Early Animal Origin of BACE1 APP/Aß Proteolytic Function.

Alzheimer's disease is characterized, in part, by the accumulation of ß-amyloid (Aß) in the brain. Aß is produced via the proteolysis of APP by BACE1 and γ-secretase. Since BACE1 is the rate-limiting enzyme in the production of Aß, and a target for therapeutics, it is of interest to know when its proteolytic function evolved and for what purpose. Here, we take a functional evolutionary approach to show that BACE1 likely evolved from a gene duplication event near the base of the animal clade and that BACE1 APP/Aß proteolytic function evolved during early animal diversification, hundreds of millions of years before the evolution of the APP/Aß substrate. Our examination of BACE1 APP/Aß proteolytic function includes cnidarians, ctenophores, and choanoflagellates. The most basal BACE1 ortholog is found in cnidarians, while ctenophores, placozoa, and choanoflagellates have genes equally orthologous to BACE1 and BACE2. BACE1 from a cnidarian (Hydra) can cleave APP to release Aß, pushing back the date of the origin of its function to near the origin of animals. We tested more divergent BACE1/2 genes from a ctenophore (Mnemiopsis) and a choanoflagellate (Monosiga), and neither has this activity. These findings indicate that the specific proteolytic function of BACE1 evolved during the very earliest diversification of animals, most likely after a gene-duplication event.

Evaluation of Metabolic and Synaptic Dysfunction Hypotheses of Alzheimer's Disease (AD): A Meta-Analysis of CSF Markers.

BACKGROUND: Alzheimer's disease (AD) is currently incurable and a majority of investigational drugs have failed clinical trials. One explanation for this failure may be the invalidity of hypotheses focusing on amyloid to explain AD pathogenesis. Recently, hypotheses which are centered on synaptic and metabolic dysfunction are increasingly implicated in AD. OBJECTIVE: Evaluate AD hypotheses by comparing neurotransmitter and metabolite marker concentrations in normal versus AD CSF. METHODS: Meta-analysis allows for statistical comparison of pooled, existing cerebrospinal fluid (CSF) marker data extracted from multiple publications, to obtain a more reliable estimate of concentrations. This method also provides a unique opportunity to rapidly validate AD hypotheses using the resulting CSF concentration data. Hubmed, Pubmed and Google Scholar were comprehensively searched for published English articles, without date restrictions, for the keywords "AD", "CSF", and "human" plus markers selected for synaptic and metabolic pathways. Synaptic markers were acetylcholine, gamma-aminobutyric acid (GABA), glutamine, and glycine. Metabolic markers were glutathione, glucose, lactate, pyruvate, and 8 other amino acids. Only studies that measured markers in AD and controls (Ctl), provided means, standard errors/deviation, and subject numbers were included. Data were extracted by six authors and reviewed by two others for accuracy. Data were pooled using ratio of means (RoM of AD/Ctl) and random effects meta-analysis using Cochrane Collaboration's Review Manager software. RESULTS: Of the 435 identified publications, after exclusion and removal of duplicates, 35 articles were included comprising a total of 605 AD patients and 585 controls. The following markers of synaptic and metabolic pathways were significantly changed in AD/controls: acetylcholine (RoM 0.36, 95% CI 0.24-0.53, p<0.00001), GABA (0.74, 0.58-0.94, p<0.01), pyruvate (0.48, 0.24-0.94, p=0.03), glutathione (1.11, 1.01- 1.21, p=0.03), alanine (1.10, 0.98-1.23, p=0.09), and lower levels of significance for lactate (1.2, 1.00-1.47, p=0.05). Of note, CSF glucose and glutamate levels in AD were not significantly different than that of the controls. CONCLUSION: This study provides proof of concept for the use of meta-analysis validation of AD hypotheses, specifically via robust evidence for the cholinergic hypothesis of AD. Our data disagree with the other synaptic hypotheses of glutamate excitotoxicity and GABAergic resistance to neurodegeneration, given observed unchanged glutamate levels and decreased GABA levels. With regards to metabolic hypotheses, the data supported upregulation of anaerobic glycolysis, pentose phosphate pathway (glutathione), and anaplerosis of the tricarboxylic acid cycle using glutamate. Future applications of meta-analysis indicate the possibility of further in silico evaluation and generation of novel hypotheses in the AD field.

Asynchronous evolutionary origins of Aß and BACE1.

Neurodegenerative plaques characteristic of Alzheimer's disease (AD) are composed of amyloid beta (Aß) peptide, which is proteolyzed from amyloid precursor protein (APP) by ß-secretase (beta-site APP cleaving enzyme [BACE1]) and γ-secretase. Although γ-secretase has essential functions across metazoans, no essential roles have been identified for BACE1 or Aß. Because their only known function results in a disease phenotype, we sought to understand these components from an evolutionary perspective. We show that APP-like proteins are found throughout most animal taxa, but sequences homologous to Aß are not found outside gnathostomes and the ß cut site is only conserved within sarcopterygians. BACE1 enzymes, however, extend through basal chordates and as far as cnidaria. We then sought to determine whether BACE1 from a species that never evolved Aß could proteolyze APP substrates that include Aß. We demonstrate that BACE1 from a basal chordate is a functional ortholog that can liberate Aß from full-length human APP, indicating BACE1 activity evolved at least 360 My before Aß.

ER-targeted Bcl-2 and inhibition of ER-associated caspase-12 rescue cultured immortalized cells from ethanol toxicity.

Alcohol abuse, known for promoting apoptosis in the liver and nervous system, is a major public health concern. Despite significant morbidity and mortality resulting from ethanol consumption, the precise cellular mechanism of its toxicity remains unknown. Previous work has shown that wild-type Bcl-2 is protective against ethanol. The present study investigated whether protection from ethanol toxicity involves mitochondrial Bcl-2 or endoplasmic reticulum (ER) Bcl-2, and whether mitochondria-associated or ER-associated caspases are involved in ethanol toxicity. Chinese hamster ovary (CHO695) cells were transiently transfected with cDNA constructs encoding wild-type Bcl-2, mitochondria-targeted Bcl-2, or ER-targeted Bcl-2. MTT assay was used to measure cell viability in response to ethanol. Ethanol treatments of 1 and 2.5 M reduced cell viability at 5, 10, and 24 h. Wild-type Bcl-2, localized both to mitochondria and ER, provided significant rescue for CHO695 cells treated with 1M ethanol for 24 h, but did not rescue toxicity at 2.5 M. ER-targeted Bcl-2, however, provided significant and robust rescue following 24 h of 1 and 2.5 M ethanol. Mitochondria-targeted Bcl-2 offered no protection at any ethanol concentration and generally reduced cell viability. To follow up these experiments, we used a peptide inhibitor approach to investigate which caspases were responsible for ethanol-induced apoptosis. Caspase-9 and caspase-12 are known to be downstream of mitochondria and the ER, respectively. CHO695 cells were treated with a pan-caspase inhibitor, a caspase-9 or caspase-12 inhibitor along with 1.5 M ethanol, followed by MTT cell viability assay. Treatment with the pan-caspase inhibitor provided significant rescue from ethanol, whereas inhibition of caspase-9 did not. Inhibition of ER-associated caspase-12, however, conferred significant protection from ethanol toxicity, similar to the pan inhibitor. These findings are consistent with our transfection data and, taken together, suggest a significant role for the ER in ethanol toxicity.

Vertical and horizontal integration of bioinformatics education: A modular, interdisciplinary approach.

Bioinformatics education for undergraduates has been approached primarily in two ways: introduction of new courses with largely bioinformatics focus or introduction of bioinformatics experiences into existing courses. For small colleges such as Kalamazoo, creation of new courses within an already resource-stretched setting has not been an option. Furthermore, we believe that a true interdisciplinary science experience would be best served by introduction of bioinformatics modules within existing courses in biology and chemistry and other complementary departments. To that end, with support from the Howard Hughes Medical Institute, we have developed over a dozen independent bioinformatics modules for our students that are incorporated into courses ranging from general chemistry and biology, advanced specialty courses, and classes in complementary disciplines such as computer science, mathematics, and physics. These activities have largely promoted active learning in our classrooms and have enhanced student understanding of course materials. Herein, we describe our program, the activities we have developed, and assessment of our endeavors in this area.

Ethanol exposure alters neurotrophin receptor expression in the rat central nervous system: Effects of prenatal exposure.

Developmental ethanol exposure produces significant central nervous system (CNS) abnormalities. The cellular mechanisms of ethanol neurotoxicity, however, remain elusive. Recent data implicate altered neurotrophin signaling pathways in ethanol-mediated neuronal death. The present study investigated ethanol-induced alterations in neurotrophin receptor proteins in the rat CNS following chronic ethanol treatment during gestation, via liquid diet to pregnant dams. Brains were dissected on P1 and P10, and Western blots for the neurotrophin receptors TrkA, TrkB, TrkC, and p75 were quantified. Such ethanol treatment produced significant changes in neurotrophin receptor levels in the hippocampus, septum, cerebral cortex, and cerebellum. Receptor levels in hippocampus, septum, and cerebellum, tended to be decreased, while levels in cortex were consistently increased. Males were generally more affected than females. While most of these alterations were transient, sustained or delayed changes were present in P10 septum, cortex, and cerebellum. These results indicate that developmental ethanol exposure produces major changes in the normal physiological levels of the neurotrophin receptors throughout the CNS. These changes in the receptor complement during critical prenatal stages could relate to the anomalous development of the CNS seen in the fetal alcohol syndrome. This relationship is discussed, together with the potential biological effects of such dramatic changes in neurotrophin receptor expression.

Ethanol exposure alters neurotrophin receptor expression in the rat central nervous system: Effects of neonatal exposure.

The detrimental effects of ethanol exposure during nervous system development have been well established. The cellular mechanisms of ethanol neurotoxicity, however, have not been clearly defined. Recent studies suggest that neurotrophin signaling pathways may be involved in ethanol-mediated neuronal death. The present investigation, therefore, was designed to examine ethanol-induced alterations in neurotrophin receptor protein levels in the developing central nervous system (CNS) following chronic ethanol treatment administered during the early neonatal period. For this study, rats were exposed to ethanol via vapor inhalation from postnatal day 4 (P4) to P10. Brains were then dissected on P10 or P21, and Western blots used to quantify expression of neurotrophin receptors TrkA, TrkB, TrkC, and p75. This early postnatal ethanol treatment produced significant alterations in receptor levels in hippocampus, septum, cerebral cortex, and cerebellum. The alterations seen were variable, with decreases generally found in hippocampus and cerebellum, increases noted in septum, and changes in both directions occurring in cortex. These alterations were generally more prevalent in males than in females. While most of the receptor changes observed were transient, sustained or delayed alterations were occasionally seen in hippocampus, cortex, and cerebellum. These results suggest that developmental ethanol exposure modulates expression of these neurotrophin receptors throughout the CNS, alterations which could have wide-ranging effects on functional CNS development. The possible linkage between such changes and abnormalities encountered in the fetal alcohol syndrome are considered.

The role of neurotrophic factors, apoptosis-related proteins, and endogenous antioxidants in the differential temporal vulnerability of neonatal cerebellum to ethanol.

BACKGROUND: Ethanol produces abnormalities in the developing nervous system, with certain regions being vulnerable during well-defined periods. Neonatal rodent cerebellum is particularly susceptible to ethanol during the early postnatal period [on postnatal days 4-5 (P4-5)], while this region is resistant to ethanol at a slightly later time (P7-9). We assessed basal levels of several substances which may be involved in differential temporal ethanol vulnerability in neonatal cerebellum, and analyzed alterations in these substances after early ethanol exposure. METHODS: Assessments were made of neurotrophic factors nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4; apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Bcl-xs, Bad, phosphorylated-Bad, phosphorylated-Akt, and phosphorylated-c-Jun N-terminal kinase; and the antioxidants superoxide dismutase, glutathione reductase, and catalase. These analyses quantified basal levels (in controls), and sequential changes following acute ethanol exposure at the vulnerable and resistant cerebellar periods (P4, P7). RESULTS: Comparisons of basal levels of the molecules assessed between P4 and P7 revealed higher levels of total proapoptotic Bad at p4, higher levels of the protective pAkt kinase at P7, and lower levels of proapoptotic pJNK at P7. Other basal levels did not differ. While ethanol-mediated alterations were found at both ages favoring both apoptosis and survival, the apoptosis-promoting changes produced on P4 exceeded those on P7, and most occurred within the first 2 hr after exposure, a critical survival/death period. The number of alterations favoring survival were similar at the two ages, but at P7 most occurred within the first 2 hr after exposure, possibly acting in a protective manner. CONCLUSIONS: Differential temporal vulnerability to ethanol in the neonatal cerebellum appears to be paralleled by cellular alterations in neurotrophic factors, apoptosis-regulatory proteins, and/or antioxidant activities which generally favor apoptosis at the most sensitive age and survival at the resistant age.

Effects of ethanol on neurotrophic factors, apoptosis-related proteins, endogenous antioxidants, and reactive oxygen species in neonatal striatum: relationship to periods of vulnerability.

The developing central nervous system is extremely sensitive to ethanol, with well-defined temporal periods of vulnerability. Many brain regions are particularly susceptible to ethanol during the early neonatal period, corresponding to the human third trimester, which represents a dynamic period of growth and differentiation. For this study, neonatal rats were acutely exposed to ethanol or control conditions at a neonatal age when the developing striatum has been shown to be vulnerable to ethanol (postnatal day 3 [P3]), and at a later age (P14), when this developing region is relatively ethanol-resistant. We then analyzed basal levels of neurotrophic factors (NTFs), and ethanol-mediated changes in NTFs, apoptosis-related proteins, antioxidants, and reactive oxygen species (ROS) generation, which may underlie this differential temporal vulnerability. Sequential analyses were made following ethanol exposure on these two postnatal days, with assessments of NTFs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4); apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Akt and c-jun N-terminal kinase (JNK); antioxidants superoxide dismutase, glutathione reductase and catalase; and ROS. The results indicated that basal levels of BDNF, and to some degree NGF, were greater at the older age, and that ethanol exposure at the earlier age elicited considerably more pro-apoptotic and fewer pro-survival changes than those produced at the later age. Thus, differential temporal vulnerability to ethanol in this CNS region appears to be related to differences in both differential levels of protective substances (e.g. NTFs), and differential cellular responsiveness which favors apoptosis at the most sensitive age and survival at the resistant age.