Cellular source of apolipoprotein E4 determines neuronal susceptibility to excitotoxic injury in transgenic mice.

The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimer's disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.

Exogenous low dose hydrogen peroxide increases hypoxia-inducible factor-1alpha protein expression and induces preconditioning protection against ischemia in primary cortical neurons.

HIF-1 is believed to play a critical role in hypoxia/ischemia (H/I) preconditioning protection in neonatal brain. Recently, it has been shown that hydrogen peroxide (H(2)O(2)) may contribute to H/I preconditioning in rat primary neurons. We hypothesize that H(2)O(2) produced during H/I preconditioning may increase HIF-1alpha protein expression and contribute to H/I preconditioning protection in the immature brain. To test this hypothesis, we used 6-8 days in vitro (DIV) primary cortical neurons from embryonic day 16 CD1 mouse brains and preconditioned them with 10 min of oxygen and glucose deprivation (OGD) or exogenous H(2)O(2) at doses from 5 to 50 microM. Both OGD and low dose H(2)O(2) (15 microM) preconditioning provided neuronal protection 24 h later against a 2 h OGD insult. Cell survival was 34.9+/-1.8% and 35.8+/-3.8% with OGD and H(2)O(2) preconditioning respectively vs. 20.0+/-0.4% without preconditioning (P<0.01). After OGD preconditioning, HIF-1alpha protein increased at 4 h and peaked at 8h, then declined at 18 h and increased again to reach another peak at 32 h. HIF-1alpha protein following H(2)O(2) preconditioning increased at 8h and peaked at 32 h. For both preconditioning paradigms, HIF-1alpha expression level declined to baseline at 72 h. Our results suggest that low levels of H(2)O(2) may up-regulate HIF-1alpha protein and thereby mediate H/I preconditioning protection.

Activated Src kinases interact with the N-methyl-D-aspartate receptor after neonatal brain ischemia.

OBJECTIVE: Neonatal stroke is associated with the N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxic brain injury. Src family kinases (SFKs) are considered to be the molecular hub for NMDAR regulation. We determined the relationship between SFKs activation and NMDAR tyrosine phosphorylation after neonatal hypoxia-ischemia (HI) and investigated the neuroprotective potential of a selective SFKs inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyramidine), against neonatal brain ischemic injury. METHODS: The Rice-Vannucci model was adapted for neonatal HI injury in postnatal day 7 CD1 mice. SFKs activity in the postsynaptic densities was measured by Western blot. NMDAR tyrosine phosphorylation and their association with SFKs were determined by coimmunoprecipitation. Brains from animals treated with PP2 or its inactive analog, PP3, were examined histologically with cresyl violet and iron stain to assess the degree of damage. RESULTS: Neonatal HI resulted in a rapid and transient increase in tyrosine phosphorylation of NMDAR subunits NR2A and NR2B. This upregulation correlated with the enhanced association of Fyn and Src with NR2A and NR2B. SFKs were activated in the postsynaptic densities after HI. Inhibition of SFKs with PP2 attenuated brain injury after neonatal HI, whereas PP3 did not protect the brain from the HI insult. INTERPRETATION: SFKs may play an important role in NMDAR-mediated excitotoxicity and downstream events leading to neuronal death after neonatal HI. Inhibition of SFKs may provide protection against neonatal stroke. Rather than blockade of NMDAR after HI in the developing brain, it may be safer and more beneficial to manipulate components of the NMDAR signaling complex at the postsynaptic density.

Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity.

Apolipoprotein (apo) E4, a 299-aa protein and a major risk factor for Alzheimer's disease, can be cleaved to generate C-terminal-truncated fragments that cause neurotoxicity in vitro and neurodegeneration and behavioral deficits in transgenic mice. To investigate this neurotoxicity, we expressed apoE4 with C- or N-terminal truncations or mutations in transfected Neuro-2a cells. ApoE4 (1-272) was neurotoxic, but full-length apoE4(1-299) and apoE4(1-240) were not, suggesting that the lipid-binding region (amino acids 241-272) mediates the neurotoxicity and that amino acids 273-299 are protective. A quadruple mutation in the lipid-binding region (I250A, F257A, W264R, and V269A) abolished the neurotoxicity of apoE4(1-272), and single mutations in the region of amino acids 273-299 (L279Q, K282A, or Q284A) made full-length apoE4 neurotoxic. Immunofluorescence staining showed that apoE4(1-272) formed filamentous inclusions containing phosphorylated tau in some cells and interacted with mitochondria in others, leading to mitochondrial dysfunction as determined by MitoTracker staining and flow cytometry. ApoE4(241-272) did not cause mitochondrial dysfunction or neurotoxicity, suggesting that the lipid-binding region alone is insufficient for neurotoxicity. Truncation of N-terminal sequences (amino acids 1-170) containing the receptor-binding region (amino acids 135-150) and triple mutations within that region (R142A, K146A, and R147A) abolished the mitochondrial interaction and neurotoxicity of apoE4(1-272). Further analysis showed that the receptor-binding region is required for escape from the secretory pathway and that the lipid-binding region mediates mitochondrial interaction. Thus, the lipid- and receptor-binding regions in apoE4 fragments act together to cause mitochondrial dysfunction and neurotoxicity, which may be important in Alzheimer's disease pathogenesis.

The complete nucleotide sequence and its organization of the genome of Barley yellow dwarf virus-GAV.

The complete nucleotide sequence of genomic RNA of BYDV-GAV was determined. It comprised 5685 nucleotides and contained six open reading frames and four un-translated regions. The size and organization of BYDV-GAV genome were similar to those of BYDV PAV-aus. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of other luteoviruses. The results showed that there was a high degree of identity between BYDV-GAV and MAV-PS1 in all ORFs except ORF5 and ORF6, which had only 87.4% and 70.2% identities respectively. The reported genomic nucleotide sequence of MAV was shorter than that of BYDV-GAV, but the comparison of the genomic nucleotide sequences for MAV-PS1 and GAV showed 90.4% sequence identity for the same region of the genome. According to the level of sequence similarities, BYDV-GAV should be closely related to BYDV-MAV.

Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice.

Apolipoprotein E (apoE) is found in amyloid plaques and neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brains, but its role in their pathogenesis is unclear. Previously, we found C-terminal-truncated fragments of apoE in AD brains and showed that such fragments can cause neurodegeneration and can induce NFT-like inclusions in cultured neuronal cells and in transgenic mice. Here, we analyzed apoE fragmentation in brain tissue homogenates from transgenic mice expressing apoE3 or apoE4 in neurons [neuron-specific enolase (NSE)-apoE] or astrocytes [glial fibrillary acidic protein (GFAP)-apoE] by Western blotting. The C-terminal-truncated fragments of apoE accumulated, in an age-dependent manner, in the brains of NSE-apoE4 and, to a significantly lesser extent, NSE-apoE3 mice; however, no fragments were detected in GFAP-apoE3 or GFAP-apoE4 mice. In NSE-apoE mice, the pattern of apoE fragmentation resembled that seen in AD brains, and the fragmentation was specific for certain brain regions, occurring in the neocortex and hippocampus, which are vulnerable to AD-related neurodegeneration, but not in the less vulnerable cerebellum. Excitotoxic challenge with kainic acid significantly increased apoE fragmentation in NSE-apoE4 but not NSE-apoE3 mice. Phosphorylated tau (p-tau) also accumulated in an age-dependent manner in NSE-apoE4 mice and, to a much lesser extent, in NSE-apoE3 mice but not in GFAP-apoE3 or GFAP-apoE4 mice. Intraneuronal p-tau inclusions in the hippocampus were prominent in 21-month-old NSE-apoE4 mice but barely detectable in NSE-apoE3 mice. Thus, the accumulation of potentially pathogenic C-terminal-truncated fragments of apoE depends on both the isoform and the cellular source of apoE. Neuron-specific proteolytic cleavage of apoE4 is associated with increased phosphorylation of tau and may play a key role in the development of AD-related neuronal deficits.

Astroglial regulation of apolipoprotein E expression in neuronal cells. Implications for Alzheimer's disease.

Although apolipoprotein (apo) E is synthesized in the brain primarily by astrocytes, neurons in the central nervous system express apoE, albeit at lower levels than astrocytes, in response to various physiological and pathological conditions, including excitotoxic stress. To investigate how apoE expression is regulated in neurons, we transfected Neuro-2a cells with a 17-kilobase human apoE genomic DNA construct encoding apoE3 or apoE4 along with upstream and downstream regulatory elements. The baseline expression of apoE was low. However, conditioned medium from an astrocytic cell line (C6) or from apoE-null mouse primary astrocytes increased the expression of both isoforms by 3-4-fold at the mRNA level and by 4-10-fold at the protein level. These findings suggest that astrocytes secrete a factor or factors that regulate apoE expression in neuronal cells. The increased expression of apoE was almost completely abolished by incubating neurons with U0126, an inhibitor of extracellular signal-regulated kinase (Erk), suggesting that the Erk pathway controls astroglial regulation of apoE expression in neuronal cells. Human neuronal precursor NT2/D1 cells expressed apoE constitutively; however, after treatment of these cells with retinoic acid to induce differentiation, apoE expression diminished. Cultured mouse primary cortical and hippocampal neurons also expressed low levels of apoE. Astrocyte-conditioned medium rapidly up-regulated apoE expression in fully differentiated NT2 neurons and in cultured mouse primary cortical and hippocampal neurons. Thus, neuronal expression of apoE is regulated by a diffusible factor or factors released from astrocytes, and this regulation depends on the activity of the Erk kinase pathway in neurons.