The 37kDa/67kDa laminin receptor as a therapeutic target in prion diseases: potency of antisense LRP RNA, siRNAs specific for LRP mRNA and a LRP decoy mutant

Prion diseases are a group of rare, fatal neurodegenerative
diseases, also known as transmissible spongiform encephalopathies
(TSEs), that affect both animals and humans and include bovine
spongiform encephalopathy (BSE) in cattle, scrapie in sheep,
chronic wasting disease in deer and elk and Creutzfeldt-Jakob
disease (CJD) in humans. TSEs are usually rapidly progressive and
clinical symptoms comprise dementia and loss of movement
coordination. A common hallmark of TSEs is the accumulation of an
abnormal isoform (PrPSc) of the host-encoded prion protein (PrPc)
in the brains of affected animals and humans. PrPc is a highly
conserved cell surface sialoglycoprotein that is expressed in
several cell types, mainly neuronal cells, but its normal
physiological function is still not known. However, PrPc is
elementary for the acquisition and the replication of prion
diseases. Several inhibitors of the PrPSc formation have been
reported, but none of them showed great potency in an in vivo
application. Thus, the identification of the 37kDa/67kDa laminin
receptor (LRP/LR) as the cell surface receptor for prions opened a
new direction for the development of a TSE therapy. Currently, no
treatment to slow down or stop the disease process in humans with
any form of CJD is established. However, several strategies have
been investigated to find an anti-prion treatment including
development of a vaccination therapy and screening for potent
chemical compounds. In scrapie-infected neuronal cells, which
represent a widely used and well characterized in vitro model for
transmissible spongiform encephalopathies, the accumulation of
PrPSc has been prevented by transfection of (i) antisense LRP RNA,
(ii) small interfering RNAs targeting the LRP mRNA and (iii)
incubation with the polyclonal anti-LRP antibody W3. Furthermore,
the knock down of surface LRP/LR resulted in a reduction of the
cellular PrP levels, suggesting an interference with the PrP
internalization process. Thus, LRP/LR is required for the PrPSc
propagation in vitro and involved in the PrPc metabolism.Due to the
existence of several LR genes, a major step to investigate the role
of the 37kDa/67kDa laminin receptor in scrapie pathogenesis in vivo
is the generation of transgenic mice exhibiting a lower level of
LRP/LR. Hemizygous transgenic mice that express LRP/LR antisense
RNA under the control of the neuron-specific enolase (NSE) promoter
were generated and showed a reduced LRP/LR protein level in the
cerebellum and the hippocampus. Intracerebral inoculation of these
transgenic mice with the scrapie agent will show, whether the
accumulation of pathogenic PrPSc in the brain is delayed or
prevented due to a reduced LRP/LR level. A further therapeutic
anti-prion approach is given by LRP/LR deletion mutants that can be
secreted to the cell culture medium and might act as decoys.
Previously, it has been demonstrated that a transmembrane deletion
mutant is able to prevent PrPc binding and internalization. In
vitro studies using an N-terminally truncated LRP mutant,
representing the extracellular domain of LRP/LR (LRP102-295::FLAG),
revealed a reduced binding of (i) recombinant cellular PrP to mouse
neuroblastoma cells, (ii) infectious moPrP 27-30 to BHK21 cells and
(iii) interfered with the PrPSc propagation in chronically
scrapie-infected mouse neuroblastoma cells. Furthermore, a cell
free binding assay demonstrated the direct binding of the
LRP102-295::FLAG mutant to both PrPc and PrPSc. These results
together with the finding that that endogenous LRP levels remain
unaffected by the expression of the mutant indicate that the
secreted LRP102-295::FLAG mutant may act in a trans-dominant
negative manner as a decoy by trapping PrP molecules. To
investigate the therapeutic potential of the LRP102-295::FLAG decoy
mutant in vivo transgenic mice were generated ectopically
expressing LRP102-295::FLAG in the brain. Animals showed no
phenotype and transgene expression was detected in cortical and
cerebellar brain regions. An intracerebral prion inoculation of
these mice will prove whether the expression of the
LRP102-295::FLAG mutant can impair the PrPSc accumulation in the
brain and can thus, act as a alternative therapeutic tool in prion
diseases. The recent finding that experimental introduction of RNA
can be used to interfere with the function of an endogenous gene
(RNA interference) provided another tool for the development of
gene-specific therapeutics. In order to evaluate a gene transfer
therapeutic TSE strategy, human immunodeficiency virus
(HIV)-derived vectors that express short hairpin RNA (shRNA)
directed against the LRP mRNA were used. Following integration of
LRP-shRNA-expressing lentiviral vectors into the genome of neuronal
cells efficient LRP/LR downregulation was observed. In scrapie
infected neuronal cells, downregulation of the LRP gene expression
resulted in a diminishment of PrPSc propagation, providing a
further therapeutic strategy in the development of a TSE treatment.