Phosphatase 2A (PP2A) is a multimeric
enzyme, containing a catalytic and a structural component associated
with a large number of regulatory subunits. PP2A exerts a wide range
of cellular effects including regulation of the cell cycle and cell fate
determination during development. Studies aimed at attributing specific
functions to PP2A are hampered by the fact that phosphatase inhibitors
like okadaic acid are not completely specific. In order to understand
the in vivo role of PP2A and to identify the in vivo substrates, we are
applying a transgenic approach which is more effective because PP2A can
be controlled in a time- and tissue-specific manner.
We are especially interested in the role of
PP2A in the brain, where in addition to heart the highest levels of PP2A
are found. PP2A has been shown to be associated with both microtubules
and neurofilaments and to dephosphorylate in vitro both neurofilaments
and tau (a microtubule-associated protein). Hyperphosphorylated tau protein
is the principal constituent of neuro-fibrillary lesions in Alzheimer's
disease. Hallmarks of ALS (Amyotrophic Lateral Sclerosis) pathology include
neurofilament inclusions and altered neurofilament phosphorylation.
As a first step, we have generated mice lacking
the major catalytic isoform of PP2A, Ca. Mice homozygous for the mutant
allele die in utero at the onset of gastrulation. The mechanisms underlying
early lethality are currently investigated.
In order to assess the role of PP2A in neurons
we have started to develop two alternative strategies :
(A) A brain-specific knockout of PP2A is achieved
by producing a mouse strain carrying a lox-flanked Ca gene which will be
mated to a mouse strain expressing cre recombinase exclusively in neurons.
Cre expression will be driven by a promoter which is turned on late in
development! Most likely, by this approach embryonic or early lethality
due to the absence of PP2A in the periphery or during early neuronal development
will be prevented. When applicable, the lox-flanked Ca mice can be used
to achieve a tissue-specific knockout in organs other than the brain.
(B) The Ca homozygous knockout mice will be
rescued using the binary tetracycline-transactivator system. An HA (hemagglutinin)-tagged
version of Ca is expressed under the control of a tetracycline-sensitive
transactivator. Upon feeding of tetracycline at any postembryonic stage,
a null phenotype can be induced and eventually reverted. We plan to establish
primary neuronal cultures where tetracycline can be added and removed from
the culture medium ad libitum.