Drug discovery for age-related diseases and their targets

Insight into biological pathways, identification of new targets and appropriate biomarkers are the prerequisites for drug discovery & development.
This PhD thesis addresses the role of fundamental research in drug discovery in age-related Alzheimer’s disease (AD), and a cancer-related target,
the cell cycle.
The large economic impact of AD, exponentially increasing in the ageing societies of industrialised countries strongly urges for discovery and
development of drugs and therapy-related tools. As a consequence, the interest for retinoids based on their confirmed involvement in the regulation
of disease-related genes in neurodegenerative ailments including AD has been revived, even though details of their molecular modes of action remain
to be elucidated.
The first project described in this PhD thesis focuses on the regulation of alpha-, beta-, and gamma-secretases mediated by all-trans-retinoic acid
(ATRA), the main metabolite of retinoids. Our findings support the hypothesis that ATRA is capable of regulating secretases in the anti-amyloidogenic
sense at the levels of transcription, translation, and activation. Apart from increased alpha-secretase activity, we show a complex chain of
regulatory events, resulting in impaired beta-secretase trafficking and its membranal localisation due to protein kinase C (PKC) activation by ATRA,
resulting in enhanced secretion of soluble APPalpha. ATRA also affected activity, transcription and localisation of Presenilin 1, the functional core of
the gamma-secretase, but had no effect on Presenilin 2. The experiments were performed in three neuronal and non-neuronal cell lines of human and
murine origin, which allowed us to determine general mechanisms and cell line-specific differences in the ATRA-mediated regulation of secretases.
Although control of gene expression by retinoids occurs mainly via genomic regulation of target genes, anti-amyloidogenic effects of retinoids have
been attributed to their PKC-stimulating ability and subsequent activation of alpha-secretases only. Therefore, contribution of genomic and
non-genomic control mechanisms to the regulation of BACE1, the main beta-secretase, by ATRA was examined in the second project.
Our findings corroborated the role of PKC stimulation in the down-regulation of BACE1 at the transcriptional and translational levels, resulting in
decrease of Abeta42 in the human embryonic kidney HEK 293 cell line. ATRA induced DNA-binding activity of its receptors RARbeta-RXR, and showed PKC-
dependent activation of the transcription factors NFkB and AP-1, as revealed by electrophoretic mobility shift assay (EMSA). We show disruption of a
PKC-modulated decrease of BACE1 mRNA and Abeta levels, when NFkB or AP-1 activity was blocked by BAY11-7082, NFkB inhibitor, or by the transfection
of inhibitory DNA-oligonucleotides. For the first time PKC-signalling induced by retinoids could be linked to the transcriptional control of BACE1
regulation.
The last project described in this PhD studied the role of ADAM17 in the the cell cycle. ADAM17 is gaining attention in the field of clinical
research as a potential outcome biomarker in breast cancer and adenocarcinoma. However, a possible implication of ADAM17 in the cell cycle has not
been investigated, since its role in cancer is usually considered in the context of epidermal growth factor receptor signalling.
First, through a combination of mass spectrometry and biochemical methods, we identified alpha- and beta-tubulins as binding partners of ADAM17.
This interaction was confirmed bidirectionally in the murine neuroblastoma N2a cell line, and the ADAM17 fragments involved in tubulin-binding were
identified. Additionally, we showed co-localisation of ADAM17 and tubulins in the spindle of mitotic cells. Furthermore, we were able to show a cell
cycle-dependent regulation of ADAM17 by over-expression of either full length or mature ADAM17 versions in N2a cell line and observed significantly
increased invasiveness due to over-expression of mature ADAM17 form. Still, biological significance of the interaction between ADAM17 and tubulins
remains to be discovered.
Altogether, these studies examined existing theories, raised new hypothesis, and contributed to the identification of new targets and a clearer
understanding of fundamental mechanisms.