Einar Hallberg

Targeting cytokine expression in glial cells by cellular delivery of an NF-κB decoy

Inhibition of nuclear factor (NF)-κB has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer’s disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-κB decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer β amyloid peptide in the presence of the inflammatory cytokine interleukin (IL) 1β. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-κB binding activity and IL-6 mRNA expression, respectively.

ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 micro m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.

Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine

The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.

Localized caspase sensors for live cell imaging of amyloid-β induced apoptosis

Background:xa0Apoptosisxa0is an evolutionary conserved cellular process important for normal development, maintenance of tissue homeostasis and an effective immune system. Cysteine-aspartic proteases, ...

P3-278: Transcription factors as targets to block inflammation in neurodegenerative disorders

by 40% at age 5 months, with most severe neuronal loss in hippocampus. Surprisingly, complete absence of phospho-epitopes of protein tau (AT100, PHF1, AT8, TG3, MC1, PG5, AD2, . . .) with exception of AT270, indicated tauopathy not to be the cause of neuronal death. Moreover, TUNEL staining remained negative, while also no activated caspase-3 was detectable. Indices of neurodegeneration (Fluoro-Jade B) and activated glia (GFAP, CD45, CD11, MHCII) were not only present early, but remained important during the entire disease process. Microgliosis peaked around age 2.5 months while astrogliosis remained intense throughout. Degenerating neurons and activated microglia and astroglia could not be separated spatially or temporally using a panel of histochemical and immunohistochemical markers. Conclusions: Inflammation, initiated by microglia and maintained by astroglia, is demonstrated as the more important contributor to neurodegeneration. The p25 mouse model thereby offers great potential to identify the responsible triggers, i.e. secreted factors originating from the degenerating neurons, and the very initial molecular events of neurodegeneration.

Analysis of apoptotic processes in live cells

Background:xa0Neuronal and synaptic loss can be observed in several neurologic disorders, like Alzheimer’s disease (AD). The mechanism behind cell death in AD has been intensively studied and apoptos ...