Uman-type neurofilament light antibodies are effective reagents for the imaging of neurodegeneration.

Recent work shows that certain antibody-based assays for the neurofilament light chain detect informative signals in the CSF and blood of human and animals affected by a variety of CNS injury and disease states. Much of this work has been performed using two mouse monoclonal antibodies to neurofilament light, UD1 and UD2, also known as Clones 2.1 and 47.3, respectively. These are the essential components of the Uman Diagnostics Neurofilament-Light™ ELISA kit, the Quanterix Simoa™ bead-based assay and others. We show that both antibodies bind to neighbouring epitopes in a short, conserved and unusual peptide in the centre of the neurofilament light Coil 2 segment of the 'rod' domain. We also describe a surprising and useful feature of Uman and similar reagents. While other well-characterized neurofilament antibodies generally show robust staining of countless cells and processes in CNS sections from healthy rats, both Uman antibodies reveal only a minor subset of profiles, presumably spontaneously degenerating or degenerated neurons and their processes. However, following experimental mid-cervical spinal cord injuries to rats, both Uman antibodies recognize numerous profiles in fibre tracts damaged by the injury administered. These profiles were typically swollen, beaded, discontinuous or sinusoidal as expected for degenerating and degenerated processes. We also found that several antibodies to the C-terminal 'tail' region of the neurofilament light protein bind undamaged axonal profiles but fail to recognize the Uman-positive material. The unmasking of the Uman epitopes and the loss of the neurofilament light tail epitopes can be mimicked by treating sections from healthy animals with proteases suggesting that the immunostaining changes we discovered are due to neurodegeneration-induced proteolysis. We have also generated a novel panel of monoclonal and polyclonal antibodies directed against the Uman epitopes that have degeneration-specific staining properties identical to the Uman reagents. Using these, we show that the region to which the Uman reagents bind contains further hidden epitopes distinct from those recognized by the two Uman reagents. We speculate that the Uman-type epitopes are part of a binding region important for higher order neurofilament assembly. The work provides important insights into the properties of the Uman assay, describes novel and useful properties of Uman-type and neurofilament light tail-binding antibodies and provides a hypothesis relevant to further understanding of neurofilament assembly.

TNFα increases tyrosine hydroxylase expression in human monocytes.

Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. Here, we report the development of a highly sensitive and reproducible Bio-ELISA to quantify picogram levels of TH in multiple model systems. We have applied this assay to monocytes isolated from blood of persons with Parkinson's disease (PD) and to age-matched, healthy controls. Our study unexpectedly revealed that PD patients' monocytes express significantly higher levels of TH protein in peripheral monocytes relative to healthy controls. Tumor necrosis factor (TNFα), a pro-inflammatory cytokine, has also been shown to be increased in the brains and peripheral circulation in human PD, as well as in animal models of PD. Therefore, we investigated a possible connection between higher levels of TH protein and the known increase in circulating TNFα in PD. Monocytes isolated from healthy donors were treated with TNFα or with TNFα in the presence of an inhibitor. Tissue plasminogen activator (TPA) was used as a positive control. We observed that TNFα stimulation increased both the number of TH+ monocytes and the quantity of TH per monocyte, without increasing the total numbers of monocytes. These results revealed that TNFα could potentially modify monocytic TH production and serve a regulatory role in peripheral immune function. The development and application of a highly sensitive assay to quantify TH in both human and animal cells will provide a novel tool for further investigating possible PD immune regulatory pathways between brain and periphery.

Erythropoietin Does Not Alter Serum Profiles of Neuronal and Axonal Biomarkers After Traumatic Brain Injury: Findings From the Australian EPO-TBI Clinical Trial.

OBJECTIVE: To determine profiles of serum ubiquitin carboxy-terminal hydrolase L1 and phosphorylated neurofilament heavy-chain, examine whether erythropoietin administration reduce their concentrations, and whether biomarkers discriminate between erythropoietin and placebo treatment groups. DESIGN: Single-center, prospective observational study. SETTING: A sub-study of the erythropoietin-traumatic brain injury clinical trial, conducted at the Alfred Hospital, Melbourne, Australia. PATIENTS: Forty-four patients with moderate-to-severe traumatic brain injury. INTERVENTIONS: Epoetin alfa 40,000 IU or 1 mL sodium chloride 0.9 as subcutaneous injection within 24 hours of traumatic brain injury. MEASUREMENTS AND MAIN RESULTS: Ubiquitin carboxy-terminal hydrolase L1, phosphorylated neurofilament heavy-chain, and erythropoietin concentrations were measured in serum by enzyme-linked immunosorbent assay from D0 (within 24 hr of injury, prior to erythropoietin/vehicle administration) to D5. Biomarker concentrations were compared between injury severities, diffuse versus focal traumatic brain injury and erythropoietin or placebo treatment groups. Ubiquitin carboxy-terminal hydrolase L1 peaked at 146.0 ng/mL on D0, significantly decreased to 84.30 ng/mL on D1, and declined thereafter. Phosphorylated neurofilament heavy-chain levels were lowest at D0 and peaked on D5 at 157.9 ng/mL. D0 ubiquitin carboxy-terminal hydrolase L1 concentrations were higher in diffuse traumatic brain injury. Peak phosphorylated neurofilament heavy-chain levels on D3 and D4 correlated with Glasgow Outcome Score-Extended, predicting poor outcome. Erythropoietin did not reduce concentrations of ubiquitin carboxy-terminal hydrolase L1 or phosphorylated neurofilament heavy-chain. CONCLUSIONS: Serum ubiquitin carboxy-terminal hydrolase L1 and phosphorylated neurofilament heavy-chain increase after traumatic brain injury reflecting early neuronal and progressive axonal injury. Consistent with lack of improved outcome in traumatic brain injury patients treated with erythropoietin, biomarker concentrations and profiles were not affected by erythropoietin. Pharmacokinetics of erythropoietin suggest that the dose given was possibly too low to exert neuroprotection.

Biochemical characterization of protein quality control mechanisms during disease progression in the C22 mouse model of CMT1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin-proteasome system (UPS), autophagosomes and heat-shock proteins (HSPs). Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins.

Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase.

The formation of primary cilia is a highly choreographed process that can be disrupted in developing neurons by overexpressing neuromodulatory G-protein-coupled receptors GPCRs or by blocking intraflagellar transport. Here, we examined the effects of overexpressing the ciliary GPCRs, 5HT6 and SSTR3, on cilia structure and the differentiation of neocortical neurons. Neuronal overexpression of 5HT6 and SSTR3 was achieved by electroporating mouse embryo cortex in utero with vectors encoding these receptors. We found that overexpression of ciliary GPCRs in cortical neurons, especially 5HT6, induced the formation of long (>30 µm) and often forked cilia. These changes were associated with increased levels of intraflagellar transport proteins and accelerated ciliogenesis in neonatal neocortex, the induction of which required Kif3a, an anterograde motor critical for cilia protein trafficking and growth. GPCR overexpression also altered the complement of signaling molecules within the cilia. We found that SSTR3 and type III adenylyl cyclase (ACIII), proteins normally enriched in neuronal cilia, were rarely detected in 5HT6-elongated cilia. Intriguingly, the changes in cilia structure were accompanied by changes in neuronal morphology. Specifically, disruption of normal ciliogenesis in developing neocortical neurons, either by overexpressing cilia GPCRs or a dominant-negative form of Kif3a, significantly impaired dendrite outgrowth. Remarkably, coexpression of ACIII with 5HT6 restored ACIII to cilia, normalized cilia structure, and restored dendrite outgrowth, effects that were not observed in neurons coexpressing ACIII and dominant-negative form of Kif3a. Collectively, our data suggest the formation of neuronal dendrites in developing neocortex requires structurally normal cilia enriched with ACIII.

Activation of phospholipase C mimics the phase shifting effects of light on melatonin rhythms in retinal photoreceptors.

Many aspects of retinal photoreceptor function and physiology are regulated by the circadian clocks in these cells. It is well established that light is the primary stimulus that entrains these clocks; yet, the biochemical cascade(s) mediating light's effects on these clocks remains unknown. This deficiency represents a significant gap in our fundamental understanding of photoreceptor signaling cascades and their functions. In this study, we utilized re-aggregated spheroid cultures prepared from embryonic chick retina to determine if activation of phospholipase C in photoreceptors in the absence of light can phase shift the melatonin secretion rhythms of these cells in a manner similar to that induced by light. We show that spheroid cultures rhythmically secrete melatonin and that these melatonin rhythms can be dynamically phase shifted by exposing the cultures to an appropriately timed light pulse. Importantly, we show that activation of phospholipase C using m-3M3FBS in the absence of light induces a phase delay in photoreceptor melatonin rhythms that mirrors that induced by light. The implication of this finding is that the light signaling cascade that entrains photoreceptor melatonin rhythms involves activation of phospholipase C.

Bicistronic lentiviruses containing a viral 2A cleavage sequence reliably co-express two proteins and restore vision to an animal model of LCA1.

The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase-1 (GC1) and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis-1 (LCA1). GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies.

Gene expression in the hippocampus: regionally specific effects of aging and caloric restriction.

We measured changes in gene expression, induced by aging and caloric restriction (CR), in three hippocampal subregions. When analysis included all regions, aging was associated with expression of genes linked to mitochondrial dysfunction, inflammation, and stress responses, and in some cases, expression was reversed by CR. An age-related increase in ubiquintination was observed, including increased expression of ubiquitin conjugating enzyme genes and cytosolic ubiquitin immunoreactivity. CR decreased cytosolic ubiquitin and upregulated deubiquitinating genes. Region specific analyses indicated that CA1 was more susceptible to aging stress, exhibiting a greater number of altered genes relative to CA3 and the dentate gyrus (DG), and an enrichment of genes related to the immune response and apoptosis. CA3 and the DG were more responsive to CR, exhibiting marked changes in the total number of genes across diet conditions, reversal of age-related changes in p53 signaling, glucocorticoid receptor signaling, and enrichment of genes related to cell survival and neurotrophic signaling. Finally, CR differentially influenced genes for synaptic plasticity in CA1 and CA3. It is concluded that regional disparity in response to aging and CR relates to differences in vulnerability to stressors, the availability of neurotrophic, and cell survival mechanisms, and differences in cell function.

Rapamycin activates autophagy and improves myelination in explant cultures from neuropathic mice.

Misexpression and cytosolic retention of peripheral myelin protein 22 (PMP22) within Schwann cells (SCs) is associated with a genetically heterogeneous group of demyelinating peripheral neuropathies. PMP22 overproducer C22 and spontaneous mutant Trembler J (TrJ) mice display neuropathic phenotypes and affected nerves contain abnormally localized PMP22. Nutrient deprivation-induced autophagy is able to suppress the formation of PMP22 aggregates in a toxin-induced cellular model, and improve locomotor performance and myelination in TrJ mice. As a step toward therapies, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could facilitate the processing of PMP22 within neuropathic SCs and enhance their capacity to myelinate peripheral axons. Exposure of mouse SCs to RM induced autophagy in a dose- and time-dependent manner and decreased the accumulation of poly-ubiquitinated substrates. The treatment of myelinating dorsal root ganglion (DRG) explant cultures from neuropathic mice with RM (25 nm) improved the processing of PMP22 and increased the abundance and length of myelin internodes, as well as the expression of myelin proteins. Notably, RM is similarly effective in both the C22 and TrJ model, signifying that the benefit overlaps among distinct genetic models of PMP22 neuropathies. Furthermore, lentivirus-mediated shRNA knockdown of the autophagy-related gene 12 (Atg12) abolished the activation of autophagy and the increase in myelin proteins, demonstrating that autophagy is critical for the observed improvement. Together, these results support the potential use of RM and other autophagy-enhancing compounds as therapeutic agents for PMP22-associated demyelinating neuropathies.

Reduction of Dicer impairs Schwann cell differentiation and myelination.

The process of Schwann cell myelination requires precisely coordinated gene expression. At the onset of myelination, there is an increase in the expression of differentiation-promoting transcription factors that regulate key Schwann cell genes. Further control of myelin gene expression occurs at the posttranscriptional level and, in part, is mediated by RNA binding proteins and micro-RNAs (miRNAs). miRNAs are small, endogenously derived RNA molecules that repress gene expression by specifically binding to their mRNA targets. In the experiments described here, we tested whether miRNAs were essential in controlling myelination by reducing the levels of Dicer, an essential endoribonuclease in miRNA biogenesis. We decreased the expression of Dicer by about 60% within Schwann cells using a lentiviral vector expressing an shRNA against Dicer. The reduced levels of Dicer led to a decrease in the steady-state expression of selected miRNAs and of the transcription factors Oct6 and Egr2/Krox20, both of which are critical for Schwann cells differentiation and myelination. In contrast, the levels of c-jun and Sox2 were up-regulated by the reduction in Dicer and were associated with an increase in Schwann cell proliferation. In dorsal root ganglion cocultures, Schwann cells transduced with Dicer shRNA synthesized less myelin, which was accompanied by significant reductions in the levels of myelin basic protein and protein zero. These findings support a critical role for Dicer and miRNAs in Schwann cell differentiation and myelination.

Lifelong calorie restriction alleviates age-related oxidative damage in peripheral nerves.

Aging is associated with protein damage and imbalance in redox status in a variety of cells and tissues, yet little is known about the extent of age-related oxidative stress in the peripheral nervous system. Previously, we showed a drastic decline in the expression of glial and neuronal proteins in myelinated peripheral nerves with age, which is significantly ameliorated by lifelong calorie restriction. The age-related decline in functional molecules is associated with alterations in cellular protein homeostatic mechanisms, which could lead to a buildup of damaged, aggregated proteins. To determine the extent of oxidative damage within myelinated peripheral nerves, we studied sciatic nerves from rats of four different ages (8, 18, 29, and 38 months) maintained on an ad libitum or a 40% calorie-restricted diet. We found a prominent accumulation of polyubiquitinated substrates with age, which are associated with the conglomeration of distended lysosomes and lipofuscin adducts. The occurrence of these structures is notably less frequent within nerves of age-matched rodents kept on a lifelong reduced calorie diet. Markers for lipid peroxidation, inflammation, and immune cell infiltration are all elevated in nerves of ad libitum-fed rats, whereas food restriction is able to attenuate such deleterious processes with age. Together these results show that dietary restriction is an efficient means of defying age-related oxidative damage and maintaining a younger state in peripheral nerves.

Intermittent fasting alleviates the neuropathic phenotype in a mouse model of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth type 1A (CMT1A) neuropathies linked to the misexpression of peripheral myelin protein 22 (PMP22) are progressive demyelinating disorders of the peripheral nervous system. In this study we asked whether dietary restriction by intermittent fasting (IF) could alleviate the neuropathic phenotype in the Trembler J (TrJ) mouse model of CMT1A. Our results show that neuropathic mice kept on a five month long IF regimen had improved locomotor performance compared to ad libitum (AL) fed littermates. The functional benefits of this dietary intervention are associated with an increased expression of myelin proteins combined with a thicker myelin sheath, less redundant basal lamina, and a reduction in aberrant Schwann cell proliferation. These morphological improvements are accompanied by a decrease in PMP22 protein aggregates, and enhanced expression of cytosolic chaperones and constituents of the autophagy-lysosomal pathway. These results indicate that dietary restriction is beneficial for peripheral nerve function in TrJ neuropathic mice, as it promotes the maintenance of locomotor performance.

Molecular architecture of myelinated peripheral nerves is supported by calorie restriction with aging.

Peripheral nerves from aged animals exhibit features of degeneration, including marked fiber loss, morphological irregularities in myelinated axons and notable reduction in the expression of myelin proteins. To investigate how protein homeostatic mechanisms change with age within the peripheral nervous system, we isolated Schwann cells from the sciatic nerves of young and old rats. The responsiveness of cells from aged nerves to stress stimuli is weakened, which in part may account for the observed age-associated alterations in glial and axonal proteins in vivo. Although calorie restriction is known to slow the aging process in the central nervous system, its influence on peripheral nerves has not been investigated in detail. To determine if dietary restriction is beneficial for peripheral nerve health and glial function, we studied sciatic nerves from rats of four distinct ages (8, 18, 29 and 38 months) kept on an ad libitum (AL) or a 40% calorie restricted diet. Age-associated reduction in the expression of the major myelin proteins and widening of the nodes of Ranvier are attenuated by the dietary intervention, which is paralleled with the maintenance of a differentiated Schwann cell phenotype. The improvements in nerve architecture with diet restriction, in part, are underlined by sustained expression of protein chaperones and markers of the autophagy-lysosomal pathway. Together, the in vitro and in vivo results suggest that there might be an age-limit by which dietary intervention needs to be initiated to elicit a beneficial response on peripheral nerve health.

Pharmacological induction of the heat shock response improves myelination in a neuropathic model.

Misexpression and intracellular retention of peripheral myelin protein 22 (PMP22) is associated with hereditary neuropathies in humans, including Charcot-Marie-Tooth disease type 1A (CMT1A). Mice expressing extra copies of the human PMP22, termed C22, display morphologic and behavioral characteristics of CMT1A. In neuropathic Schwann cells, the turnover of the newly-synthesized PMP22 is decreased, leading to the formation of cytosolic protein aggregates. To aid the processing of PMP22 and alleviate the associated myelin defects, we pharmacologically stimulated the expression of protein chaperones by synthetic small-molecule inhibitors of heat shock protein 90 (HSP90). The exposure of Schwann cells to these compounds enhanced the levels of cytosolic chaperones in a time- and dose-dependent manner, with minimal cytotoxicity. Treatment of dorsal root ganglion (DRG) explants from neuropathic mice improved myelin formation and the processing of PMP22. These results warrant further studies with HSP90 inhibitors as potential therapeutic candidates for hereditary demyelinating neuropathies.

The formation of peripheral myelin protein 22 aggregates is hindered by the enhancement of autophagy and expression of cytoplasmic chaperones.

The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Peripheral myelin protein 22 (PMP22) is a hereditary neuropathy-linked, short-lived molecule that forms aggresomes when the proteasome is inhibited or the protein is mutated. We previously showed that the removal of pre-existing PMP22 aggregates is assisted by autophagy. Here we examined whether the accumulation of such aggregates could be suppressed by experimental induction of autophagy and/or chaperones. Enhancement of autophagy during proteasome inhibition hinders protein aggregate formation and correlates with a reduction in accumulated proteasome substrates. Conversely, simultaneous inhibition of autophagy and the proteasome augments the formation of aggregates. An increase of heat shock protein levels by geldanamycin treatment or heat shock preconditioning similarly hampers aggresome formation. The beneficial effects of autophagy and chaperones in preventing the accumulation of misfolded PMP22 are additive and provide a potential avenue for therapeutic approaches in hereditary neuropathies linked to PMP22 mutations.

Effects of acute ethanol exposure on regulatory mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat cerebellum: differential effects during vulnerable and resistant developmental periods.

BACKGROUND: Prenatal alcohol exposure produces anatomical and behavioral abnormalities associated with fetal alcohol syndrome (FAS). Animal FAS models have demonstrated temporal windows of vulnerability in the developing cerebellum, with substantial ethanol (EtOH)-mediated apoptotic activation during these periods. In rodents, the cerebellum is most sensitive to EtOH on postnatal days 4 to 6 (P4 to P6). At slightly later ages (P7 and later), this region is less vulnerable to EtOH. The present study investigated EtOH effects on mechanisms related to activities of Bad, a proapoptotic member of the Bcl-2 gene family, to further characterize processes underlying these disparate EtOH sensitivities. In healthy cells, Bad is retained in the cytosol by association with 14-3-3, a primarily cytosolic protein. Bad promotes apoptosis by disassociating from 14-3-3 and sequestering Bcl-xL through heterodimerization. This dimerization prevents the neutralizing association of Bcl-xL with Bax, freeing Bax to perform in a prodeath manner. Caspase-dependent cleavage of Bad to a 15-kDa fragment increases its proapoptogenic capacity. METHODS: Two hours following EtOH exposure of P4 and P7 animals via inhalation, we determined how exposure affects intracellular localization and proteolytic cleavage of Bad and expression of cerebellar 14-3-3, using subcellular fractionation and Western blot techniques. Ethanol effects on interactions between Bad and 14-3-3 or Bcl-xL at the more vulnerable and less vulnerable ages were determined using an enzyme-linked immunosorbent assay-based technique to detect native protein-protein interactions. RESULTS: At P4, EtOH increased mitochondrial localization of Bad, expression of a 15-kDa fragment recognized by Bad antibody, and formation of Bad:Bcl-xL complexes. At that more vulnerable age, EtOH also decreased formation of Bad:14-3-3 complexes. At P7, EtOH increased Bad:14-3-3 complexes and reduced Bad:Bcl-xL complexes. Cytosolic 14-3-3 remained unchanged by EtOH at P4 and P7. CONCLUSIONS: Ethanol-induced alterations of Bad-related mechanisms at P4 favor a prodeath response. EtOH does not influence these same mechanisms in a manner that promotes cell death at P7. Divergent Bad-related responses at these 2 developmental ages likely contribute to their differential EtOH vulnerability.

Effect of bax deletion on ethanol sensitivity in the neonatal rat cerebellum.

The developing cerebellum is highly sensitive to ethanol during discrete neonatal periods. This sensitivity has been linked to ethanol-induced alterations in molecules of the Bcl-2 survival-regulatory gene family. Ethanol exposure during peak periods of cerebellar sensitivity, for example, results in increased expression of proapoptotic proteins of this family, while overexpression of the antiapoptotic Bcl-2 protein in the nervous system protects against ethanol neurotoxicity. For the present study, neonatal mice with a targeted deletion of the proapoptotic bax gene were used to determine whether elimination of this protein would mitigate ethanol toxicity. bax knock-out and wild-type mice pups were exposed to ethanol via vapor inhalation during the maximal period of neonatal cerebellar ethanol sensitivity and cerebellar tissue was subsequently assessed for Purkinje and granule cell number and ethanol-mediated generation of reactive oxygen species (ROS). The results revealed that: (1) ethanol exposure during the peak period of cerebellar vulnerability resulted in substantial loss of Purkinje cells in wild-type animals, but not in bax knock-outs; (2) granule cells in the bax gene-deleted animals were not similarly protected from ethanol effects; and (3) levels of ROS following acute ethanol exposure were appreciably enhanced in the wild-type animals but not in the bax knock-outs. These results imply that Bax is important to ethanol-induced Purkinje cell death during critical neonatal periods, but that ethanol effects on granule cells may function at least partially independent of this apoptosis agonist. Amelioration of ethanol-mediated increases in ROS production in the knock-outs may contribute to the observed effects.

Functional mechanisms of apoptosis-related proteins in neonatal rat cerebellum are differentially influenced by ethanol at postnatal days 4 and 7.

Exposure of the developing nervous system to ethanol (EtOH) produces neurological aberrations associated with fetal alcohol syndrome. During a well-defined period, cerebellar neurons are highly susceptible to EtOH-induced death, primarily through apoptosis. Neonatal rodent cerebellum is exquisitely sensitive to EtOH on postnatal days 4-6 (P4-6); however, at slightly later developmental ages (P7 and later), EtOH effects are minimal. We have previously shown that EtOH differentially influences expression of apoptosis-related proteins of the Bcl-2 survival-regulatory gene family in P4 and P7 cerebellum. In the present study, the effects of EtOH on multiple functional mechanisms of Bcl-2, Bcl-xL, and Bax were investigated to characterize further the processes underlying these disparate EtOH sensitivities. For these analyses, we addressed the following questions, by using P4 and P7 cerebellar tissue following in vivo exposure: 1) Are there differential patterns of expression of antiapoptotic Bcl-2 or proapoptotic Bax in EtOH-vulnerable Purkinje cells that could contribute to the different degrees of temporal EtOH vulnerability? 2) How does EtOH affect intracellular localization of apoptosis-related proteins? 3) Does cleavage of Bax contribute to EtOH sensitivity? 4) Does EtOH differentially modulate cerebellar protein-protein interactions of Bcl-2, Bcl-xL, and Bax at the vulnerable vs. the resistant ages? Overall, we show that, at P4, the EtOH-mediated effects on Bcl-2, Bcl-xL, and Bax favor a prodeath response, whereas most of the intracellular responses to EtOH exposure at P7 promote survival. Such differential responsiveness likely plays a major role in the disparate ethanol vulnerability at these two postnatal ages.

Mitochondrially targeted vitamin E and vitamin E mitigate ethanol-mediated effects on cerebellar granule cell antioxidant defense systems.

Ethanol (EtOH) disrupts the structure and function of the developing nervous system, sometimes leading to birth defects associated with fetal alcohol syndrome (FAS). Animal FAS models indicate that cellular membrane peroxidation, intracellular oxidant accumulation, and suppression of endogenous antioxidant enzymes contribute to the toxic effects of EtOH. Mitochondrially targeted vitamin E (MitoVit E), a chemically engineered form of vitamin E (VE) designed to accumulate in the mitochondria, has been shown to inhibit intracellular oxidant accumulation and cell death more effectively than VE. In previous investigations, we have shown that, in vivo, VE reduces neuronal death in the developing cerebellum of EtOH-exposed animals, and, in vitro, VE prevents apoptotic and necrotic death of EtOH-exposed cerebellar granule cells (CGCs). The present investigation shows that, in a FAS CGC model, 1 nM MitoVit E renders significant neuroprotection against EtOH concentrations as high as 1600 mg/dL. The present study also demonstrates that, in this same model, MitoVit E mitigates EtOH-induced accumulation of intracellular oxidants and counteracts suppression of glutathione peroxidase/glutathione reductase (GSH-Px/GSSG-R) functions, protein expression of gamma-glutamylcysteine synthetase (gamma-GCS), and total cellular glutathione (GSH) levels. In the presence and absence of EtOH, VE amplifies the protein expression levels of gamma-GCS, an enzyme that performs the rate-limiting step for GSH synthesis, and total GSH levels. These results suggest that MitoVit E and VE ameliorate EtOH toxicity through non-oxidant mechanisms-modulations of endogenous cellular proteins-and antioxidant means.