Anke Schmidt

Cerebral amyloid-β proteostasis is regulated by the membrane transport protein ABCC1 in mice

In Alzheimer disease (AD), the intracerebral accumulation of amyloid-β (Aβ) peptides is a critical yet poorly understood process. Aβ clearance via the blood-brain barrier is reduced by approximately 30% in AD patients, but the underlying mechanisms remain elusive. ABC transporters have been implicated in the regulation of Aβ levels in the brain. Using a mouse model of AD in which the animals were further genetically modified to lack specific ABC transporters, here we have shown that the transporter ABCC1 has an important role in cerebral Aβ clearance and accumulation. Deficiency of ABCC1 substantially increased cerebral Aβ levels without altering the expression of most enzymes that would favor the production of Aβ from the Aβ precursor protein. In contrast, activation of ABCC1 using thiethylperazine (a drug approved by the FDA to relieve nausea and vomiting) markedly reduced Aβ load in a mouse model of AD expressing ABCC1 but not in such mice lacking ABCC1. Thus, by altering the temporal aggregation profile of Aβ, pharmacological activation of ABC transporters could impede the neurodegenerative cascade that culminates in the dementia of AD.

E2F1 in Melanoma Progression and Metastasis

Metastases are responsible for cancer deaths, but the molecular alterations leading to tumor progression are unclear. Overexpression of the E2F1 transcription factor is common in high-grade tumors that are associated with poor patient survival. To investigate the association of enhanced E2F1 activity with aggressive phenotype, we performed a gene-specific silencing approach in a metastatic melanoma model. Knockdown of endogenous E2F1 via E2F1 small hairpin RNA (shRNA) expression increased E-cadherin expression of metastatic SK-Mel-147 melanoma cells and reduced their invasive potential but not their proliferative activity. Although growth rates of SK-Mel-147 and SK-Mel-103 xenograft tumors expressing E2F1 shRNA or control shRNA were similar, mice implanted with cells expressing E2F1 shRNA had a smaller area of metastases per lung than control mice (n = 3 mice per group; 5% vs 46%, difference = 41%, 95% confidence interval = 15% to 67%; P = .01; one-way analysis of variance). We identified epidermal growth factor receptor as a direct target of E2F1 and demonstrated that inhibition of receptor signaling abrogates E2F1-induced invasiveness, emphasizing the importance of the E2F1-epidermal growth factor receptor interaction as a driving force in melanoma progression that may serve as a paradigm for E2F1-induced metastasis in other human cancers.

Selective Targeting of Adenoviral Vectors to Neural Precursor Cells in the Hippocampus of Adult Mice: New Prospects for In Situ Gene Therapy

The adult brain contains neural precursor cells (NPC) that are attracted to brain lesions, such as areas of neurodegeneration, ischemia, and cancer. This suggests that NPC engineered to promote lineage‐specific differentiation or to express therapeutic genes might become a valuable tool for restorative cell therapy and for targeting therapeutic genes to diseased brain regions. Here we report the identification of NPC‐specific ligands from phage display peptide libraries and show their potential to selectively direct adenovirus‐mediated gene transfer to NPC in adult mice. Identified peptides mediated specific virus binding and internalization to cultured neurospheres. Importantly, peptide‐mediated adenoviral vector infection was restricted to precursor cells in the hippocampal dentate gyrus of pNestin‐green fluorescent protein transgenic or C57BL/6 mice. Our approach represents a novel method for specific manipulation of NPC in the adult brain and may have major implications for the use of precursor cells as therapeutic delivery vehicles in the central nervous system.

Efficient Near-Infrared In Vivo Imaging of Amyoid-β Deposits in Alzheimer's Disease Mouse Models

The development of early diagnostic and prognostic tools for the visualization of amyloid-β (Aβ) deposits is one important focus of current imaging research. In patients with Alzheimers disease (AD), non-invasive and efficient detection of soluble and aggregated Aβ is important to determine the immediate success of intervention trails. The novel near infrared-fluorescence (NIRF) probe THK-265 efficiently penetrates the blood-brain barrier and has a strong and efficient binding to cerebral Aβ. Ex vivo microscopy of i) THK-265-labeling of plaques in paraffin-embedded tissue and ii) cerebral cryo-sections after intravenous injection of THK-265 confirmed a systematic increase of the NIRF signal corresponding to Aβ plaque number and size during disease progression. Furthermore, we investigated different stages of plaque formation in amyloid-β protein precursor transgenic mice in vivo after intravenous application of THK-265 to evaluate different aggregation levels with NIRF signals. The intensity of the NIRF signal correlated well with the plaque burden, indicating its utility for direct monitoring of Aβ aggregation progression. In summary, our results support the use of the NIRF probe THK-265 for the diagnosis and direct visualization of amyloid deposits and open the possibility for efficient, pre-symptomatic monitoring of Aβ deposition in the aging brain.

Novel subventricular zone early progenitor cell-specific adenovirus for in vivo therapy of central nervous system disorders reinforces brain stem cell heterogeneity

Neural stem/progenitor cells (NSPCs) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our previous targeting strategies have indicated precursor cell heterogeneity between different brain regions that warrants the development of NSPC-specific delivery vehicles. Here, we demonstrate a target-specific adenoviral vector system for the in vivo manipulation of progenitor cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity, SNQLPQQ, was expressed in conjunction with a bispecific adaptor molecule. To verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPCs in the SVZ was achieved, whereas hippocampal NSPCs were negative. Our results offer an expeditious and simple tool to produce retargeted viral vectors for a specific and direct in vivo manipulation of these progenitor cells. This powerful technique provides an opportunity to develop innovative strategies and express therapeutic genes in specific types of neural progenitor cells to allow success in treatment of brain disorders.

106. Novel Subventricular Zone Early Progenitor Cell Specific Adenovirus for In Situ Brain Stem Cell Bioengineering Unveils Cellular Heterogeneity

Neural stem/progenitor cells (NSPC) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our recent endeavors in the generation of Ad vectors that specifically target SPC in situ have focused on linking selective peptides to viral particles that direct the vector to yet unknown receptors on the surface of stem cells. Using an Ad-PEGylation approach, we demonstrated that a short artificial peptide, which specifically binds neural precursor cells established from the hippocampal area of adult mice or rats, was highly effective in targeting NSPC after vector administration into the brain. These studies also revealed that long-term transgene expression was restricted to precursor cells located in dentate gyrus, and was not detected in other brain regions such as the SVZ and the ependyma of the lateral ventricle. This new indirect evidence for precursor cell heterogeneity in different brain regions that warrants the development of NSPC-specific delivery vehicles was further investigated. Here, we demonstrate a target-specific adenoviral vector system for in vivo manipulation of stem cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity was expressed in conjunction with a bispecific adaptor molecule. In order to verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPC in the SVZ was achieved, whereas hippocampal NSPC were negative. Our results offer an expeditious and simple tool to produce target-specific viral vectors for selective and direct bioengineering of stem cells in situ. This powerful technique provides an excellent opportunity to gain new insight into the fundamental mechanisms that control stem cell fate and to develop innovative strategies for a successfull treatment of brain disorders.This work was supported by the German Research Foundation (DFG) grant PU 188/8-1, WR 20/2-1 and Research funding provided by the Medical Faculty of Rostock University (FORUN program).