2022 Undergraduate Research Symposium: Sana Latif Learning and memory are critical functions that allow organisms to adapt to and respond to their environment. Long-term potentiation (LTP) is an important model for memory and involves the persistent strengthening of synapses. Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a kinase that has been identified as essential for the establishment of LTP. Due to its critical role in synaptic plasticity and memory formation, the enhancement of CaMKIIα function has been studied as a mechanism to improve memory formation. The novel intrabody VHH Anti-GluN1 (VHHAN1) developed by our lab has been shown to localize to and bind to endogenous N-methyl-D-aspartate receptors (NMDARs) in mouse models. The influx of Ca2+ was expected to activate the downstream target CaMKIIα which has been implicated in LTP. Previously, we showed that VHHAN1 could bind endogenous NMDARs in mouse brains and that we could bring CaMKIIα to the NMDAR location, where LTP initiates. This molecular approach was termed CLEVIR (CaMKIIα Local Enrichment by VHH for Improvement of memory). This local enrichment of CaMKIIα by the intrabody in the hippocampus improved contextual memory. Nevertheless, many questions remain. Since long-term memory is linked to the cerebral cortices, it is possible that applying the CLEVIR approach to broader regions of the brain can improve contextual memory better. We hypothesized that global expression of the CLEVIR (VHHAN1-CaMKIIα) approach in CaMKIIα expressing neurons of the hippocampal CA3, dentate gyrus, and cortices could improve memory processes more effectively as compared to CaMKIIα alone.

1. Synaptic local enrichment of CaMKIIa for memory improvement in mice
Background Methods
AAV-PHP.eB enables systemic expression of CLEVIR
Localization of CaMKIIa and CLEVIR
Conclusion
SANA LATIF1,, ANTHONY CHIFOR2,, JEONGYOON CHOI2, JOONGKYU PARK1,2
1Department of Pharmacology and 3Department of Neurology, Wayne State University School of Medicine
Detroit, MI 48201, USA
Long-term potentiation (LTP) is critical for memory formation.
Ca2+/calmodulin-dependent protein kinase II (CaMKIIa) is a
kinase that has been identified as essential for the
establishment of LTP1. This role of CaMKIIa has led to
studies on its use for memory improvement. Previously,
expression of activated CaMKIIa in the hippocampus
impaired avoidance memory and context discrimination in
rodents2. However, our prior work recognized the possibility
of administering CaMKIIa in postsynaptic regions proximal to
N-methyl-D-aspartate receptors (NMDARs) to improve
memory. The novel intrabody VHH Anti-GluN1 (VHHAN1)
developed by our lab has been shown to localize to and bind
to endogenous NMDARs in mouse models, leading to an
influx of Ca2+ and activation of CaMKIIa. This approach was
termed CLEVIR (VHHAN1-CaMKIIa).3 Local enrichment of
CaMKIIa by the intrabody in the hippocampus improved
contextual memory3.
References
• VHHAN1 can be globally expressed in the cortex, CA3, and
dentate gyrus using AAV expression
• CaMKIIa expression in these regions is broader and more
diffused, while the expression of CLEVIR is more localized
to the excitatory postsynaptic region
• VHHAN1 may potentially orient exogenous wild-type
CaMKIIa to excitatory postsynaptic regions in areas outside
of the hippocampus as well and can be potentially used to
improve contextual fear memory
1. Nicoll, R. A. A brief history of long-term potentiation. Neuron 93, 281–290 (2017)
2. Ye, S., Kim, J. I., Kim, J. & Kaang, B. K. Overexpression of activated CaMKII in the
CA1 hippocampus impairs context discrimination, but not contextual conditioning. Mol
Brain 12, 32 (2019)
3. Chifor, A., Choi, J. & Park J. NMDA receptor-targeted enrichment of CaMKIIa improves
fear memory. bioRxiv doi: https://doi.org/10.1101/2021.11.06.467164.
4. Morabito, G., Giannelli S., Ordazzo G., Bido S., Castoldi, V., Indrigo M., Cabassi T.,
Cattaneo S., Luoni M., Cancellieri C., Sessa A., Bacigaluppi M., Taverna S., Leocani L.,
Lanciego J. & Broccoli V. AAV-PHP.B-dedicated global-scale expression in the mouse
nervous system enables GBA1 gene therapy for wide protection from synucleinopathy.
Mol Ther 25: 2727-2742.
HA-CaMKIIa
VHHAN1-
HA-CaMKIIa
CaMKIIa
CLEVIR
Figure 2: Systemic expression of HA-CaMKIIa in the
hippocampal CA3, dentate gyrus, and cortex in comparison
to VHHAN1-HA-CaMKIIa
Scale bar = 50 mm
Hippocampal CA3 Cortex
Dentate Gyrus
anti-HA
anti-HA anti-Homer1
Hypothesis
Since memory is linked to the cerebral cortices, it
is possible that applying the CLEVIR approach to broader
regions of the brain beyond the hippocampus can
improve memory better. This was accomplished using an
AAV-DJ serotype known as AAV-PHP.eB​ which can infect
entire brains4. This AAV serotype was Cre-dependent. We
hypothesized that systemic expression of CLEVIR
(VHHAN1-CaMKIIa) in the cortex and hippocampus could
better improve contextual memory as compared to the
CaMKIIa condition alone. In order to test this hypothesis,
we had to confirm AAV-mediated systemic expression of
CLEVIR in the hippocampal CA3, dentate gyrus, and cortex
and then compare localization patterns of CaMKIIa alone
vs. CLEVIR.
Global AAV
injection of
CLEVIR
or CaMKIIa
Minimum
3 weeks of
expression
Brain extraction
and tissue
fixation
Tissue
sectioning, IHC,
and confocal
microscopy
Figure 1: Mice were injected with AAV-PHP.eB expressing either
CLEVIR (VHHAN1-CAMKIIa) or CAMKIIa alone (control condition).
Then, the brains were extracted so we could obtain tissue
sections, conduct immunostaining via IHC, and visualize our
results with confocal microscopy.
Figure 3: Differences
in expression of
CaMKIIa and
CLEVIR. While
CaMKIIa shows
diffused expression,
the expression of
CLEVIR is punctated
to the postsynaptic
regions of cells
Scale bar = 50 mm