selectively degrade several cancer‐related client proteins. Additionally, Hsp90 inhibitors also function as molecular chaperone inducers. Several previous studies have suggested that Hsp90 inhibitors could be applied to non‐oncological diseases as neuroprotective agents based on their induction of molecular chaperones. Here, we examined the efficacy and safety of 17‐AAG in a mouse model of SBMA and its ability to degrade polyQ‐expanded mutant AR, since Hsp90 inhibitors have two major activities, preferential client protein degradation and molecular chaperone induction.
Methods: SH‐SY5Y cells were plated in six‐cm dishes, and each dish was transfected with 8 µg of the vector containing AR24, AR97, or mock (negative control). For cultured cell models, a 1.8 mM stock solution of 17‐AAG in DMSO was diluted into fresh medium to give final concentrations of 18–360 nM. For mouse models, 17‐AAG treatments were started when mice attained the age of five weeks, and continued until the age of 25 weeks. Male normal littermates, the mice expressing full‐length human AR with 24 (AR‐24Q mice, 5‐5 line) and 97‐polyQ tract (AR‐97Q mice, 7‐8 line) received 50 µl, intraperitoneal injections of 2.5 or 25 mg/kg 17‐AAG three times a week on alternate days.
Results: Administration of 17‐AAG markedly ameliorated motor impairments in the SBMA transgenic mouse model without detectable toxicity, by reducing amounts of monomeric and nuclear accumulated mutant AR. The mutant AR showed a higher affinity to Hsp90‐p23 and preferentially formed an Hsp90 chaperone complex compared with wild‐type AR; mutant AR was preferentially degraded in the presence of 17‐AAG in both the cell and transgenic mouse models compared with wild‐type AR. 17‐AAG also mildly induced Hsp70 and Hsp40.
Discussion and conclusions: We reported that administration of 17‐AAG significantly ameliorated polyQ‐mediated motor neuron degeneration by preferential proteasome degradation of mutant AR. The ability of 17‐AAG to preferentially degrade mutant protein would be directly applicable to SBMA and other neurodegenerative diseases. Thus, modulation of Hsp90 function by 17‐AAG has emerged as a candidate of molecular targeted therapy for SBMA and probably for other neurodegenerative diseases as well.