Deregulation of ZPR1 causes respiratory failure in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the low levels of survival motor neuron (SMN) protein and is characterized by motor neuron degeneration and muscle atrophy. Respiratory failure causes death in SMA but the underlying molecular mechanism is unknown. The zinc finger protein ZPR1 interacts with SMN. ZPR1 is down regulated in SMA patients. We report that ZPR1 functions downstream of SMN to regulate HoxA5 levels in phrenic motor neurons that control respiration. Spatiotemporal inactivation of Zpr1 gene in motor neurons down-regulates HoxA5 and causes defects in the function of phrenic motor neurons that results in respiratory failure and perinatal lethality in mice. Modulation in ZPR1 levels directly correlates and influences levels of HoxA5 transcription. In SMA mice, SMN-deficiency causes down-regulation of ZPR1 and HoxA5 that result in degeneration of phrenic motor neurons. Identification of ZPR1 and HoxA5 as potential targets provides a paradigm for developing strategies to treat respiratory distress in SMA.

Genetic inhibition of JNK3 ameliorates spinal muscular atrophy.

Mutation of the Survival Motor Neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder that occurs in early childhood. Degeneration of spinal motor neurons caused by SMN deficiency results in progressive muscle atrophy and death in SMA. The molecular mechanism underlying neurodegeneration in SMA is unknown. No treatment is available to prevent neurodegeneration and reduce the burden of illness in SMA. We report that the c-Jun NH2-terminal kinase (JNK) signaling pathway mediates neurodegeneration in SMA. The neuron-specific isoform JNK3 is required for neuron degeneration caused by SMN deficiency. JNK3 deficiency reduces degeneration of cultured neurons caused by low levels of SMN. Genetic inhibition of JNK pathway in vivo by Jnk3 knockout results in amelioration of SMA phenotype. JNK3 deficiency prevents the loss of spinal cord motor neurons, reduces muscle degeneration, improves muscle fiber thickness and muscle growth, improves motor function and overall growth and increases lifespan of mice with SMA that shows a systemic rescue of phenotype by a SMN-independent mechanism. JNK3 represents a potential (non-SMN) therapeutic target for the treatment of SMA.

Adaptation of arcuate insulin receptor, estrogen receptor-alpha, estrogen receptor-beta, and type-II glucocorticoid receptor gene profiles to chronic intermediate insulin-induced hypoglycemia in estrogen-treated ovariectomized female rats.

Insulin and corticosterone signal energy surfeit and deficiency, respectively, to metabolic structures in the brain, including the hypothalamic arcuate nucleus (ARH). This peripheral input may be subject to ovarian control, since ovariectomy (OVX) increases insulin receptor transcripts and decreases glucocorticoid receptor protein in the hypothalamus. The present studies examined the hypothesis that estradiol regulates basal and hypoglycemic patterns of ARH insulin and glucocorticoid receptor mRNA expression, and governs habituation of these gene profiles to recurring intermediate insulin administration. The premise that estrogen receptor-alpha (ERalpha) and beta (ERbeta) gene profiles may be regulated differentially during acute and chronic hypoglycemia in the presence of estradiol was also evaluated. Insulin receptor-beta chain (InsRb), type-II glucocorticoid receptor (GR), ERalpha, and ERbeta mRNA levels in ARH tissue microdissected from estradiol benzoate (EB)- and oil-implanted OVX rats after single or serial sc neutral protamine Hagedorn insulin (NPH) injection were measured by quantitative real-time RT-PCR. ARH InsRb gene profiles were decreased, relative to baseline, after either one or four NPH injections in OVX + EB rats; mean mRNA levels were significantly lower after serial dosing since basal InsRb transcripts were diminished by precedent NPH treatment. InsRb transcription rates did not differ among OVX + oil treatment groups. Acute insulin elevated ARH GR mRNA relative to baseline in both EB- and oil-implanted rats. Prior NPH injections increased basal GR gene expression and suppressed transcriptional reactivity to a fourth dose of NPH in OVX + EB, but not OVX + oil animals. ARH ERalpha and ERbeta mRNA levels were increased or decreased, respectively, after one insulin dose in OVX + EB rats. Baseline expression of these genes was correspondingly augmented or suppressed after precedent NPH treatment, but ERalpha and ERbeta transcripts were not modified relative to these adjusted baselines after a fourth NPH dose. In the presence of estradiol, ARH InsRb and GR gene profiles exhibit divergent modifications during acute NPH-induced hypoglycemia, as well as opposite adjustments in baseline expression after serial NPH dosing. GR transcriptional acclimation to recurring NPH administration was also estrogen-dependent. Further research is needed to characterize potential effects of adjustments in ARH neuronal sensitivity to insulin and corticosterone on ARH metabolic neurotransmitter release during and after intermediate insulin-induced hypoglycemia in females. The current evidence for converse effects of NPH on ARH ERalpha and ERbeta gene profiles in the presence of estradiol supports the need to identify ARH-directed metabolic activities governed by each ER subtype, including metabolic hormone receptor expression, and to assess the impact of NPH-induced habituation of ER gene profiles on those functions.

Sex differences in acclimation of hypothalamic arcuate glucokinase gene and protein expression to repeated intermediate insulin administration.

BACKGROUND: Repeated intermediate-acting insulin administration attenuates genomic reactivity of neurons in key autonomic metabolic structures in the male, but not female rat brain - results that support a central neural component of sex-specific response desensitization. The glucokinase (GK) enzyme functions as a glucose sensor in a body-wide system of metabolic monitoring structures, including the brain, and is expressed at high levels in the hypothalamic arcuate nucleus (ARH). METHOD: Quantitative real-time RT-PCR was used to investigate the hypothesis that habituation of ARH GK gene expression to neutral protamine Hagedorn insulin (NPH) injection differs among sexes. In lieu of evidence for region-based functional heterogeneity in this structure, effects of NPH on in situ GK protein-staining patterns were evaluated at different rostrocaudal levels of the ARH by immunocytochemistry. RESULTS: Basal ARH GK mRNA levels were equivalent in sham-operated (SHAM) and orchidectomized (ORDX) male rats. SHAM males exhibited augmented GK gene profiles in response to acute NPH injection, as well as elevated numbers of GK-immunoreactive (-ir) neurons in the rostral ARH. ORDX abolished this stimulatory transcriptional response, but did not prevent increased GK labeling throughout this structure. Stimulatory effects of precedent insulin administration on baseline GK mRNA were reversed by ORDX. Serial dosing of SHAM males with NPH elicited no change in ARH GK transcription, but decreased GK-ir in the rostral ARH. Acute NPH injection had no impact on GK gene profiles in estradiol benzoate (EB)- or oil-implanted ovariectomized (OVX) female rats, but diminished GK-ir cell counts in the OVX + EB caudal ARH. Precedent NPH treatment did not modify baseline GK mRNA levels in either group of females, but resulted in decreased or elevated GK gene and protein expression during recurring injection in the presence or absence of EB, respectively. CONCLUSION: These results provide novel evidence for sex-specific patterns of acclimation of GK mRNA and protein expression within the rat ARH to serial NPH injection, and support the need to elucidate the physiological ramifications of these adaptations regarding behavioral and physiological responses to recurring intermediate insulin administration.

Adaptation of glucokinase gene expression in the rat dorsal vagal complex in a model for recurrent intermediate insulin-induced hypoglycemia: impact of gender.

Standard therapeutic management of insulin-dependent diabetes mellitus with intermediate-acting insulin poses a significant risk for iatrogenic hypoglycemia and associated hypoglycemia-associated autonomic failure. Gender differences in preservation of counterregulatory function during recurring hypoglycemia have been documented in the clinical setting. In rats, repeated induction of prolonged hypoglycemia by neutral protamine Hagadorn insulin (NPH) results in diminished neuronal transcriptional activation in several key metabolic loci in male, but not female rat brain, including the hindbrain dorsal vagal complex (DVC). Glucose is committed to glycolytic catabolism by hexokinase-mediated phosphorylation. The low-affinity, high K (m) hexokinase, glucokinase (GCK), monitors intracellular glucose levels in pancreatic beta cells, and is purported to fulfill a similar function in the CNS. GCK is expressed in the rat DVC, where mRNA is localized to neurons that exhibit electrophysiological sensitivity to glucose imbalance. The current study investigated the hypothesis that DVC GCK gene expression acclimates to RIIH in a gender-dependent manner. Quantitative real-time RT-PCR was used to evaluate GCK mRNA levels in microdissected DVC tissue obtained from male and female rats before or after one or serial doses of NPH. Basal DVC GCK transcripts were equal between ovariectomized (OVX) female rats implanted with estradiol benzoate (EB) or oil (O). Tissue mRNA levels were increased following a single NPH injection in both groups, but this response was greater in the presence of estrogen. Basal GCK gene expression was elevated by precedent insulin dosing in both O- and EB-implanted rats; however, transcripts were not modified relative to this adjusted baseline during subsequent NPH treatment in either group of females. While acute NPH treatment did not modify tissue transcript levels in orchidectomized (ORDX) or sham-ORDX rats, precedent NPH dosing increased basal GCK mRNA levels and further enhanced gene expression during ensuring hypoglycemia in sham males. These studies provide novel evidence for gender-dimorphic DVC GCK gene responses to single and serial intermediate-acting insulin administration. Further research is required to determine if and how stimulatory versus inhibitory transcriptional adaptation of this gene profile in male and female rats, respectively, impacts glucose-sensing functions in the DVC, and whether such adjustments may contribute to gender differences in magnitude of precedent hypoglycemic impairment of counterregulation.