Bryostatin%20and%20lactacystin%20affect%20PKC%20activation

1
Bryostatin and lactacystin affect PKC
activation and Long Term Memory
Christopher J Gagliardi, Alan Kuzirian, Herman
Epstein, Daniel Alkon and Christie
Parish Roger Williams University, One Old Ferry
Road, Bristol, Rhode Island 02809 Marine
Biological Laboratory, 7 MBL street, Woods Hole,
Massachusetts 02543 Blanchette Rockefeller
Neurosciences Institute, 9610 Medical Center
Drive, Rockville, Maryland 20850
Effect of 4 Hours of lactacystin plus Bryostatin
Followed by Two Training Events
Nudibranch mollusk Hermissenda crassicornis
Abstract It has recently been shown that protein
synthesis plays an important role in the
acquisition of sensory learning and the retention
of memory. It has also been demonstrated that
particular stages of protein synthesis can be
manipulated by altering specific components of an
established calcium-activated pathway. In this
Ca2 activated pathway, protein kinase C (PKC) is
known to function as a key regulator through a
direct relationship between PKC activation and
the acquisition and retention of memory.
bryostatin, a macrocyclic lactone, has been shown
to be a potent activator of PKC in sub-molar
concentrations. Pavlovian conditioning of the
nudibranch mollusk, Hermissenda crassicornis,
generated by pairing two natural stimuli (light
and agitation) is known to produce an
unconditioned response when animals are tested
with light alone. Hermissenda have further
demonstrated both biochemically and behaviorally,
that multiple treatments with bryostatin
(0.25ng/mL) administered over several days can
induce long-term memory (LTM) from training
regimes that typically produce only short-term
memory (STM) in untreated animals. Furthermore,
to test for the possible relationship between
elevated levels of PKC and increased memory
retention, we inhibited the ubiquitin-mediated
degradation of PKC using the ubiquitin lipase
inhibitor lactacystin. Recent results indicate
that bryostatin administration, when coupled with
lactacystin, increased memory retention by a
factor of four compared to animals administered
bryostatin (0.25ng/mL) alone. And finally, to
demonstrate that protein synthesis was needed for
the retention of memory, anisomycin, a protein
synthesis inhibitor was used to block mRNA
translation. It was found that memory retention
dropped significantly when animals were given
anisomycin (1ug/mL) immediately following
associative training and bryostatin (0.25ng/mL)
administration. This present work may have
important implications for studies associated
with memory acquisition and retention in
Alzheimer's patients as the Ca2 activated, PKC
pathway operating in Hermissenda is equivalent in
humans.

Introduction The focus of our research has been
on understanding the process of memory and memory
retention. The organism we used to carry out our
studies is a small shell-less mollusk called a
Hermissenda Crassicornis. Hermissenda are
bio-chemically and bio-physically identical and
structurally similar to larger vertebrate
systems, making them ideal organisms for the
study neurological disorders such as Alzheimers
disease. In fact, Hermissenda possess an
identical calcium pathway that is interrupted in
Alzheimers patients. In this pathway, we focus
on protein kinace C (PKC) as a major modulator.
There exists a direct relationship between PKC
activation and the acquisition and retention of
memory. Our Hypothesis is that if the necessary
proteins are produced through drug induced PKC
activation and are present in subsequent days,
the organisms should exhibit long term memory
more readily than untreated organisms. The drug
we used to test the activation of PKC is
Bryostatin (bryo), a cancer drug in stage one
trials that is known to activate PKC in small
amounts. Bryo increases the translocation of PKC
leading to increased levels of calexcitin (CE).
We use the immunocytochemical intensity levels of
CE as a measure of both PKC and CE. Results show
that the short-term effects of repeated bryo
exposure down regulates the amount of CE present
in the cell. We hypothesized that this down
regulation of PKC was a result of the ubiquitin
pathway. To test the demonstrated effects of the
ubiquitin mediated degradation of PKC, we used
the ubiquitin lipace inhibitor lactacystine to
inhibit PKC degradation. To test our working
bio-chemical pathway, demonstrate that protein
synthesis is needed for the retention of memory
and to prove that PKC and CE are a part of this
pathway we used an mRNA translation inhibitor
called Anisomycin (ANI) to inhibit protein
synthesis.
Established Ca2 Activated Pathway
Time dependant addition of mRNA translation
inhibitor Anisomycin (1ug/mL)
Effects of Bryostatin (0.25ng/L) on Memory
Retention
A.M. Kuzirian, H.T. Epstein
H
H
CHO
CHO
Br
Br
Methods and Materials We used classic Pavlovian
conditioning to train our Hermissenda using two
natural stimuli paired to cause a conditioned
response. Hermissenda are naturally
photopositive, and move towards light.
Hermissenda are also inter-tidal organisms, when
exposed to a stimulus that mimics the ebb and
flow of the tide, like a shaking, Hermissenda
contract as to secure them self to a substrate.
With these two stimuli paired together, the
Hermissenda are trained to contract when exposed
to light. Each exposure to light paired with a
shaking is called a training event (TE). We are
able to determine how well an animal remembers
training events by exposing the animals to light
only and than measuring there body length change
by direct measurement. If an animal contracts, it
is said to display recall (retention of memory)
however, if it does not move, or if it elongates,
it is said to display no recall (no retention of
memory). Drugs in our experiments are
administered by immersion. The drugs are mixed
with seawater and than injected into the lanes of
Plexiglas trays fixed with openings for syringes.
These trays are used for both training and
testing the animals.
CH3
CH3

• References
• 1. Alkon, D. L., Epstein, H. T., Kuzirian, A.
  M.,Bennett, C.M., Nelson, T. J. 2005. PNAS
  10216432-16437.
• 2. Etcheberrigaray, R., Tan, M., Dewachter, I.,
  Kuiperi, C., Van der Auwera, I., Wera, S., Qiao,
  L., Bank, B.,
• Nelson,T.J., Kozikowski, A.P., et al. 2004.
  PNAS 10111141-11146.
• 3. Alkon, D. L., Nelson, T. J., Zhao, W.,
  Cavallaro, S. 1998. TINS 21529-537.
• 4. Olds, J.L., Golski, S., McPhie, D. L.,
  Olton,D., Mishkin,M., Alkon, D. L. 1990. J
  Neurosci 103707-3713
• 5. Muzzio et al., 2001. J Neurophysiol.
  851639-1647.
• 6. Mc Phie, D. L., Matzel,L. D., Olds, J. L.,
  Lester, D. S., Kuzirian, A.M. Alkon, D. L. 1993
  J Neurochem. 60646-651.
• 7. Bank, B., DeWeer,A., Kuzirian, A. M.,
  Rasmussen, H., Alkon, D. L. 1988. PNAS
  851988-1992.
• 8. Scharenberg et al., 1991. PNAS 886637-6641.
• 9. Farley, J., Schuman, E. 1991. PNAS
  882016-2020.
• 10. Wender et al., 1998. PNAS 956624-6629
• 11. Crow, T. 2004. Learn Mem 1131-39.
• 12. Ramirez, R. R., Gandhi, C.C., Muzziio,I. A.,
  Matzel, L. D. 1998. Learn Mem 4462-477.

Cl
Results/Discussion Our working bio-chemical
memory pathway begins with the postsynaptic
influx of Ca2, which when coupled with
co-factors archidonic acid and inositol
triphosphate, induce the autophosphorylation and
subsequent translocation of PKC to the cell
membrane. The autophosphorylation of PKC causes
the phosphorylation of calexcitin (CE) and its
subsequent translocation to the cell membrane.
CEs translocation to the cell membrane inhibits
potassium channels, which when coupled with
internal calcium release, leads to a long lasting
depolarization, increasing the resting potential
of the cell. This long lasting depolarization is
analogous to long-term potentiatian (LTP) in
vertebrate systems. Behavioral tests show
sub-optimal training normally producing
short-term memory (STM) in untreated animals can
be induced into long-term memory (LTM) when
organisms are treated with bryostatin. Organisms
given 6 TEs with no bryo demonstrae no recall,
but with 9TEs demonstrate significant recall.
However, 6 TEs given with bryo showed an
extended memory equal to untreated animals given
9 TEs. Tests given with the ubiquitin lipase
inhibitor lactacystin and bryo paired together
have shown far greater memory retention than
tests run with bryo alone, further suggesting
that PKC plays a major role in the acquisition
and retention of memory. Tests given with the
mRNA translation inhibitor anisomycin (ANI) and
bryo demonstrated recall equal to that of
untreated animals, compared to animals given bryo
alone with no ANI, which demonstrated significant
recall. This adds significant support to our
current working model.
NH2
OH
l
OH
Acknowledgments Financial support for travel form
RWU A special thank you to Alan Kuzirian and
Herman Epstein for being terrific mentors,and
brilliant scientists Janice Simmons and Nancy
Roderic for making my experience at MBL one I
will never forget Roxanna Smolowitz for letting
me us use her office and microscope for hours on
end All of the MRC staff and interns Dr. Cliff
Timpson for helping me compile this poster The
Marine Biological Laboratory summer internship
for making this poster possible