Thomas D. Alexander

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.

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.

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 neuritinor 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.

Gonadal steroid enhancement of facial nerve regeneration: Role of heat shock protein 70

Over the past decade, our laboratory has been investigating the now well-established neurotrophic capabilities of gonadal steroids in the context of peripheral nerve injury and repair. The focus of our work has been on the hamster facial motoneuron (FMN) system (Kujawa & Jones, 1995), although we have recently begun to explore two additional motoneuron injury paradigms, the rat sciatic and hamster rubrospinal systems (Kujawa et al., 1993). In this brief review, we will discuss the effects of androgens and estrogens on the regenerative properties and the molecular programming of injured hamster FMN. This will be followed by a discussion of the effects of androgens on the glial response to injury in the facial nucleus. Finally, a working model of the mechanism by which gonadal steroids enhance neuronal reparative processes will be advanced. Our proposed mechanism involves a neuroprotective role for gonadal steroids, in that we hypothesize that exposure to these agents at the time of injury reduces the need for the injured neuron to mount a classical “stress response” involving the production of heat shock protein 70. Finally, a series of future directions for our work will be presented.

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.

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 mSOD1G93A 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. J. Comp. Neurol. 522:2349–2376, 2014.

Axotomy-induced target disconnection promotes an additional death mechanism involved in motoneuron degeneration in ALS 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 mSOD1G93A 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. J. Comp. Neurol. 522:2349–2376, 2014.

Axotomy-induced target disconnection promotes an additional death mechanism involved in motoneuron degeneration in amyotrophic lateral sclerosis transgenic mice: MN Death mechanisms involved in target disconnection

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 mSOD1G93A 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. J. Comp. Neurol. 522:2349–2376, 2014.