The rate of production of uric acid by hepatocytes is a sensitive index of compromised cell ATP homeostasis.

Plasma levels of uric acid, the final product of purine degradation in humans, are elevated in metabolic syndrome and are strongly associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD). Hepatic and blood levels of purine metabolites (inosine, hypoxanthine, and xanthine) are also altered in pathophysiological states. We optimized a rat hepatocyte model to test the hypothesis that the production of uric acid by hepatocytes is a potential marker of compromised homeostasis of hepatocellular inorganic phosphate (Pi) and/or ATP. The basal rate of uric acid production from endogenous substrates in rat hepatocytes was comparable to that in human liver and was <10% of the maximum rate with saturating concentrations of purine substrates. It was marginally (~20%) decreased by insulin and increased by glucagon but was stimulated more than twofold by substrates (fructose and glycerol) that lower both cell ATP and Pi, and by inhibitors of mitochondrial respiration (complexes I, III, and V) that lower ATP but raise cell Pi. Clearance of inosine and its degradation to uric acid were also inhibited by cell Pi depletion. Analysis of gene expression in NAFLD biopsies showed an association between mRNA expression of GCKR, the glucokinase regulatory protein that is functionally linked to uric acid production, and mRNA expression of the phosphate transporters encoded by SLC17A1/3. Uric acid production by hepatocytes is a very sensitive index of ATP depletion irrespective of whether cell Pi is lowered or raised. This suggests that raised plasma uric acid may be a marker of compromised hepatic ATP homeostasis.

CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model.

BACKGROUND: The production of neurotrophic factors, such as BDNF, has generally been considered an important mechanism of immune-mediated neuroprotection. However, the ability of T cells to produce BDNF remains controversial. METHODS: In the present study, we examined mRNA and protein of BDNF using RT-PCR and western blot, respectively, in purified and reactivated CD4(+) T cells. In addition, to determine the role of BDNF derived from CD4(+) T cells, the BDNF gene was specifically deleted in T cells using the Cre-lox mouse model system. RESULTS: Our results indicate that while both mRNA expression and protein secretion of BDNF in reactivated T cells were detected at 24 h, only protein could be detected at 72 h after reactivation. The results suggest a transient up-regulation of BDNF mRNA in reactivated T cells. Furthermore, in contrast to our hypothesis that the BDNF expression is necessary for CD4(+) T cells to mediate neuroprotection, mice with CD4(+) T cells lacking BDNF expression demonstrated a similar level of facial motoneuron survival compared to their littermates that expressed BDNF, and both levels were comparable to wild-type. The results suggest that the deletion of BDNF did not impair CD4(+) T cell-mediated neuroprotection. CONCLUSION: Collectively, while CD4(+) T cells are a potential source of BDNF after nerve injury, production of BDNF is not necessary for CD4(+) T cells to mediate their neuroprotective effects.

Androgen regulates neuritin mRNA levels in an in vivo model of steroid-enhanced peripheral nerve regeneration.

Following crush injury to the facial nerve in Syrian hamsters, treatment with androgens enhances axonal regeneration rates and decreases time to recovery. It has been demonstrated in vitro that the ability of androgen to enhance neurite outgrowth in motoneurons is dependent on neuritin-a protein that is involved in the re-establisment of neuronal connectivity following traumatic damage to the central nervous system and that is under the control of several neurotrophic and neuroregenerative factors--and we have hypothesized that neuritin is a mediator of the ability of androgen to increase peripheral nerve regeneration rates in vivo. Testosterone treatment of facial nerve-axotomized hamsters resulted in an approximately 300% increase in neuritin mRNA levels 2 days post-injury. Simultaneous treatment with flutamide, an androgen receptor blocker that is known to prevent androgen enhancement of nerve regeneration, abolished the ability of testosterone to upregulate neuritin mRNA levels. In a corroborative in vitro experiment, the androgen dihydrotestosterone induced an approximately 100% increase in neuritin mRNA levels in motoneuron-neuroblastoma cells transfected with androgen receptors, but not in cells without androgen receptors. These data confirm that neuritin is under the control of androgens, and suggest that neuritin is an important effector of androgen in enhancing peripheral nerve regeneration following injury. Given that neuritin has now been shown to be involved in responses to both central and peripheral injuries, and appears to be a common effector molecule for several neurotrophic and neurotherapeutic agents, understanding the neuritin pathway is an important goal for the clinical management of traumatic nervous system injuries.

Effects of single versus combinatorial treatment strategies on beta II-tubulin gene expression in axotomized hamster rubrospinal motoneurons.

PURPOSE: betaII-tubulin, a regeneration-associated gene, is upregulated in injured peripheral neurons, but significantly less so in injured central neurons. Using a hamster dorsal spinal cord injury (SCI), the ability of single versus combinatorial treatment strategies to alter betaII-tubulin mRNA expression in rubrospinal motoneurons (RSMN) was examined. We have shown that systemic testosterone propionate (TP) treatment in combination with peripheral nerve grafting into a SCI site produces a peripheral-like pattern of betaII-tubulin mRNA expression in injured RSMN. In the present study, selected single- and combinatorial-therapy strategies were tested for their ability to promote a sustained upregulation of betaII-tubulin mRNA levels in injured RSMN. METHODS: Single treatments of olfactory ensheathing cells (OEC), brain-derived neurotrophic factor (BDNF), or Schwann cells (SC) vs combinatorial treatments (SC+TP, OEC+TP, and OEC+BDNF) were administered to hamsters following a dorsal SCI. Quantitative in situ hybridization in conjunction with a betaII-tubulin cDNA probe was accomplished. RESULTS: All of the single-therapy treatments tested were able to prevent the downregulation of betaII-tubulin mRNA that occurred a week after injury alone, but only BDNF maintained high levels of betaII-tubulin mRNA. In contrast, all combinatorial treatments tested maintained the upregulation of betaII-tubulin mRNA expression in injured RSMN 1 week post-SCI. CONCLUSIONS: Targeting both intrinsic and extrinsic components of CNS injury can re-program elements of the molecular response of injured central motoneurons.

Motoneuron injury and repair: New perspectives on gonadal steroids as neurotherapeutics.

In this review, we will summarize recent work from our laboratory on the role of gonadal steroids as neuroprotective agents in motoneuron viability following cell stress. Three motoneuron models will be discussed: developing axotomized hamster facial motoneurons (FMNs); adult axotomized mouse FMNs; and immortalized, cultured mouse spinal motoneurons subjected to heat shock. New work on two relevant motoneuron proteins, the survival of motor neuron protein, and neuritin or candidate plasticity-related gene 15, indicates differential steroid regulation of these two proteins after axotomy. The concept of gonadal steroids as cellular stress correction factors and the implications of this for acute neurological injury situations will be presented as well.

Axotomy-induced target disconnection promotes an additional death mechanism involved in motoneuron degeneration in amyotrophic lateral sclerosis transgenic mice.

The target disconnection theory of amyotrophic lateral sclerosis (ALS) pathogenesis suggests that disease onset is initiated by a peripheral pathological event resulting in neuromuscular junction loss and motoneuron (MN) degeneration. Presymptomatic mSOD1(G93A) mouse facial MN (FMN) are more susceptible to axotomy-induced cell death than wild-type (WT) FMN, which suggests additional CNS pathology. We have previously determined that the mSOD1 molecular response to facial nerve axotomy is phenotypically regenerative and indistinguishable from WT, whereas the surrounding microenvironment shows significant dysregulation in the mSOD1 facial nucleus. To elucidate the mechanisms underlying the enhanced mSOD1 FMN loss after axotomy, we superimposed the facial nerve axotomy model on presymptomatic mSOD1 mice and investigated gene expression for death receptor pathways after target disconnection by axotomy vs. disease progression. We determined that the TNFR1 death receptor pathway is involved in axotomy-induced FMN death in WT and is partially responsible for the mSOD1 FMN death. In contrast, an inherent mSOD1 CNS pathology resulted in a suppressed glial reaction and an upregulation in the Fas death pathway after target disconnection. We propose that the dysregulated mSOD1 glia fail to provide support the injured MN, leading to Fas-induced FMN death. Finally, we demonstrate that, during disease progression, the mSOD1 facial nucleus displays target disconnection-induced gene expression changes that mirror those induced by axotomy. This validates the use of axotomy as an investigative tool in understanding the role of peripheral target disconnection in the pathogenesis of ALS.

Use of laser microdissection in the investigation of facial motoneuron and neuropil molecular phenotypes after peripheral axotomy.

The mechanism underlying axotomy-induced motoneuron loss is not fully understood, but appears to involve molecular changes within the injured motoneuron and the surrounding local microenvironment (neuropil). The mouse facial nucleus consists of six subnuclei which respond differentially to facial nerve transection at the stylomastoid foramen. The ventromedial (VM) subnucleus maintains virtually full facial motoneuron (FMN) survival following axotomy, whereas the ventrolateral (VL) subnucleus results in significant FMN loss with the same nerve injury. We hypothesized that distinct molecular phenotypes of FMN existed within the two subregions, one responsible for maintaining cell survival and the other promoting cell death. In this study, we used laser microdissection to isolate VM and VL facial subnuclear regions for molecular characterization. We discovered that, regardless of neuronal fate after injury, FMN in either subnuclear region respond vigorously to injury with a characteristic "regenerative" profile and additionally, the surviving VL FMN appear to compensate for the significant FMN loss. In contrast, significant differences in the expression of pro-inflammatory cytokine mRNA in the surrounding neuropil response were found between the two subnuclear regions of the facial nucleus that support a causative role for glial and/or immune-derived molecules in directing the contrasting responses of the FMN to axonal transection.

Local delivery of a collagen-binding FGF-1 chimera to smooth muscle cells in collagen scaffolds for vascular tissue engineering.

We investigated the delivery of R136K-CBD (a collagen-binding mutant chimera of fibroblast growth factor-1) with a type I collagen scaffold as the delivery vehicle to smooth muscle cells (SMCs) for vascular tissue engineering. The binding affinity of R136K-CBD to 3-D collagen scaffolds was investigated both in the presence and absence of cells and/or salts. 2-D and 3-D visualization of delivery of R136K-CBD into SMCs were accomplished by combined fluorescent and reflection confocal microscopy. The mitogenic effect of collagen-immobilized R136K-CBD on SMCs in 3-D collagen was studied by Cyquant assay at different time intervals. In the group devoid of salt and cells, no detectable release of R136K-CBD into overlying culture media was found, compared with burst-and-continuous release of R136K and FGF-1 over a 14-day period in all other groups. The release rate of R136K-CBD was 1.7 and 1.6-fold less than R-136K and FGF-1 when media was supplemented with 2m salt (P<0.0001), and 2.6 and 2.5-fold less in cell-populated collagen hydrogels (P<0.0001), respectively. R136K-CBD showed essentially uniform binding to collagen and its distribution was dependent on that of the collagen scaffold. Internalization of R136K-CBD into SMCs was documented by confocal microscopy. 3-D local delivery of collagen-immobilized R136K-CBD increased the proliferation of SMCs in the collagen matrix to significantly greater levels and for a significantly greater duration than R136K or FGF-1, with 2.0 and 2.1-fold more mitogenicity than R136K and FGF-1 respectively (P<0.0001) at day 7. The results suggest that our collagen-binding fusion protein is an effective strategy for growth factor delivery for vascular tissue engineering.