Jens Brodbeck

Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons

Convergent evidence has revealed an association between insulin resistance and Alzheimers disease (AD), and the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, an insulin sensitizer and mitochondrial activator, improves cognition in patients with early or mild-to-moderate AD. Apolipoprotein (apo) E4, a major genetic risk factor for AD, exerts neuropathological effects through multiple pathways, including impairment of dendritic spine structure and mitochondrial function. Here we show that rosiglitazone significantly increased dendritic spine density in a dose-dependent manner in cultured primary cortical rat neurons. This effect was abolished by the PPAR-γ-specific antagonist, GW9662, suggesting that rosiglitazone exerts this effect by activating the PPAR-γ pathway. Furthermore, the C-terminal-truncated fragment of apoE4 significantly decreased dendritic spine density. Rosiglitazone rescued this detrimental effect. Thus, rosiglitazone might improve cognition in AD patients by increasing dendritic spine density.

Intron-3 Retention/Splicing Controls Neuronal Expression of Apolipoprotein E in the CNS

Neuronal expression of apolipoprotein (apo) E4 may contribute to the pathogenesis of Alzheimers disease (AD). In studying how apoE expression is regulated in neurons, we identified a splicing variant of apoE mRNA with intron-3 retention (apoE-I3). ApoE-I3 mRNA was detected in neuronal cell lines and primary neurons, but not in astrocytic cell lines or primary astrocytes, from humans and mice by reverse transcription (RT)-PCR. In both wild-type and human apoE knock-in mice, apoE-I3 was found predominantly in cortical and hippocampal neurons by in situ hybridization. Cell fractionation and quantitative RT-PCR revealed that over 98% of the apoE-I3 mRNA was retained in the nucleus without protein translation. In transfected primary neurons, apoE expression increased dramatically when intron-3 was deleted from a genomic DNA construct and decreased markedly when intron-3 was inserted into a cDNA construct, suggesting that intron-3 retention/splicing controls apoE expression in neurons. In response to excitotoxic challenge, the apoE-I3 mRNA was markedly increased in morphologically normal hippocampal neurons but reduced in degenerating hippocampal neurons in mice; apoE mRNA showed the opposite pattern. This apparent precursor–product relationship between apoE-I3 and apoE mRNA was supported by a transcriptional inhibition study. Thus, neuronal expression of apoE is controlled by transcription of apoE-I3 under normal conditions and by processing of apoE-I3 into mature apoE mRNA in response to injury.

Small Molecule Structure Correctors Abolish Detrimental Effects of Apolipoprotein E4 in Cultured Neurons

Background: Apolipoprotein E4 (apoE4), the major gene involved in Alzheimer disease, has a unique structure, intramolecular domain interaction, that is associated with neuropathology. Results: Potent small molecule structure correctors block apoE4 domain interaction and reverse apoE4 detrimental effects in cultured neurons. Conclusion: Structure correctors negate the detrimental effects of apoE4 in neurons. Significance: ApoE4 structure correctors could represent a therapeutic approach for treating apoE4-associated neuropathology. Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC50 of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.

Structure-dependent Impairment of Intracellular Apolipoprotein E4 Trafficking and Its Detrimental Effects Are Rescued by Small-molecule Structure Correctors

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

Arf4 Determines Dentate Gyrus-Mediated Pattern Separation by Regulating Dendritic Spine Development

The ability to distinguish between similar experiences is a critical feature of episodic memory and is primarily regulated by the dentate gyrus (DG) region of the hippocampus. However, the molecular mechanisms underlying such pattern separation tasks are poorly understood. We report a novel role for the small GTPase ADP ribosylation factor 4 (Arf4) in controlling pattern separation by regulating dendritic spine development. Arf4+/− mice at 4–5 months of age display severe impairments in a pattern separation task, as well as significant dendritic spine loss and smaller miniature excitatory post-synaptic currents (mEPSCs) in granule cells of the DG. Arf4 knockdown also decreases spine density in primary neurons, whereas Arf4 overexpression promotes spine development. A constitutively active form of Arf4, Arf4-Q71L, promotes spine density to an even greater extent than wildtype Arf4, whereas the inactive Arf4-T31N mutant does not increase spine density relative to controls. Arf4′s effects on spine development are regulated by ASAP1, a GTPase-activating protein that modulates Arf4 GTPase activity. ASAP1 overexpression decreases spine density, and this effect is partially rescued by concomitant overexpression of wildtype Arf4 or Arf4-Q71L. In addition, Arf4 overexpression rescues spine loss in primary neurons from an Alzheimers disease-related apolipoprotein (apo) E4 mouse model. Our findings suggest that Arf4 is a critical modulator of DG-mediated pattern separation by regulating dendritic spine development.

O3-03-03: Apolipoprotein E4 and its fragment impair mitochondrial dynamics in neuronal cultures

internalization and cell viability in CBVs were increased when they were coincubated with conditioned media with high concentrations of apoE and with A . No internalization of A was observed in astrocytes, however. Conclusions: These results implicate that apoE has a more important role in regulating A internalization and A -mediated cytotoxicity in CBVs, than in astrocytes, which may be relevant to the process of A clearance across the blood brain barrier.

Arf4 regulates dendritic spine development in mouse primary neurons

co-immunoprecipitated with DISC1 at the endogenous protein level. Knockdown of DISC1 (lentivirus-mediated shRNA to DISC1) in mature primary cortical neurons, elicited no change in the levels of full-length APP (both mature and immature isoforms). In contrast, knockdown of DISC1 led to a significant increase in the levels of intracellular APP-C-terminal fragments C83 and C99. Knockdown of DISC1 also significantly enhanced sAPPa levels. Interestingly, we observed that DISC1 knockdown significantly decreased the levels of Ab42 and Ab40. The knockdown effects of DISC1 by RNAi on APP-CTFs, sAPPa and Ab 42/40 were successfully rescued by co-expression of wild-type DISC1. In the next step, we addressed whether DISC1 influences APP processing by using DISC1 knockout (KO) mice. We observed a significant increase in both C83 and C99 APP CTFs, with no change in the total APP levels in the DISC1 KO mice, as was the case in the primary neurons. Conclusions: DISC1 affects the processing of APP and generation of Ab in primary neuron cultures. Our goal is to characterize a novel mechanism of the regulation of APP processing/metabolism by DISC1.