Toxic Advanced Glycation End-Products Inhibit Axonal Elongation Mediated by ß-Tubulin Aggregation in Mice Optic Nerves.

Advanced glycation end-products (AGEs) form through non-enzymatic glycation of various proteins. Optic nerve degeneration is a frequent complication of diabetes, and retinal AGE accumulation is strongly linked to the development of diabetic retinopathy. Type 2 diabetes mellitus is a major risk factor for Alzheimer's disease (AD), with patients often exhibiting optic axon degeneration in the nerve fiber layer. Notably, a gap exists in our understanding of how AGEs contribute to neuronal degeneration in the optic nerve within the context of both diabetes and AD. Our previous work demonstrated that glyceraldehyde (GA)-derived toxic advanced glycation end-products (TAGE) disrupt neurite outgrowth through TAGE-ß-tubulin aggregation and tau phosphorylation in neural cultures. In this study, we further illustrated GA-induced suppression of optic nerve axonal elongation via abnormal ß-tubulin aggregation in mouse retinas. Elucidating this optic nerve degeneration mechanism holds promise for bridging the knowledge gap regarding vision loss associated with diabetes mellitus and AD.

Decreased Hepatic Functional Reserve Increases the Risk of Piperacillin/Tazobactam-Induced Abnormal Liver Enzyme Levels: A Retrospective Case-Control Study.

BACKGROUND: Piperacillin/tazobactam (PIPC/TAZ), which is a combination of a beta-lactam/beta-lactamase inhibitor, often causes liver enzyme abnormalities. The albumin-bilirubin (ALBI) score is a simple index that uses the serum albumin and total bilirubin levels for estimating hepatic functional reserve. Although patients with low hepatic reserve may be at high risk for drug-induced liver enzyme abnormalities, the relationship between PIPC/TAZ-induced abnormal liver enzymes levels and the ALBI score remains unknown. OBJECTIVE: This study aimed to elucidate the relationship between PIPC/TAZ-induced abnormal liver enzyme levels and the ALBI score. METHODS: This single-center retrospective case-control study included 335 patients. The primary outcome was PIPC/TAZ-induced abnormal liver enzyme levels. We performed COX regression analysis with male gender, age (≥75 years), alanine aminotransferase level (≥20 IU/L), and ALBI score (≥-2.00) as explanatory factors. To investigate the influence of the ALBI score on the development of abnormal liver enzyme levels, 1:1 propensity score matching between the ≤-2.00 and ≥-2.00 ALBI score groups was performed using the risk factors for drug-induced abnormal liver enzyme levels. RESULTS: The incidence of abnormal liver enzyme levels was 14.0% (47/335). COX regression analysis revealed that an ALBI score ≥-2.00 was an independent risk factor for PIPC/TAZ-induced abnormal liver enzyme levels (adjusted hazard ratio: 3.08, 95% coefficient interval: 1.207-7.835, P = 0.019). After 1:1 propensity score matching, the Kaplan-Meier curve revealed that the cumulative risk for PIPC/TAZ-induced abnormal liver enzyme levels was significantly higher in the ALBI score ≥-2.00 group (n = 76) than in the <-2.00 group (n = 76) (P = 0.033). CONCLUSION AND RELEVANCE: An ALBI score ≥-2.00 may predict the development of PIPC/TAZ-induced abnormal liver enzyme levels. Therefore, frequent monitoring of liver enzymes should be conducted to minimize the risk of severe PIPC/TAZ-induced abnormal liver enzyme levels in patients with low hepatic functional reserve.

Effect of Ceftriaxone Dosage and Albumin-Bilirubin Score on the Risk of Ceftriaxone-Induced Liver Injury.

The albumin-bilirubin (ALBI) score is an index of hepatic functional reserve and is calculated from serum albumin and total bilirubin levels. However, the relationship between ceftriaxone (CTRX)-induced liver injury and ALBI score remains unknown. Therefore, we aimed to elucidate the risk of CTRX-induced liver injury based on the ALBI scores and CTRX dosage. This was a single-center, retrospective, case-control study of 490 patients and the primary outcome was CTRX-induced liver injury. We performed a COX regression analysis using age ≥75 years, male sex, alanine aminotransferase levels, ALBI score, and CTRX dosage regimen (4 ≥2 or 1 g/d) as explanatory factors. We also performed 1 : 1 propensity score matching between non-liver injury and liver injury groups. The incidence of liver injury was 10.0% (49/490). In COX regression analysis, CTRX 4 g/d was an independent risk factor for liver injury (95% coefficient interval: 1.05-6.96, p = 0.04). Meanwhile, ALBI score ≥-1.61 was an independent factor for liver injury (95% coefficient interval: 1.03-3.22, p = 0.04) with the explanatory factor of ≥2 and 1 g/d. The Kaplan-Meier curve indicated that the cumulative risk for CTRX-induced liver injury was significantly higher in the ALBI score ≥-1.61 group than in the ALBI score <-1.61 group before propensity score matching (p = 0.032); however, no significant differences were observed after propensity score matching (p = 0.791). These findings suggest that in patients treated with CTRX with ALBI score ≥-1.61, frequent liver function monitoring should be considered.

Toxic Advanced Glycation End-Products-Dependent Alzheimer's Disease- Like Alternation in the Microtubule System.

Type 2 diabetes mellitus (T2DM) is a risk factor for Alzheimer's Disease (AD). However, the detailed mechanism underlying T2DM-related AD remains unknown. In DM, many types of advanced glycation end-products (AGEs) are formed and accumulated. In our previous study, we demonstrated that Glyceraldehyde (GA)-derived Toxic Advanced Glycation End-products (Toxic AGEs, TAGE) strongly showed cytotoxicity against neurons and induced similar alterations to those observed in AD. Further, GA induced dysfunctional neurite outgrowth via TAGE-ß-- tubulin aggregation, which resulted in the TAGE-dependent abnormal aggregation of ß-tubulin and tau phosphorylation. Herein, we provide a perspective on the possibility that T2DM increases the probability of AD onset and accelerates its progression.

Effects of Toxic AGEs (TAGE) on Human Health.

The habitual and excessive consumption of sugar (i.e., sucrose and high-fructose corn syrup, HFCS) is associated with the onset and progression of lifestyle-related diseases (LSRD). Advanced glycation end-products (AGEs) have recently been the focus of research on the factors contributing to LSRD. Approaches that inhibit the effects of AGEs may be used to prevent and/or treat LSRD; however, since the structures of AGEs vary depending on the type of reducing sugars or carbonyl compounds to which they respond, difficulties are associated with verifying that AGEs are an etiological factor. Cytotoxic AGEs derived from glyceraldehyde, a triose intermediate in the metabolism of glucose and fructose, have been implicated in LSRD and are called toxic AGEs (TAGE). A dietary imbalance (the habitual and excessive intake of sucrose, HFCS, or dietary AGEs) promotes the generation/accumulation of TAGE in vivo. Elevated circulating levels of TAGE have been detected in non-diabetics and diabetics, indicating a strong relationship between the generation/accumulation of TAGE in vivo and the onset and progression of LSRD. We herein outline current findings on "TAGE as a new target" for human health.

Pyridoxamine and Aminoguanidine Attenuate the Abnormal Aggregation of ß-Tubulin and Suppression of Neurite Outgrowth by Glyceraldehyde-Derived Toxic Advanced Glycation End-Products.

Diabetes mellitus (DM) has been identified as a risk factor for the onset and progression of Alzheimer's disease (AD). In our previous study, we demonstrated that glyceraldehyde (GA)-derived toxic advanced glycation end-products (toxic AGEs, TAGE) induced similar alterations to those observed in AD. GA induced dysfunctional neurite outgrowth via TAGE-ß-tubulin aggregation, which resulted in the TAGE-dependent abnormal aggregation of ß-tubulin and tau phosphorylation in human neuroblastoma SH-SY5Y cells. However, the effects of inhibitors of AGE formation on dysfunctional neurite outgrowth caused by GA-induced abnormalities in the aggregation of ß-tubulin and tau phosphorylation remain unknown. Aminoguanidine (AG), an AGE inhibitor, and pyridoxamine (PM), a natural form of vitamin B6 (VB6), are effective AGE inhibitors. Therefore, the present study investigated whether AG or PM ameliorate TAGE-ß-tubulin aggregation and the suppression of neurite outgrowth by GA. The results obtained showed that AG and PM inhibited the formation of TAGE-ß-tubulin, mitigated the GA-induced suppression of neurite outgrowth, and reduced GA-mediated increases in tau phosphorylation levels. Collectively, these results suggest the potential of AG and PM to prevent the DM-associated onset and progression of AD.

Elevated Hydrostatic Pressure Causes Retinal Degeneration Through Upregulating Lipocalin-2.

Elevation of intraocular pressure is a major risk factor for glaucoma development, which causes the loss of retinal ganglion cells (RGCs). Lipocalin 2 (Lcn2) is upregulated in glaucomatous retinae; however, whether Lcn2 is directly involved in glaucoma is debated. In this study, retinal explant cultures were subjected to increased water pressure using a two-chamber culture device, and Lcn2 protein levels were examined by immunoblotting. In situ TdT-mediated dUTP nick and labeling (TUNEL) and glial fibrillary acidic protein (GFAP) immunohistochemical assays were performed to assess apoptosis and gliosis, respectively. The neurotoxicity of Lcn2 in the retinal explant culture was determined with exogenous administration of recombinant Lcn2. The Lcn2 protein levels, percentage of TUNEL-positive cells, and GFAP-positive area were significantly higher in retinae cultured under 50 cm H2O pressure loads compared to those cultured under 20 cm H2O. We found that Lcn2 exhibited neurotoxicity in retinae at dose of 1 µg/ml. The negative effects of increased hydrostatic pressure were attenuated by the iron chelator deferoxamine. This is the first report demonstrating the direct upregulation of Lcn2 by elevating hydrostatic pressure. Modulating Lcn2 and iron levels may be a promising therapeutic approach for retinal degeneration.

Intracellular Toxic AGEs (TAGE) Triggers Numerous Types of Cell Damage.

The habitual intake of large amounts of sugar, which has been implicated in the onset/progression of lifestyle-related diseases (LSRD), induces the excessive production of glyceraldehyde (GA), an intermediate of sugar metabolism, in neuronal cells, hepatocytes, and cardiomyocytes. Reactions between GA and intracellular proteins produce toxic advanced glycation end-products (toxic AGEs, TAGE), the accumulation of which contributes to various diseases, such as Alzheimer's disease, non-alcoholic steatohepatitis, and cardiovascular disease. The cellular leakage of TAGE affects the surrounding cells via the receptor for AGEs (RAGE), thereby promoting the onset/progression of LSRD. We demonstrated that the intracellular accumulation of TAGE triggered numerous cellular disorders, and also that TAGE leaked into the extracellular space, thereby increasing extracellular TAGE levels in circulating fluids. Intracellular signaling and the production of reactive oxygen species are affected by extracellular TAGE and RAGE interactions, which, in turn, facilitate the intracellular generation of TAGE, all of which may contribute to the pathological changes observed in LSRD. In this review, we discuss the relationships between intracellular TAGE levels and numerous types of cell damage. The novel concept of the "TAGE theory" is expected to open new perspectives for research into LSRD.

TC10, a Rho family GTPase, is required for efficient axon regeneration in a neuron-autonomous manner.

Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock-out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild-type and TC10 knock-out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron-intrinsic.

The Effect of Glyceraldehyde-Derived Advanced Glycation End Products on ß-Tubulin-Inhibited Neurite Outgrowth in SH-SY5Y Human Neuroblastoma Cells.

Nutritional factors can affect the risk of developing neurological disorders and their rate of progression. In particular, abnormalities of carbohydrate metabolism in diabetes mellitus patients lead to an increased risk of neurological disorders such as Alzheimer's disease (AD). In this study, we investigated the relationship between nervous system disorder and the pathogenesis of AD by exposing SH-SY5Y neuroblastoma cells to glyceraldehyde (GA). We previously reported that GA-derived toxic advanced glycation end products (toxic AGEs, TAGE) induce AD-like alterations including intracellular tau phosphorylation. However, the role of TAGE and their target molecules in the pathogenesis of AD remains unclear. In this study, we investigated the target protein for TAGE by performing two-dimensional immunoblot analysis with anti-TAGE antibody and mass spectrometry and identified ß-tubulin as one of the targets. GA treatment induced TAGE-ß-tubulin formation and abnormal aggregation of ß-tubulin, and inhibited neurite outgrowth in SH-SY5Y cells. On the other hand, glucose-derived AGEs were also involved in developing AD. However, glucose did not make abnormal aggregation of ß-tubulin and did not inhibit neurite outgrowth. Understanding the underlying mechanism of TAGE-ß-tubulin formation by GA and its role in neurodegeneration may aid in the development of novel therapeutics and neuroprotection strategies.

A novel activation mechanism of cellular Factor XIII in zebrafish retina after optic nerve injury.

Cellular Factor XIII (cFXIII) mRNA is rapidly upregulated in the fish retina after optic nerve injury (ONI). Here, we investigated the molecular mechanism of cFXIII gene activation using genetic information from the A-subunit of cFXIII (cFXIII-A). Real-time PCR that amplified the active site (exons 7-8) of cFXIII-A showed increased cFXIII-A mRNA in the retina after ONI, whereas the PCR that amplified the activation peptide (exons 1-2) showed no change. RT-PCR analysis that amplified exons 1-8 showed two bands, a faint long band in the control retina and a dense short band in the injured retina. Therefore, we conclude that activated cFXIII-A mRNA after ONI is shorter than that of the control retina. Western blot analysis also confirmed an active form of 65 kDa cFXIII-A protein in the injured retina compared to the control 84 kDa protein. 5'-RACE analysis using injured retina revealed that the short cFXIII-A mRNA lacked exons 1, 2 and part of exon 3. Exon 3 has two sites of heat shock factor 1 (HSF-1) binding consensus sequence. Intraocular injection of HSF inhibitor suppressed the expression of cFXIII-A mRNA in the retina 1 day after ONI to 40% of levels normally seen after ONI. Chromatin immunoprecipitation provides direct evidence of enrichment of cFXIII-A genomic DNA bound with HSF-1. The present data indicate that rapid HSF-1 binding to the cFXIII-A gene results in cleavage of activation peptide and an active form of short cFXIII-A mRNA and protein in the zebrafish retina after ONI without thrombin.

Protein Carbonylation-Dependent Photoreceptor Cell Death Induced by N-Methyl-N-nitrosourea in Mice.

Retinal degenerative diseases, such as retinitis pigmentosa, are characterized by night blindness and peripheral vision loss caused by the slowly progressive loss of photoreceptor cells. A comprehensive molecular mechanism of the photoreceptor cell death remains unclear. We previously reported that heat shock protein 70 (HSP70), which has a protective effect on neuronal cells, was cleaved by a calcium-dependent protease, calpain, in N-methyl-N-nitrosourea (MNU)-treated mice retina. Carbonylated HSP70 is much more vulnerable than noncarbonylated HSP70 to calpain cleavage. However, it was not known whether protein carbonylation occurs in MNU-treated mice retina. In this study, we clearly show protein carbonylation-dependent photoreceptor cell death induced by MNU in mice. Therefore, protein carbonylation and subsequent calpain-dependent cleavage of HSP70 are key events in MNU-mediated photoreceptor cell death. Our data provide a comprehensive molecular mechanism of the photoreceptor cell death.

Alternative Splicing for Activation of Coagulation Factor XIII-A in the Fish Retina After Optic Nerve Injury.

Factor XIII-A (FXIII-A), which has become known as cellular transglutaminase, plays important roles in mediating cross-linking reactions in various tissues. FXIII-A acts as one of the regeneration molecules in the fish retina and optic nerve after optic nerve injury and becomes activated at the site of injury within a few hours. Previous research has shown that activated FXIII-A induces neurite outgrowth from injured retinal ganglion cells and supports elongation of the regenerating optic nerve. However, the activation mechanism of FXIII-A remains unknown. Furthermore, the injured tissues do not express thrombin, a known activator of plasma FXIII. Here, we investigated the mRNA expression of FXIII-A based on two different regions, one encoding the activation peptide and the other encoding the enzymatic active site. We found that expression of the region encoding the activation peptide was markedly suppressed compared with the region encoding the active site. An overexpression study with a short-type FXIII-A cDNA lacking the activation peptide revealed induction of long neurite outgrowth in fish retinal explant cultures compared with full-length FXIII-A cDNA. The present findings suggest that alternative splicing may occur in the FXIII-A gene, resulting in deletion of the region encoding the activation peptide and thus allowing direct production of activated FXIII-A protein in the fish retina and optic nerve after optic nerve injury.

Talaumidin Promotes Neurite Outgrowth of Staurosporine-Differentiated RGC-5 Cells Through PI3K/Akt-Dependent Pathways.

Talaumidin, a tetrahydrofuran neolignan isolated from the root of Aristolochia arcuata, was an interesting small molecule with neurotrophic activity in the cultured neuron. Talaumidin can promote neurite outgrowth from neurons. However, the mechanism by which talaumidin exerts its neurotrophic actions on retinal neurons has not been elucidated to date. In this study, we describe that talaumidin has neurotrophic properties such as neurite outgrowth in neuroretinal cell line, RGC-5. Talaumidin promotes staurosporine-induced neurite outgrowth in RGC-5 cells. The neurite outgrowth effect of talaumidin was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, but not by Erk inhibitor, PD98059. These data suggest that talaumidin promotes neurite outgrowth through PI3K/Akt pathway and that the potential of talaumidin serves as a promising lead compound for the treatment of retinal degenerative disorders.

Structure-activity relationships of talaumidin derivatives: Their neurite-outgrowth promotion in vitro and optic nerve regeneration in vivo.

(-)-Talaumidin (1), a 2,5-biaryl-3,4-dimethyltetrahydrofuran lignan, shows potent neurotrophic activities such as neurite-outgrowth promotion and neuroprotection. Previously, we found that (-)-(1S,2R,3S,4R)-stereoisomer 2 exhibited more significant activity than did the natural product talaumidin (1). However, the preparation of optically active (-)-2 requires a complicated synthetic route. To explore new neurotrophic compounds that can be obtained on a large scale, we established a short step synthetic route for talaumidin derivatives and synthesized fourteen analogues based on the structure of (-)-2. First, we synthesized a racemic compound of (-)-2 (2a) and assessed its neurotrophic activity. We found that the neurotrophic property of racemic 2a is similar in activity to that of (-)-2. Using the same synthetic methodology, several talaumidin derivatives were synthesized to optimize the oxy-functionality on aromatic rings. As a result, bis(methylenedioxybenzene) derivative 2b possessed the highest neurotrophic activity. Furthermore, examination of the structure-activity relationships of 2b revealed that the 2,5-diphenyl-tetrahydrofuran structure was an essential structure and that two methyl groups on THF ring could enhance neurotrophic activity. In addition, compounds 2a and 2b were found to induce mouse optic nerve regeneration in vivo.

Early Gene Expression Profile in Retinal Ganglion Cell Layer After Optic Nerve Crush in Mice.

Purpose: Optic nerve crush (ONC) induces retinal ganglion cell (RGC) death, which causes vision loss in glaucoma. To investigate early events leading to apoptosis of RGCs, we performed gene expression analysis of injured retinas in the period before RGC loss. Methods: The temporal changes of gene profiles at 0, 1, and 4 days after ONC were determined by DNA microarray. To verify the gene expression changes in RGCs, we enriched RGCs by laser-captured microdissection and performed real-time RT-PCR of 14 selected genes. In situ localization study was performed by immunohistochemistry. Results: At 1 day and 4 days after ONC, 1423 and 2010 retinal genes were changed compared with 0 day, respectively; these genes were mainly related to apoptotic process, immune process, regulation of cell cycle, and ion transport. RT-PCR analysis revealed that expression levels of Activating transcription factor 3 (Atf3), Lipocalin 2 (Lcn2), and tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) were remarkably changed in RGC-enriched fraction within 4 days postcrush. Immunohistochemical analysis confirmed that all of these genes expressed highly in the ganglion cell layer of crushed retinas. Conclusions: In response to ONC, the expression of apoptotic genes was stimulated soon after crush. Atf3, Lcn2, and Tnfrsf12a might be key molecules responsible for RGC loss in glaucoma.

S-Nitrosylation Regulates Cell Survival and Death in the Central Nervous System.

Nitric oxide (NO), which is produced from nitric oxide synthase, is an important cell signaling molecule that is crucial for many physiological functions such as neuronal death, neuronal survival, synaptic plasticity, and vascular homeostasis. This diffusible gaseous compound functions as an effector or second messenger in many intercellular communications and/or cell signaling pathways. Protein S-nitrosylation is a posttranslational modification that involves the covalent attachment of an NO group to the thiol side chain of select cysteine residues on target proteins. This process is thought to be very important for the regulation of cell death, cell survival, and gene expression in the central nervous system (CNS). However, there have been few reports on the role of protein S-nitrosylation in CNS disorders. Here, we briefly review specific examples of S-nitrosylation, with particular emphasis on its functions in neuronal cell death and survival. An understanding of the role and mechanisms underlying the effects of protein S-nitrosylation on neurodegenerative/neuroprotective events may reveal a novel therapeutic strategy for rescuing neurons in neurodegenerative diseases.

Mechanisms of RhoA inactivation and CDC42 and Rac1 activation during zebrafish optic nerve regeneration.

When axons of the mammalian central nervous system (CNS) are injured, they fail to regenerate, while those of lower vertebrates undergo regeneration after injury. Wingless-type MMTV integration site family (Wnt) proteins play important roles in the CNS, and are reported to be activated after mammalian spinal cord or brain injury. Moreover, for axon growth to proceed, it is thought that small G-proteins, such as CDC42 and Rac1, need to be activated, whereas RhoA must be inactivated. However, the cell and molecular mechanisms involved in optic nerve regeneration remain unclear. In this study, we investigated axonal regeneration after injury using the zebrafish optic nerve as a model system. We sought to clarify the role of Wnt proteins and the mechanisms involved in the activation and inactivation of small G-proteins in nerve regeneration. After optic nerve injury, mRNA levels of Wnt5b, TAX1BP3 and ICAT increased in the retina, while those of Wnt10a decreased. These changes were associated with a reduction in ß-catenin in nuclei. We found that Wnt5b activated CDC42 and Rac1, leading to the inactivation of RhoA, which appeared to be dependent on increased TAX1BP3 mRNA levels. Furthermore, we found that mRNA levels of Daam1a and ARHGEF16 decreased. We speculate that the decrease in ß-catenin levels, which also further reduces levels of active RhoA, might contribute to regeneration in the zebrafish. Collectively, our novel results suggest that Wnt5b, Wnt10a, ICAT and TAX1BP3 participate in the activation and inactivation of small G-proteins, such as CDC42, Rac1 and RhoA, during the early stage of optic nerve regeneration in the zebrafish.

A novel function of neuroglobin for neuroregeneration in mice after optic nerve injury.

Neuroglobin (Ngb) is a recently discovered heme protein in the vertebrate brain that can bind to oxygen molecules. Mammalian Ngb plays a crucial role in neuroprotection under conditions of oxidative stress. To investigate other potential functions of Ngb, we investigated the mouse retinal Ngb system following optic nerve injury. In the retina of control mice, Ngb immunoreactivity was limited to the retinal ganglion cell (RGC) layer, and this immunoreactivity rapidly decreased to less than 50% of the control level 5 days after optic nerve injury. On the basis of this decrease, we designed in vivo experiments with enhanced expression of Ngb using adult mouse retina. The enhanced expression of Ngb was achieved by injecting chimeric human Ngb protein, which included the cell membrane-penetrating module of fish Ngb. One-day pretreatment with chimeric Ngb increased immunoreactivity levels of Ngb two-fold in mouse RGCs and increased the number of surviving RGCs three-fold by 14 days after optic nerve injury compared with vehicle controls. Furthermore, in the mouse retinas showing enhanced Ngb expression, several regenerating central optic axons exhibited outgrowth and were found to pass through the nerve crush site 14 days after nerve injury. No such regenerating optic axons were observed in the control mouse optic nerve during the same time frame. The data obtained from in vivo experiments strongly indicate that mammalian Ngb has neuroprotective and neuroregenerative properties.