Interferon gamma induces protective non-canonical signaling pathways in primary neurons.

The signal transduction molecule, Stat1, is critical for the expression of type I and II interferon (IFN)-responsive genes in most cells; however, we previously showed that primary hippocampal mouse neurons express low basal Stat1, with delayed and attenuated expression of IFN-responsive genes. Moreover, IFNγ-dependent resolution of a neurotropic viral challenge in permissive mice is Stat1-independent. Here, we show that exogenous IFNγ has no deleterious impact on neuronal viability, and staurosporine-induced apoptosis in neurons is significantly blunted by the addition of IFNγ, suggesting that IFNγ confers a pro-survival signal in neurons. To identify the pathways induced by IFNγ in neurons, the activation of alternative signal transducers associated with IFNγ signaling was assessed. Rapid and pronounced activation of extracellular signal regulated kinase (Erk1/2) was observed in neurons, compared to a modest response in fibroblasts. Moreover, the absence of Stat1 in primary fibroblasts led to enhanced Erk activation following IFNγ addition, implying that the cell-specific availability of signal transducers can diversify the cellular response following IFN engagement.

Pre-synaptic C-terminal truncated tau is released from cortical synapses in Alzheimer's disease.

The microtubule-associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimer's disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal-specific antibodies show that in many synaptosome samples tau lacks a C-terminus. Flow cytometry experiments to quantify the extent of C-terminal truncation reveal that only 15-25% of synaptosomes are positive for intact C-terminal tau. Potassium-induced depolarization demonstrates release of tau and tau fragments from pre-synaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well-positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the pre-synaptic compartment in AD. Results demonstrate the abundance of tau, mainly C-terminal truncated tau, in synaptic terminals in aged control and in Alzheimer's disease (AD) samples. Tau fragments and dimers/oligomers are prominent in AD synapses. Following depolarization, tau release is potentiated in AD nerve terminals compared to aged controls. We hypothesize (i) endosomal release of the different tau peptides from AD synapses, and (ii) together with phosphorylation, fragmentation of synaptic tau exacerbates tau aggregation, synaptic dysfunction, and the spread of tau pathology in AD. Aß = amyloid-beta.

Isolation of synaptic terminals from Alzheimer's disease cortex.

Amyloid beta (Aß) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aß, but the Aß and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aß-positive synaptosomes with the goal of understanding the nature of Aß and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aß were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aß-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aß oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aß and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.

Alzheimer amyloid beta-peptide A-beta25-35 blocks adenylate cyclase-mediated forms of hippocampal long-term potentiation.

Progressive memory loss and deposition of amyloid beta (Abeta) peptides throughout cortical regions are hallmarks of Alzheimer's disease (AD). Several studies in mice and rats have shown that overexpression of amyloid precursor protein (APP) or pretreatment with Abeta peptide fragments results in the inhibition of hippocampal long-term potentiation (LTP) as well as impairments in learning and memory of hippocampal-dependent tasks. For these studies we have investigated the effects of the Abeta(25-35) peptide fragment on LTP induced by adenylate cyclase stimulation followed immediately by application of Mg(++)-free aCSF ("chemLTP"). Treatment of young adult slices with the Abeta(25-35) peptide had no significant effect on basal synaptic transmission in area CA1, but treatment with the peptide for 20 min before inducing chemLTP with isoproterenol (ISO; 1 microM) or forskolin (FSK;10 microM) + Mg(++)-free aCSF resulted in complete blockade of LTP. In contrast, normal ISO-chemLTP was observed after treatment with the control peptide Abeta(35-25). The ability of the Abeta(25-35) peptide fragment to block this and other forms of synaptic plasticity may help elucidate the mechanisms underlying hippocampal deficits observed in animal models of AD and/or AD individuals.

AD synapses contain abundant Aß monomer and multiple soluble oligomers, including a 56-kDa assembly.

Much evidence indicates that soluble amyloid beta (Aß) oligomers are key mediators of early cognitive loss, but the localization and key peptide species remain unclear. We have used flow cytometry analysis to demonstrate that surviving Alzheimer's disease (AD) synapses accumulate both Aß and phosphorylated tau (p-tau). The present experiments use peptide-specific X-map assays and Western blot analyses to identify the Aß peptide species in synaptosome-enriched samples from normal human subjects, neurologic controls, and AD cases. Aß40 peptide levels did not vary, but both Aß42 and Aß oligomers were increased in soluble AD extracts, with oligomer levels 20-fold higher in aqueous compared with detergent extracts. In Western blot analysis, a ladder of sodium dodecyl sulfate (SDS)-stable oligomers was observed in AD cases, varying in size from monomer, the major peptide observed, to larger assemblies up to about 200 kDa and larger. Multiple oligomers, including monomer, small oligomers, a 56-kDa assembly, and amyloid precursor protein (APP) were correlated with the Aß level measured in flow cytometry-purified synaptosomes. These results suggest that multiple amyloid precursor protein processing pathways are active in AD synapses and multiple soluble oligomeric assemblies may contribute to synaptic dysfunction.

Extensive p-tau pathology and SDS-stable p-tau oligomers in Alzheimer's cortical synapses.

Like amyloid beta (Aß) oligomers, tau aggregates are increasingly recognized as potential key toxic intermediates in Alzheimer's disease (AD) and as therapeutic targets. P-tau co-localizes with Aß in cortical AD synapses and may contribute to synapse dysfunction and loss. Flow cytometry analysis of synaptosomes from AD compared with aged cognitively normal cortex demonstrates increased immunolabeling for three p-tau antibodies (AT8, PHF-1 and pS422), indicating phosphorylation at multiple tau epitopes. Sequential extraction experiments show increased soluble p-tau in AD synapses, but a sizable pool of p-tau requires detergent solubilization, suggesting endosomal/lysosomal localization. P-tau is co-localized with Aß in individual synaptosomes in dual labeling experiments, and flow cytometry sorting of Aß-positive synaptosomes from an AD case reveals a marked enrichment of p-tau aggregates. The p-tau enrichment, a 76-fold increase over the initial homogenate, is consistent with sequestration of p-tau in internal synaptic compartments. Western analysis of a series of AD and normal cases shows SDS-stable tau oligomers in the dimer/trimer size range in AD samples. These results indicate that widespread synaptic p-tau pathology accompanies Aß accumulations in surviving synaptic terminals, particularly in late-stage AD.