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.

IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection.

We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4+ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4+ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4+ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

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.

Delayed functional recovery in presymptomatic mSOD1G93A mice following facial nerve crush axotomy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving progressive loss of motoneurons (MN). Axonal pathology and presynaptic deaf-ferentation precede MN degeneration during disease progression in patients and the ALS mouse model (mSOD1). Previously, we determined that a functional adaptive immune response is required for complete functional recovery following a facial nerve crush axotomy in wild-type (WT) mice. In this study, we investigated the effects of facial nerve crush axotomy on functional recovery and facial MN survival in presymptomatic mSOD1 mice, relative to WT mice. The results indicate that functional recovery and facial MN survival levels are significantly reduced in presymptomatic mSOD1, relative to WT, and similar to what has previously been observed in immunodeficient mice. It is concluded that a potential immune system defect exists in the mSOD1 mouse that negatively impacts neuronal survival and regeneration following target disconnection associated with peripheral nerve axotomy.

Differential gene expression in the axotomized facial motor nucleus of presymptomatic SOD1 mice.

Previously, we compared molecular profiles of one population of wild-type (WT) mouse facial motoneurons (FMNs) surviving with FMNs undergoing significant cell death after axotomy. Regardless of their ultimate fate, injured FMNs respond with a vigorous pro-survival/regenerative molecular response. In contrast, the neuropil surrounding the two different injured FMN populations contained distinct molecular differences that support a causative role for glial and/or immune-derived molecules in directing contrasting responses of the same cell types to the same injury. In the current investigation, we utilized the facial nerve axotomy model and a presymptomatic amyotrophic lateral sclerosis (ALS) mouse (SOD1) model to experimentally mimic the axonal die-back process observed in ALS pathogenesis without the confounding variable of disease onset. Presymptomatic SOD1 mice had a significant decrease in FMN survival compared with WT, which suggests an increased susceptibility to axotomy. Laser microdissection was used to accurately collect uninjured and axotomized facial motor nuclei of WT and presymptomatic SOD1 mice for mRNA expression pattern analyses of pro-survival/pro-regeneration genes, neuropil-specific genes, and genes involved in or responsive to the interaction of FMNs and non-neuronal cells. Axotomized presymptomatic SOD1 FMNs displayed a dynamic pro-survival/regenerative response to axotomy, similar to WT, despite increased cell death. However, significant differences were revealed when the axotomy-induced gene expression response of presymptomatic SOD1 neuropil was compared with WT. We propose that the increased susceptibility of presymptomatic SOD1 FMNs to axotomy-induced cell death and, by extrapolation, disease progression, is not intrinsic to the motoneuron, but rather involves a dysregulated response by non-neuronal cells in the surrounding neuropil.

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.

Toll-like receptor 2 and facial motoneuron survival after facial nerve axotomy.

We have previously demonstrated that CD4(+) Th2 lymphocytes are required to rescue facial motoneuron (FMN) survival after facial nerve axotomy through interaction with peripheral antigen presenting cells, as well as CNS resident microglia. Furthermore, the innate immune molecule, toll-like receptor 2 (TLR2), has been implicated in the development of Th2-type immune responses and can be activated by intracellular components released by dead or dying cells. The role of TLR2 in the FMN response to axotomy was explored in this study, using a model of facial nerve axotomy at the stylomastoid foramen in the mouse, in which blood-brain-barrier (BBB) permeability does not occur. After facial nerve axotomy, TLR2 mRNA was significantly upregulated in the facial motor nucleus and co-immunofluorescence localized TLR2 to CD68(+) microglia, but not GFAP(+) astrocytes. Using TLR2-deficient (TLR2(-/-)) mice, it was determined that TLR2 does not affect FMN survival levels after axotomy. These data contribute to understanding the role of innate immunity after FMN death and may be relevant to motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

Effects of facial nerve axotomy on Th2- and Th1-associated chemokine expression in the facial motor nucleus of wild-type and presymptomatic mSOD1 mice.

We have previously demonstrated a neuroprotective mechanism of facial motoneuron (FMN) survival after facial nerve axotomy that is dependent on CD4(+) Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS resident microglia. To investigate this mechanism, we chose to study the Th2-associated chemokine, CCL11, and Th1-associated chemokine, CXCL11, in wild-type and presymptomatic mSOD1 mice after facial nerve axotomy. In this report, the results indicate that CCL11 is constitutively expressed in the uninjured facial motor nucleus, but CXCL11 is not expressed at all. Facial nerve axotomy induced a shift in CCL11 expression from FMN to astrocytes, whereas CXCL11 was induced in FMN. Differences in the number of CCL11- and CXCL11-expressing cells were observed between WT and mSOD1 mice after facial nerve axotomy.

Effects of facial nerve axotomy on Th2-associated and Th1-associated chemokine mRNA expression in the facial motor nucleus of wild-type and presymptomatic SOD1 mice.

The authors have previously demonstrated a neuroprotective mechanism of facial motoneuron (FMN) survival after facial nerve transection that is dependent on CD4(+)T helper 2 (Th2) cell interactions with peripheral antigen presenting cells, as well as central nervous system (CNS) resident microglia. Pituitary adenylyl cyclase activating polypeptide is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these data suggest a model involving CD4(+) Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. In this study, the authors tested the hypothesis that Th2-associated chemokine expression occurs in the facial motor nucleus after facial nerve axotomy at the stylomastoid foramen. Initial microarray analysis of Th2-associated and Th1-associated chemokine mRNA levels was accomplished after facial nerve axotomy in wild type (WT) and presymptomatic mutant superoxide dismutase 1 (mSOD1) [model of familial amyotrophic lateral sclerosis (ALS)] mice. Based on that initial microarray analysis, the Th2-associated chemokine, CCL11, and Th1-associated chemokine, CXCL11, were further analyzed by RT-PCR. The results indicate that facial nerve injury predominantly increases Th2-associated chemokine, but not Th1-associated chemokine mRNA levels in the mouse facial motor nucleus. Interestingly, no differences were detected between WT and mSOD1 mice for CCL11 and CXCL11 after injury. These data provide a basis for further investigation into Th2-associated chemokine expression in the facial motor nucleus after FMN injury, which may lead to more specifically targeted therapeutics in motoneuron diseases, such as ALS.

Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice.

We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron) survival after facial nerve axotomy that is dependent on CD4(+) Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system)-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide) is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4(+) Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type), a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3(-/-) mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2(-/-) (recombination activating gene-2-deficient) mice adoptively transferred CD4(+) T-cells isolated from CCR3(-/-) mice, but not in CCR3(-/-) mice adoptively transferred CD4(+) T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4(+) T-cell- and CCR3-mediated neuroprotection after FMN injury.